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The role of arginine as the inducer of bacterial luciferase in Achromobacter fischeri ND

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Title:
The role of arginine as the inducer of bacterial luciferase in Achromobacter fischeri ND
Creator:
Quarles, Thomas Stephen, 1941-
Publication Date:
Language:
English
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vii, 77 leaves : illus. ; 28 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Bacteria ( jstor )
Bioluminescence ( jstor )
Cultured cells ( jstor )
Enzymes ( jstor )
In vitro fertilization ( jstor )
Luminescence ( jstor )
Molecules ( jstor )
Nitrates ( jstor )
Protein synthesis ( jstor )
Achromobacter fischeri ( lcsh )
Arginine ( lcsh )
Dissertations, Academic -- Zoology -- UF
Luciferase ( lcsh )
Zoology thesis Ph. D
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida, 1970.
Bibliography:
Bibliography: leaves 73-76.
General Note:
Manuscript copy.
General Note:
Vita.

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This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator(ufdissertations@uflib.ufl.edu) with any additional information they can provide.
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THE ROLE OF ARGININE AS THE INDUCER

OF BACTERIAL LUCIFERASE IN

ACHROMOBACTER FISCHERI ND













By
THOMAS STEPHEN QUARLES













A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY














UNIVERSITY OF FLORIDA 1970













ACKNOWLEDGMENTS


I wish to thank Dr. P. R. Elliott in the Department of Zoology and the Office of Radiation Control for their generosity in providing equipment and instrumentation for use in the performance of the research reported here. Appreciation is also expressed to Drs. James Gregg, William Carr, L. Berner and A.S. Bleiweis for the many helpful suggestions they offered and the materials they so frequently supplied during the course of this work.







































ii












TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . . . . ....... ii

LIST OF TABLES . . . . . . . . . . . . v

LIST OF FIGURES . . . . . . . . . . . . vi

INTRODUCTION . . . . . . . . . . . . . I

MATERIALS AND METHODS . . . . . . . . ....... 6

Culture Techniques and Procedure for Arginine Addition . 6 Light Measurement . . . .. . ......... . . . 6

Chromatographic Procedures . . . . . . . . 7

Quantitative Procedures . . . . . . . . . 8

Special Procedures .. . . . . . . . . . ... 9

RESULTS AND DISCUSSION . . . ... . . . . . . 11

Luminescent Response to Arginine . . . . . . . 11

In vitro estimation of enzyme activity .. .. .' . 15 Lag period . . . . . . . . . ... . 19

Arginine Specificity . . ....... . . ... .... 21

Stimulation by other compounds . . . . .. . 21

Arginine Uptake . . . . . . .. .. .... 26

Induced Periodicity . . . . ......... . 28

Arginine Concentrating Ability . . . . ..... 28

Arginine Metabolism . . . . . .. ... . . 31

Mechanism of Arginine Action . . . . . ... . 38

Effects of the nitrate ion . . . . . . . 38

Effect of arginine on growth . . . . . .. . 43


iii







Arginine incorporation into luciferase ........ 50 Effect of inhibitors of bioluminescence response . . 50 Effect of initial arginine concentration ........ 54 In vitro effects of arginine ............. 62

In vivo effector function of arginine ........ 66

Discussion of Mechanistic Interpretations .. ........ 68

SUMMARY AND CONCLUSION ................... .. 71

BIBLIOGRAPHY . . . . . . . . . . . . . 73

BIOGRAPHICAL SKETCH ................... .... 77















































iv













LIST OF TABLES

Table Page

1. Compounds Tested for Stimulation of In Vivo
4 Luminescence .......... ... ... ....... 22

2. Arginine Sparing of Canavanine Inhibition of Growth. ... 25

3. Long-term Effect of Various Nitrogenous Compounds on
Luminescence and Growth ..... . ........... 47














































V











LIST OF FIGURES

Figure Page

1. Relationship between growth of culture, argininestimulated bioluminescence and bioluminescence
in the absence of arginine (constituitive luminescence) ... .. ... . . . ..... . 13

2. Relationship of luminescence and growth in maximum
stationary phase cultures. .............. 16

3. Relationship of in vivo and in vitro measurement of
luminescence following addition of arginine to
a culture . . . ... . . . . . . . 18

4. Onset of luminescence following addition of arginine .. 20 5. Arginine uptake .. ............... ... 27

6. Periodicity in growth, luminescence and arginine uptake 29

7. Puromycin inhibition of 14C-argini.ne incorporation into
acid-insoluble material ............... 36

8. Effect of adding puromycin two minutes after adding
arginine ................ .... 37

9. Proposed electron transport scheme for nitrate
reduction in A. fischeri ......... . ... .. 39

10. Effect of high nitrate ion concentration on luminescence ................... .. 41

11. Effect of added nitrate in low concentration on arginine-stimulated luminescence ........ ... 42

12. Effect of added nitrate in low concentration on luminescence in A. fischeri ............. 44

13. Short-term effect of arginine on growth ......... 46

14. Effect of mitomycin on the luminescence response to added arginine . . . . . . . . . . 52

15. Effect of concentration of added arginine on in vivo luminescence . . . . . . . . . . . 56

16. Hill plot of arginine concentration effect ....... 58


vi









Figure Page

17. Hill plot of data for arginine requiring mutant ..... 59 18. Effect of second addition of arginine ........ . 61

19. Effect of arginine on in vitro luciferase activity .... 64

20. Effect of arginine on in vitro luciferase activity
in the absence of bovine serum albumin ..... . ..... 65

21. Effect of arginine on second in vitro flash ....... 67

22. Effect of puromycin addition to cultures containing
different concentrations of arginine ......... 69













































vii












INTRODUCTION


An arginine-inducible bacterial luciferase has been described recently (1). Two aspects of the phenomenon are unusual compared to previously described inducible bacterial enzyme systems. The first of these is that no known relationship exists between arginine and the reaction catalyzed by the induced enzyme. The other unusual feature is that these bacteria arecapable of de novo synthesis of the inducer and, in fact, do produce sufficient amounts of arginine to support growth under the conditions necessary for induction. This raises the question as to how the addition of exogenous arginine results in the observed increase in luciferase activity.

In 1961, Jacob and Monod (2) suggested a mechanism by which protein biosynthesis could be genetically controlled. Briefly, their model proposes a set of three regions on the genome associated with the synthesis of a protein. These regions are 1) the structural gene, which dictates the amino acid sequence of the specific enzyme protein, 2) the operator region, which controls the initiation of transcription of the structural gene, and 3) the repressor region, which produces a product that, by combining with the operator region, prevents initiation of protein biosynthesis. Another property of the repressor product, in an inducible system, is its ability to combine specifically with a small molecule, the inducer, such as a sugar or amino acid.

When combined with the inducer, the repressor material loses its

capacity to attach to the operator region of the genome. This frees the operator, permitting the initiation of enzyme synthesis. Alternately,


1






2

in a repressible system, the small molecule (termed co-repressor) combines with the repressor product and attaches to the operator to prevent transcription of the structural gene. The system under consideration in this study has been considered to be inducible rather than repressible (1). In bacterial inducible enzyme systems, the inducer molecule often is related to the induced enzyme as the substrate or as'a homologue of the substrate. However, this does not appear to be an absolute requirement (3).

An entirely different means for controlling enzyme activity has

been suggested by Monod, Changeux and Jacob (4), involving an "allosteric effector". The molecule serving as the "effector" is not necessarily a component in the reaction catalyzed by the affected enzyme, but may elicit a quantitative change in enzyme activity. According to the proposed model, combination of the effector with its target enzyme causes a structural modification of the protein which may result in an increase (activation) in the rate or a decrease (inactivation) in the rate of the catalyzed reaction, or in a stabilization of the enzyme protein (5-10). Since,under certain conditions, data for an enzyme exhibiting allosteric properties in vitro demonstrate an in vivo behavior very similar to an inducible system (7) both models will be considered here.

The organism used in the present study is the ND strain of the marine bacterium Achromobacter fischeri. Originally isolated for its ability to utilize nitrate as its sole nitrogen source, it was found to be essentially non-luminescent when cultured in a minimal nitrate medium (nitrate dark). P. R. Elliott and A. H. Farghaly (personal communication) reported that the strain luminesced normally when grown in the presence of peptone, and that the component responsible for this effect was specifically L-arginine. Early studies of the system indicated that the virtual






3

absence of light production in cultures grown with nitrate as the sole nitrogen source was due to the lack of functional enzyme required for luminescence.

Through the extensive use of various inhibitors of protein synthesis, Coffey (1) has demonstrated that the addition of L-arginine to a log phase culture growing on minimal nitrate medium results in the synthesis of new luciferase. He has found that new mRNA synthesis is required to effect this response. Elliott and Farghaly (personal communication) had previously shown that added arginine did not affect the level of activity of six other randomly selected enzymes, thus arguing against a generalized enzyme induction. These data, combined with those of Coffey, strongly suggest that exogenously added arginine is an inducer for the specific synthesis of luciferase.

The investigations of Coffey (1) indicated a very high specificity of L(+)-arginine as the inducer of luciferase in his strain of bacteria. Other amino acids, structural analogues of arginine, and ammonium salts were ineffective as inducers with the exceptions of L-proline, L-aspartic acid, and the arginine precursors L-ornithine, L-citrulline and L-argininosuccinic acid. The most efficient of these, L-citrulline, elicited a response that was only 37.3 percent as strong as that produced by L-arginine.

-On the basis of this and 14C-arginine experiments, Coffey concluded that arginine is the specific inducer molecule, and that the positive response to the other compounds results from intracellular conversions to or sparing of arginine. He also stated that the growth rate of the culture is not altered during the first two hours after treatment with arginine.

The first studies of the influence of arginine on bacterial growth and luminescent systems were carried out by McElroy and Farghaly (11) in a series of experiments on A. fischeri mutants isolated after exposure




4

to ultraviolet light. Among these were two strains which exhibited a specific nutritional requirement for arginine. Both mutants showed a linear growth response to low arginine concentrations. No luminescence was evident, however, unless the cell density after 48 hours exceeded 30 percent of that attained by the wild type A. fischeri in argininefree medium after the same incubation period. As the arginine concentration was increased beyond the level satisfying this condition, the slope of the growth versus arginine plot diminished and luminescence developed rapidly. Both growth and luminescence were measured only after an incubation period of forty-eight hours. No attempt to determine the short-term effect of added arginine was reported. The only other growth-supporting amino acid, ornithine, gave results similar to those reported for arginine. The workers concluded that the growth and luminescent systems were competing for the common factor arginine or some component containing arginine. The also pointed out that the wild type A. fischeri exhibited a distinct lag in the production of light during the early stages of rapid growth. This suggested that strong competition existed for some component common to both luminescent and growth systems even in the normal strain.

In a later study, Farghaly (12) determined the influence of various amino acids on light production and growth in wild type A. fischeri. Arginine proved no more effective than any other amino acid for stimulating luminescence and was less so than either lysine, methionine, histidine or ammonium salts.

The requirements for in vitro luminescence by the bacterial system have been well defined. Reduced flavine mononucleotide (FMNH2), luciferase, a long-chain saturated aldehyde in a buffer, and oxygen are sufficient to produce a flash of light (13). Work by Hastings and






5

his group (14) has indicated that FMNH2 reduces the enzyme directly and that no substrate in which arginine could be a component is involved in the reaction. It would appear, therefore, that arginine, as an inducer, does not bear a typical relationship to the induced enzyme.

The work reported here was carried out in an attempt to answer the following questions: Is arginine an inducer of luciferase or is some arginine metabolite responsible for the phenomenon? Does the luminescent response to arginine occur as a result of an induction process or as a result of some other mechanism? Finally, does arginine serve a double function by acting both as an inducer and as a stabilizer of the luciferase molecule?












MATERIALS AND METHODS


Culture Techniques and Procedure for Arginine Addition

In all experiments involving stimulation of luciferase activity, the cultures were grown in the minimal liquid medium described by Farghaly (12) modified to contain 0.76 grams per liter (g/1) NaNO3 (approximately 9 millimolar) to replace the (NH4)H2PO4. Complete broth contained both (NH4)H2P04, 0.5 g/l, and 1.0 percent peptone (weight per volume) in addition to minimal salts and glycerol (12).

Cultures were incubated 18 to 20 hours at 230C with reciprocal

shaking of 80 cycles per minute. Cell density was determined turbidimetrically with a Klett-Summerson colorimeter using a number 42 filter. Arginine was added to the culture when a reading was obtained of 80 to 100 Klett units. If the culture had overgrown this density, it was diluted with fresh medium to a reading of about 40 units and allowed to grow back to the usual cell concentration before introducing arginine.


Light Measurement

In.vivo light measurements were carried out in a light-excluded

tank maintained at 230C by a circulating water bath. An RCA 1P21 photomultiplier tube was positioned below a one-inch diameter window located in the bottom of the chamber. Mounted inside the tank was a rack for six 125 ml Erlenmeyer flasks, any one of which could be rotated into a fixed position over the phototube window. Aeration of the culture was maintained by reciprocal shaking of the rack at 80 cycles per minute. Normal operation of the photomultiplier was at a regulated 1000 volts potential. Cathodal current signal was measured on a strip-chart

6








servo-recorder with variable sensitivity by use of precision resistors for signal attenuation. The instrumentation gave linear response across the full range of light measurements.

In vitro bioluminescence measurements were carried out in 10 x 75 mm

test tubes placed in a fixed geometry relative to an RCA 1P21 photomultiplier tube in a light-excluded chamber. Reaction components were injected with a hypodermic syringe and needle without admission of extraneous light. The photomultiplier was operated at 1000 volts and the output signal traced by a recorder with 5 milliseconds response time.


Chromatographic Procedures

Thin-layer chromatography was performed using plastic sheets precoated with silica gel G (Eastman Distillation Products Industries) and a sandwich style developing chamber. Routine detection of amino acids was obtained with a 0.25 percent ninhydrin in acetone spray followed by heating at 1000C for about five minutes. Ehrlich's Reagent was sprayed for detection of ureides and Sakaguchi reagent for the identification of arginine (15). Tricarboxylic acid cycle intermediates were located by spraying with bromocresol green reagent (16). In addition to identification by specific color reactions, each unknown was required to co-chromatograph with a known standard in at least three solvent systems before being considered identified (15, 16). A similar procedure was employed to ascertain the chemical purity of compounds used to stimulate luminescence in the cultures.

Ion exchange chromatography columns were prepared and operated

according to the procedure of Moore, Spackman and Stein (17) with the exceptions that Dowex 50-X-8 resin, 200-400 mesh, was used without additional particle sizing, and the dimensions of the columns were





8

11 mm by 100 cm and 11 mm by 20 cm. A fraction collecter was used to collect 2 ml samples for ninhydrin analysis or 10 ml fractions for isolation and subsequent identification of 14C-labeled compounds.

Samples to be de-salted were applied to a Dowex 50-X-8 resin column (15 mm x 150 mm) in the acid form, eluted with concentrated NH40H and evaporated. For one series of experiments, small organic molecules were isolated from macromolecules and salts by water elution of the samples from a 9 mm x 150 mm column of Sephadex G-10.


;Iuantitative Procedures

The ninhydrin method of Moore and Stein (18) was employed for

measuring free amino acids in membrane (Millipore Corporation, 0.22 micron pore size) filtered samples of medium and effluent fractions collected from column chromatography. A spectrophotofluorimetric procedure specific for guanido groups (19) was used to estimate arginine concentration in these samples.

Uniformly labeled 14C-L-arginine was employed in all experiments

involving isotopes. Radiochemical purity of each new supply was checked by thin-layer chromatography in two solvents. For most experiments, radioactive arginine was added to a final concentration of approximately 10 microcuries per liter and unlabeled arginine used as a carrier to provide a sufficient concentration for the stimulation of luminescence. For each experiment, the specific activity of the radioactive arginine is indicated.

Radioactivity was measured in a liquid scintillation counter using

Bray's solution as the scintillation fluid (20). All samples were counted for a minimum of ten minutes, or until a significant number of counts above background were obtained (21). Absolute quantification was made




9

using the internal standard method (22). The following calculations were made for each vial:

cpmu

(cpmu + cpms) cpmu = dpmu

dpms

where cpmu and cpms are counts per minute due to the unknown and standard respectively and dpmu and dpms are similar expressions for disintegrations per minute.


Special Procedures

The ability of the bacteria to concentrate arginine was determined by measuring the difference in radioactivity per unit volume in samples of membrane-filtered medium and simultaneously collected samples of membrane-filtered culture containing trichloroacetic acid to lyse the bacteria and precipitate the proteins. The number of bacteria per ml of culture was determined by direct microscopic counts. Using the average dimensions of a cell of this species of bacteria as indicated in Bergey's Manual (23) and confirmed by measurements taken from electron photomicrographs, the volume of cells (including the cell walls) per ml of culture was calculated. By appropriate corrections for dilutions, the disintegrations per minute per unit volume of bacterial cells and per unit volume of extracellular medium were determined.

Cell-free extracts of the bacteria were prepared by osmotic lysis in distilled water. Luciferase was partially purified by the method of Hastings and McElroy (24). Enzyme activity was determined in a 10 mm x 75 mm test tube containing 1.5 ml 0.1 M phosphate buffer, pH 7.4; 0.5 ml 1 percent bovine serum albumin in water; 0.1 ml partially purified enzyme; and 0.5 ml dodecanal-saturated water solution. This tube with the assay





10

mixture was placed in the photometer and 0.2 ml FMNH2, 0.1 mg per ml, injected by syringe through a rubber cap. The peak intensity of the resulting light flash was used to express enzyme activity in millivolts (mv). It should be noted that these units are not directly comparable to similar units of in vivo luminescence since the geometry and electronic components of the two systems are not identical. Protein concentration was estimated by the method of Lowry, et al. (25).












RESULTS AND DISCUSSION


Luminescent Response to Arginine

The primary question concerns the role of arginine in the bioluminescent response evoked by the addition of this amino acid to a log phase culture of Achromobacter fischeri ND grown on minimal nitrate medium. Figure 1 shows the relationship of growth, the small amount of measurable luminescence of an untreated culture (constituitive luminescence), and the luminescence observed after adding arginine (final concentration

2.4 x 10 Molar) to early log phase cells. In Figure 1, and all succeeding figures containing data on light production, luminescence is plotted as readings taken from continuous recordings at one minute intervals in those regions of particular concern and at five minute intervals over the remainder of the curve.

As may be seen in Figure 1, the addition of arginine results in a 2,000-fold increase in in vivo luminescence compared to an untreated culture. The constituitive luminescence is measured at 12 to 15 my compared to approximately thirty volts following treatment with arginine. Maximum luminescence was observed to vary by as much as a factor of five, ranging from about ten volts to fifty volts in repeated experiments. Attempts to grow and treat cultures under uniform conditions did not improve the reproducibility of the measured maximum luminescence after adding arginine. Careful examination revealed that results from parallel samples of the same culture were comparable and findings from repeated experiments were consistent relative to controls, irrespective of the variation in maximum luminescence intensity.


11
























Figure 1: Relationship between growth of culture, arginine-stimulated bioluminescence and bioluminescence in the absence of arginine (constituitive luminescence). L-arginine in final concentration of 2.4 x 10-4M is added at arrow. Growth is determined turbidimetrically and expressed in Klett-Summerson (K-S) units. Luminescence curves are plotted from continuous recordings and expressed in millivolts (my).












3.0 Growth 600


"- "i-- Constituitive luminescence

2.5 -Stimulated luminescence 500




>2.0 400 '




1.5 5
300



1.0 200




0.5 100





14 16 18 20 22 24 26 28 30 Time (hours)





14

The cause for this variation in susceptibility to arginine stimulation of light production is not known at this time. Several factors

thought to be potentially responsible were investigated. Cultures grown on successive days in aliquots of medium taken from the same

large batch preparation did not respond equally to added arginine. Addition of arginine from the same stock solution did not insure a reproducible

maximum luminescence of the treated culture, independent of whether the

solution had been stored at room temperature, 4 0C or frozen at -150C

between use. The age of the culture from which subsequent inoculations were taken did not appear to bea factor since variations were noted in

cultures inoculated with log phase, stationary phase, or death phase

cells. Although culture age does influence the sensitivity to arginine

stimulation, as will be discussed later, the maximum response varied whether the amino acid was added after the same period of incubation or at the same cell density (as estimated turbidimetrically). Other

factors controlled prior to the addition of arginine included the adjustment of pH of the medium to 7.0, adding a fresh supply of glycerol and

* supplementing the nitrate concentration. Only one variable showed any

-correlation to--the problem of reproducibility; with increasing serial transfers in minimal nitrate medium, there was a tendency toward a

decrease in maximal luminescence when all other factors were held constant.

Sub-culturing in complete medium usually resulted in an increase of

sensitivity to arginine stimulation in subsequent minimal nitrate cultures.

In his work on arginine stimulation of bioluminescence in similar cultures grown on minimal nitrate medium, Coffey (1) reported this same difficulty with peak response to added arginine but offered no suggestions as to the

cause of the problem.





15

By the time a culture has reached the late log phase or early

stationary phase of its growth cycle, the constituitive luminescence increases. The onset of this increase may be seen in Figure 1 and, in Figure 2, it is shown in relation to culture age and luminescence resulting from added arginine. Although not shown in Figure 2, the constituitive luminescence maintains a relatively constant level for about seven hours and then begins to decline before the culture enters the death phase (indicated by viable cell counts performed every four hours). This is observed independent of previous arginine treatment. The addition of arginine to a culture during this period results in an increase in light production; however, the response is not as extensive as that elicited by an equal concentration of the amino acid added in early log phase.

In Vitro Estimation of Enzyme Activity

The increased luminescence of a culture following addition of

arginine to the medium reflects an increase in the intracellular concentration of functional luciferase, as measured by in vitro assays of enzyme activity. Cell-free crude extracts of aliquots removed from a culture at timed intervals after the introduction of arginine into the medium were analyzed for luciferase activity and protein concentration. In vitro light production per mg of protein (specific activity) in these extracts is plottedas a logarithmic function against the time at which the aliquots were removed from the culture (Figure 3).

Use of the peak flash intensity as a measurement of enzyme activity, as described in the Methods section, is a standard procedure in such assays. Flash height has been shown to be directly proportional to the total light output measured as the area under the response curve (12, 26). Moreover, this area (rather than a rate) is believed to represent enzyme












700




600


4J
0
o 1. 500






o Growth gy---s f 1 Stimulated
0.5 -- Luminescence Constituitive Luminescence


28 30 32 34 36 38 40 42 Time (hours Figure 2: Relationship of luminescence and growth in maximum stationary phase cultures. (Data are plotted as in Figure 1. Stimulated luminescence data are from culture treated with L-arginine at time indicated in Figure 1.

4
0"

























Figure 3: Rela ionship of in vivo and in vitro measurement of luminescence following addition of arginine (2.4 x 10 M) to a culture at time zero. In vitro luminescence is expressed in millivolts per milligram protein in crude cell-free extract. In vivo luminescence is expressed in millivolts per ml of culture.

















3.5




3.0

00 to 2.5




0 2.0

in vivo luminescence


1.5 in vitro luminescence




1.0 I I I I I
20 40 60 80 100 120 Time (minutes)
CO




19

activity since all excess reduced flavin mononucleotide (FMNH2) not utilized in the reaction within the first second is removed from the system by autoxidation (27). In view of the luminescent reaction kinetics, the time limitations imposed by FMNH2 availability have led Hastings et al.(28) to conclude that only a single catalytic cycle occurs in vitro.

Observation of the increase in specific activity of luciferase in

crude extracts shown in Figure 3 indicates an increase in the concentration of functional luciferase relative to total intracellular protein concentration. (Because of the extraction procedure, the unlysed cells and cellular debris were removed by centrifugation to yield a clear enzyme supernatant fraction.) This increase implies a differential rate of apparent synthesis of luciferase; that is, addition of arginine to a culture appears to promote a greater increase in the rate of luciferase synthesis specifically than it does in general protein synthesizing activity of the cell.

The in vivo luminescence observed in the culture from which the

enzyme assays were taken is also indicated in Figure 3. The slope, or rate of increase, of both curves is essentially parallel, deviating only at the extremes. Since the chemical environment involved in the estimation of enzyme activity under these two conditions is so different, some variation was expected.

Lag Period

A distinct lag of approximately 12 minutes is evident between the addition oi arginine and the first measurable increase in luminescence. Although it may be seen in Figure 1, the expanded scale of Figure 4 makes it more obvious. Increasing the arginine concentration ten-fold does not





20

















500

" Constituitive luminescence


400 Arginine-Stimulated luminescence

Arginine added at time 0 r 300




200




100





10 20 30 Time (minutes) Figure 4: Onset of luminescence following addition of arginine.






21

reduce the length of this lag; however, the lag begins to increase as
-4
the concentration is lowered below 1 x 10-4 M arginine. Coffey (1) has dealt rather extensively with this aspect of the problem and has concluded that approximately eight of the twelve minutes represent a period of "activation" of the enzyme, the primary structure of the protein being completed in the first four minutes after adding arginine to the medium. This lag period and Coffey's interpretation will be given further consideration later.


Arginine Specificity

Stimulation by Other Compounds

The stimulation of bioluminescence in A. fischeri ND shows a high degree of specificity for L-arginine. A number of compounds were tested for their ability to serve as stimulators; these are listed in Table 1. With the exceptions of L-citrulline and L-proline, as discussed below, they were uniformly ineffective in stimulation of in vivo bioluminescence over control levels.

The groups of compounds in-Table 1 were selected to provide some

insight into the mechanism by which arginine exerts its stimulating effect on the bioluminescence system. Because of the participation of arginine in the cycle leading to urea biosynthesis, the various intermediates in that pathway (Table 1, Group 1) were tested for their ability to stimulate luminescence in this strain of bacteria. In view of the lag period discussed above, which might represent the time required to effect a conversion of arginine to another compound, this approach seemed particularly promising. Also included in this group are L-glutamic acid and L-proline which are related to the urea cycle, and are derivable from arginine in Sacchromyces cerevisiae by the following initial steps:






22







TABLE I



Compounds Tested for Stimulation of In Vivo Luminescence



GROUP 1: Urea Precursors

L-Glutamic acid L-Ornithine Carbamyl Phosphate L-Citrulline
L-Proline L-Aspartic acid Urea


GROUP 2: Other "Amino" Compounds

Glycine L-Cysteine
L-Alanine L-Methionine
L-Valine L-Lysine
L-Leucine L-Histidine
L-Isoleucine L-Proline
L-Serine Ammonium Chloride
--L-Tyrosine Ammonium Phosphate L-Tryptophan Ammonium Sulphate
L-Threonine Adenine
L-Phenylalanine .Cytosine


GROUP 3: Non-amino Compounds

D-Glucose Citrate (Na+ salt)
Pyruvate (Na+ salt) Dodecanal Nitrite


GROUP 4: Arginine Analogues

Agmatine L-Canavanine
L-Homoserine L-Homoarginine
Guanidoacetic acid D-Arginine






23

arginine ornithine 0 glutamic 7 semialdehyde
+
urea

(29). The glutamic-a-semialdehyde may then be oxidized to glutamic acid or cyclized (via dehydration) to lead to proline synthesis (30). Of the compounds listed in Group 1, Table 1, two resulted in increased light production by the log phase cultures to which they had been added: L-citrulline and L-proline produced 400-fold and 20-fold increases respectively in in vivo luminescence compared to the luminescence level of an untreated culture. L-ornithine failed to show any stimulatory effect; however, within the limits of sensitivity of the ninhydrin procedure, no ornithine uptake could be detected. The response to added citrulline and proline is believed to result from intracellular conversion of these amino acids to arginine or as arginine sparing intermediates. This is acceptable as a working hypothesis, since citrulline and proline are involved in the general pathway leading to arginine synthesis and their effectiveness is directly related to their remoteness from arginine in that pathway.

In view of the apparent specificity for arginine, knowledge of the effectiveness of arginine analogues (Group 4, Table 1) should yield some insight into the specific functional group requirements for stimulation of bioluminescence in this organism. The importance of the guanido group was examined by supplying it in. the form of guanidoacetic acid, by replacing it with an hydroxyl group in the arginine structure (homoserine), and by adding both guanidoacetic acid and homoserine simultaneously to the culture. Agmatine was tested to determine the effectiveness of the guanido and amino groups in combination. None of these compounds, either singly or in combination, demonstrated any stimulatory effect on the luminescence reaction.






24

Specificity for the three-dimensional structure of arginine was also investigated (Group 4, Table 1). An increase of one carbon atom in the chain length (L-homoarginine) and the substitution of an oxygen for a carbon atom in the L-arginine chain (L-canavanine) were tested. Each of these changes alters the fundamental structure of L-arginine sufficiently to make it ineffective as a stimulator of the luminescence reaction.

D-arginine was tested to determine the influence of optical isomerism on arginine specificity. Addition of this compound to a culture produced no change in luminescence intensity relative to controls. However, ninhydrin analysis of the medium indicated that D-arginine does not enter the cells. For this reason, no conclusion may be drawn in terms of the effect of optical isomerism on the intracellular molecular specificity of the bioluminescence response.

It should be noted that the addition of L-canavanine to one of two cultures containing equal amounts of L-arginine (2.4 x 10-4 M) resulted in approximately 36 percent inhibition of the luminescence response compared to the arginine control. Canavanine was also found to inhibit growth of the organism. This inhibition could not be completely reversed by the addition of arginine to the medium in 10 times the canavanine concentration. The arginine sparing of growth inhibition by canavanine is shown in Table 2. Because of this'general inhibition of growth, and indications in other bacteria that canavanine interferes with the utilization of arginine for protein biosynthesis (31), the significance of the failure of canavanine to stimulate luminescence is somewhat difficult to assess. It is not known whether the specificity requirements are such that canavanine is not suitable as an arginine substitute or if its






25













TABLE 2



ARGININE SPARING OF CANAVANINE INHIBITION OF GROWTH



Flask Number' Arginine Concentration % Control Growth x 10-4 M Rate Control* 100

1 17.4 2 1.0 22.9 3 2.5 26.7 4 5.0 32.1 5 25 57.2





Each experimental flask contains 2.5 x 104M Canavanine

* Standard nitrate medium with no additions





26


effect on the general physiology of the cell prevents the appearance of an increase in luminescence. It is possible,for example, that luciferase is synthesized, but is non-functional due to incorporation of canavanine rather than arginine. This might be clarified by looking for the formation of protein which can be precipitated by anti-luciferase antibody.


Arginine Uptake

The specificity for exogenously added L-arginine and the delay in the onset of the increase in in vivo luminescence suggested a possible lag in arginine uptake by the cells, as might be occasioned by the requirement for induction of a permease or by a slow process of simple diffusion. Therefore a study of the rate of arginine uptake was attempted.

The rate of disappearance of ninhydrin-positive material from the medium after adding arginine was used as a measure of the uptake of the amino acid (Figure 5). Treatment with permutit (0.4 g/ml) to remove ammonia produced no significant change in values obtained from treated and untreated parallel samples. Although numerical values did not correspond exactly from one experiment to another, data plotted on the basis of triplicate ninhydrin assays of each sample in ten experiments yielded essentially identical curves. Two additional methods more specific for arginine were also used to measure the uptake of this amino acid. One was a quantitative fluorescence procedure reported to be specific for guanido and ureide groups (19); the other was the disappearance of uniformly labeled 14C-arginine. Results of three experiments involving each of these methods demonstrated uptake kinetics different from those determined using the ninhydrin reaction. Figure 5 illustrates data taken from a single experiment in which all three procedures were employed. Guanido-group and 14C-label assays show close agreement in their variation in time, but the ninhydrin results are seen to be out of phase.










1.0 4Uniformly 1
Uniformly labeled C-arginine SNinhydrin-positive material


0.9 T Guanido-group




0.8




0.7
.4



0.6




0.5




0.4
10 20 30 40 50 60 70 80 90 Time (minutes) Figure 5: Arginine uptake. (For each of the three methods plotted, the values were obtained from membrane-filtered samples of medium removed from a culture at the indicated times.
-j





28


Induced Periodicity

The regular fluctuation in the arginine uptake studies, shown in Figure 5, suggests a rhythmic behavior of the culture. Examination of other factors revealed a similar periodicity in both growth and in in vivo luminescence following the addition of arginine to a culture (Figure 6). Arginine uptake is taken from the 14C-label information presented in Figure 5. Growth is indicated in Klett-Summerson turbidity units measured at ten-minute intervals, and luminescence is plotted as points read from a recording of in vivo light production. A similar periodicity exists in all three factors measured.

There does not appear to be an inherent periodicity in either

bacterial luminescence or cell division: continuous recordings of light intensity produced by the wild type A. fischeri or by the strain being studied here (when grown in luminescence-supporting medium) reveal only a uniform increase as the culture develops. In addition, studies of cultures in complete or minimal nitrate medium (prior to adding arginine) indicate no periodicity in growth rate. Therefore, the rhythmicity observed in the factors shown in Figure 6 is believed to be due to the addition of arginine. Further work will be required to define more carefully the nature of the periodicity and to clarify the relationship shown in Figure 6.


Arginine Concentrating Ability

The rapid initial rate of arginine removal from the medium suggested that some system was operating to take up the amino acid at a rate greater than that expected for simple diffusion. Experiments were conducted to determine if this strain of bacteria was capable of accumulating intracellular arginine against a concentration gradient based on the relative intra- and extra-cellular concentration of 14C-label (introduced as uniformly









o 1.0 4 0.9 o 0.8 .- 0.7

n 0.6 H 0.5
4-J

0.4 120 110 -W 100

90


300 80 U

200

100



10 20 30 40 50 60 70 80 90 Time (minutes) Figure 6: Periodicity in growth, luminescence and arginine uptake. (Arginine[2.4 x 10"4 M added at time 0).





30

labeled 14C-arginine, specific activity 0.6 mC per mM, final concentration 1 x 10-5M). The procedure is somewhat complicated by the fact that arginine is utilized in protein synthesis and thus care must be exercised to measure only free arginine as the internal pool.

Interpretation of the above experiment must be in terms of the radioactivity per unit volume of cytoplasm and medium. To do this, the number of cells per ml of culture was determined directly by microscopic counts. The volume of a single cell was calculated as the volume of a cylinder having dimensions of the average cell size determined from electron photomicrographs of bacteria taken from log phase cultures. The dimensions used were found to compare favorably with those listed for wild type A. fischeri in Bergey's Manual (23).

Several sources of error in this method must be considered. The first is that direct counting of cells, in addition to sampling and dilution errors, does not distinguish viable cells. Since viable counts correspond very closely to direct microscopic counts of cells in early log phase cultures, and these experiments were performed on such cultures, it was assumed that direct counts would accurately represent the number of viable cells. A second error source is that the cells are not perfect cylinders; therefore the volume calculations are not exact. Deviation from cylindrical form in these bacilli occurs primarily as arounding of the ends, thus introducing little variation from the volume of a cylinder. Thirdly, the use of average cell dimensions introduces error since all the cells are not the same size. By using the mean value, it was assumed that variation about that measurement would be compensated. Further, it was assumed that the cells used to determine the average dimensions of these bacteria represented a random sample. Finally, error in volume calculations is occasioned by inclusion of the cell wall volume. No estimation






31

is available for this volume. However, the influence of this error is to decrease the apparent intracellular concentration of 14C-labeled material. Since the actual volume of cytoplasm in which the 14C-label is concentrated is less than the estimated volume, the real concentration gradient is greater than the calculated one. The error introduced by including any non-viable cells in the calculations would have a similar influence on the estimated concentration gradient.

Within these limitations, calculations indicated concentration

gradients of 1,180-foldafter 5 minutes and 1,870-fold after 10 minutes. In light of the above discussion of errors, these estimates are probably low. According to A.G. DeBusk (personal communication) a 500-fold concentration gradient estimated by these methods is sufficient to indicate a concentrating mechanism. Therefore, these values strongly suggest the existance of a mechanism for taking up arginine against a concentration gradient.


Arginine Metabolism

Figure 5 shows data for arginine disappearance from the medium in time. Although the curves for the measurements of guanido groups and 14
C-arginine show close agreement at all stages, the assay for ninhydrin positive material was out of phase with these two. Since the data for all three curves were obtained from the same samples, the presence of ninhydrin-positive materials other than arginine was indicated. Therefore, an examination of the medium by chromatography was carried out.

Proline was identified as the only ninhydrin-positive compound present in detectable quantities in minimal nitrate medium of a log phase culture prior to the addition of arginine. Chromatographed sample concentrations sufficient to yield a readily discernible yellow spot for





32

proline after spraying with ninhydrin revealed no other distinct area having a positive reaction. Thirty minutes after adding uniformly labeled 14C-arginine (specific activity 0.4 mC per mM; final concentration of
-4
2.4 x 10 M) to a culture, proline was chromatographically isolated from the medium, but the eluted material contained no radioactivity. However 14C-label was isolated in the forms of citrulline and urea as well as arginine. The findings implicated these materials in the discrepancy between the ninhydrin and 14C-label assays depicted in Figure 5.

A series of experiments were designed to elucidate the intracellular fate of arginine. For this work, an early log phase culture was exposed to uniformly labeled 14C-arginine (specific activity 6 mC per mM; final
-4
concentration 2.4 x 10 M) for 90 minutes. A trichloracetic acid (5 percent) extract of washed cells was chromatographed on a heated ion exchange column using a series of buffers for elution. Ten ml fractions were collected and assayed for radioactivity. Those fractions containing 14C-label were compared to an elution pattern achieved with known amino acids. Having thus made a tentative identification of most of the 14C-labeled compounds, confirmation was obtained by thin-layer co-chromatography with standards in two dimensions with two solvent pairs. The radioactive material was required to chromatograph with and only with the standard in all instances.

Compounds identified as bearing 14C-label (which had been introduced

as uniformly labeled 14C-arginine) were as follows: arginine, urea, ornithine, citrulline, arginino-succinic acid, proline, glutamic acid, oK-keto-glutaric acid, succinic acid, fumaric acid and malic acid. The latter four compounds demonstrated very low specific activity and do not appear to constitute a pathway of great importance in the early stages of arginine metabolism in this organism. Compounds showing the highest specific activity (other than arginine) were ornithine and citrulline.






33

Evidence from additional experiments does not indicate the presence of urease in this strain of bacteria. Aliquots of A. fischeri N D cultures were incubated in sealed vials in the presence of uniformly labeled 14C-arginine and hyamine hydroxide (Packard) for 20 minutes. At the end of this period, concentrated sulfuric acid was injected into the vial to kill the cells and to drive dissolved C002 out of solution. Subsequent analysis of the hyamine hydroxide for 14CO2 did not demonstrate any of this gas.

From the above results, it is clear that early arginine metabolism in this strain of bacteria does not lead to intermediates which were not also tried as stimulators of luminescence (Table 1). This argues against the hypothesis that arginine is converted to some other compound which then acts to stimulate the bioluminescence reaction. If such a hypothesis were correct, one of the arginine metabolites would be expected to be at least as effective as arginine for stimulating the apparent synthesis of luciferase. It can be argued that the preceding statement does not apply to ornithine, since there appears to be virtually no uptake of this amino acid.

A final series of experiments was carried out in an effort to confirm arginine specificity. It was essential to determine the time at which intracellular arginine conversion to other compounds begins. This was done by collecting one ml samples of .the culture at one minute intervals following the addition of uniformly labeled 14C-arginine. Each sample was washed with cold minimal salts and then each filter was placed in 5 ml of cold distilled water with gentle shaking for 20 minutes. Microscopic examination showed very few whole cells remaining after this treatment. The extracts were then concentrated under vacuum at room temperature







34

(about 270C) to a volume less than 1 ml. These concentrated samples were applied to a Sephadex G-10 column (1 cm x 12 cm) to separate the small organic molecules from macromolecules and inorganic salts by elution with distilled water. The isolated compounds were then co-chromatographed with 10 pg arginine in two dimensions, and in two solvent pairs (n-butanol: acetic acid:water, 3:1:1; ethanol:ammonia:water, 8:1:1; t-butanol:2-butanone: formic acid: water, 8:6:3:3; and n-butanol:pyridine: water, 1:1:1). Ninhydrin was used to locate the arginine, and the adsorbant in that area was scraped into vials for quantification of radioactivity. The remaining adsorbant was also analyzed for radioactivity. Results of these experiments indicated 14
C-label associated only with arginine for the first eight minutes. The nine-minute sample showed 20 counts per minute above background which were located in areas other than that associated with arginine. Based on the products of arginine catabolism indicated above, and on published Rf values for these compounds in the solvents used, the area containing arginine was believed to be isolated from areas containing other potentially labeled compounds. Therefore, within the sensitivity limits of the present methods, xnoconversion of arginine occurs for eight minutes after introducing it into a culture.

Puromycin acts by interrupting the growth of polypeptides (32).

Preliminary work on the amino acid composition of luciferase has indicated the presence of at least five arginyl residues per molecule (J.W. Hastings, 14
personal communication). Therefore, the cessation of C-arginine incorporation into TCA insoluable material following treatment with puromycin is felt to reflect the blockage of luciferase synthesis. This represents a maximum limit; biosynthesis of some proteins appears to be more susceptible than others to puromycin inhibition (33). The degree to which luciferase synthesis is sensitive to puromycin is not known, but the






35

apparent cessation to total protein synthesis implies the halt of luciferase synthesis. The time required for puromycin inhibition of total protein synthesis was estimated to be six minutes (Figure 7).

The factors to be considered in the final experiment in this series are three-fold: 1) there is an apparent lag of eight minutes between the addition of arginine to a log phase culture and the occurance of any arginine metabolites in the cytoplasm of the bacteria; 2) evidence indicates that puromycin completely inhibits protein synthesis within six minutes after its addition to a log phase culture; and 3) in vivo luminescence may be considered as a measure of intracellular luciferase concentration. If the first two considerations are valid, then it should be possible to add arginine to a log phase culture, and after a period of two minutes, add puromycin with the result that protein synthesis is completely stopped at about the same time that arginine metabolites become available to the cells. The appearance of any increase in luciferase concentration (implying de novo synthesis of the enzyme) in a culture treated in this manner would most probably be attributable to the presence of arginine rather than an arginine metabolite. Results of such an experiment were interpreted in this manner (Figure 8).

Although the time element is critical to the interpretation of this experiment, three considerations suggest that this approach is valid with respect to time. First, independent of the mechanism by which an arginine metabolite could affect luciferase synthesis, a finite time interval should be required to exert this influence. Second, and also independent of the mechanism, there must be a sufficient concentration of the molecular species responsible for the effect: this may reflect an affinity factor, or the element of probability that the proper interaction will occur. Lastly, there must be enough time for the synthesis of complete luciferase












4.0





-


C,
S3.0



p..




2.0-
Puromycin added at time 0

--- 20 ug/ml puromycin )..--... No puromycin


1 I I I I
-1 0 1 2 3 4 5 6 7 8 Time (minutes) Figure 7: Puromycin inhibition of 14C-arginine incorporation into acid-insoluble material. (Arginine added ten minutes before adding puromycin. Puromycin added at arrow.)















30 25

20 Puromycin
0
Q 15
Uo
i0
10 Arginine

5

0 I I I I I I I I
-5 0 5 10 15 20 25 30 35 Time (minutes)






Figure 8: Effect of adding puromycin two minutes after adding arginine.





-4





38

molecules: since puromycin interrupts growing polypeptide chains (32), any luciferase molecules synthesized in response to the added arginine, must be completed prior to the inhibition resulting from the presence of puromycin. Collectively, the hypothesis that a metabolite of arginine is responsible for the bioluminescence response following the addition of arginine to the medium of log phase cultures requires that the metabolite be present in sufficient concentration to exert its effect early enough to permit the synthesis of complete luciferase molecules, but still be below the limit of detection by the procedure employed. This seems rather unlikely.

Using a somewhat similar approach to the problem of arginine

specificity, Coffey (1) concluded that the stimulation of bioluminescence in a nitrate-utilizing strain of A. fischeri results from the presence of arginine specifically rather than a metabolite of that amino acid. The evidence presented here is offered in support of that conclusion.


Mechanism of Arginine Action

Effects of the Nitrate Ion

Studies of the enzyme nitrate reductase from wild type A. fischeri

led to the proposal of the electron transport scheme shown in Figure 9 (34). Knowledge that FMNH2 serves as the reducing agent in the bacterial luminescence system suggested to McElroy (35) that the addition of nitrate ions to a culture would inhibit light production. The rationale for this depends on the assumption that the supply of electrons to the cytochrome system is constant. If this is true, addition of nitrate should add a pathway for electron removal, thus diminishing the FMNH2 available to the light reaction. Therefore, one would expect luminescence to be reduced in a culture containing nitrate as compared to one without







39












+++ Bacterial DPNH(TPNH)------+ FMN(FAD)--- Fe Cytochrome---- 02



Nitrate Reductase NO3









Abbreviations used are: DPNH(TPNH) Reduced di-(tri) phosphopyridine nucleotide FAD Flavin adenine dinucleotide
+++
Fe Ferric ion 02.- Molecular oxygen NO Nitrate ion














Figure 9: Proposed electron transport scheme for nitrate reduction in A. fischeri (34).






40

that ion. In fact the simultaneous addition of large quantities of nitrate and arginine (2.4 x 10 -4M final concentration) to a log phase culture growing on nitrate medium does reduce the intensity of the light produced when measured 60 minutes after the time of the addition (Figure 10). Although a pronounced effect on growth (estimated turbidimetrically) is detected only after several hours, the reduction in luminescence intensity is probably related to growth inhibition rather than nitrate ions, since the addition of NaCl in high concentration produces a similar dimming of luminescence.

The addition sE a much lower concentration of nitrate ions (9 x 10-4M) results in an enhancement of luminescence (Figure 11). This suggests that the presumed drain on FMNH2 supplies which is imposed by nitrate is not solely responsible for the greatly reduced light production. If it had been found otherwise, i.e., that the presence of nitrate in the concentrations used in the medium did inhibit luminescence, then the addition of amino-nitrogen rich arginine could be interpreted as relieving the nitrate reduction pathway so that a normal flow of electrons to the bioluminescence reaction would be restored. This interpretation is, however, not consistent with the results presented.

Additional evidence against the argument that nitrate reduction depletes the FMNH2 pool at the expense of bioluminescence is found in several other observations. If growth on minimal nitrate medium results in amino-nitrogen limiting conditions, as discussed above, then other amino acids, and especially ammonium salts, would be expected to be at least as effective as arginine in stimulating light production; they were not.

The role of the step in which nitrate is reduced to nitrite is also questionable. Observations of cultures grown with.nitrite as the sole





41



















200




150



0
100




( 50





0.1 0.2 0.3
NaNO3 () Figure 10: Effect of high nitrate ion concentration on luminescence.
(Data taken 60 minutes after simultaneous addition of large concentration of nitrate ions and 2.4 x 10-4M arginine.)




42


















2.0
Arginine (2.4 x 10-4M) added at time 0

- Arginine (2.4 x 10-4 M) + NaN03(9 x 10 4M) added at time 0
c 1.5
/


0 1.0




~0.5





1 2 .* 3 Time (hours)









Figure 11: Effect of added nitrate in low concentration on arginine-stimulated luminescence.






43

nitrogen source (nitrite present in equal molarity to the nitrate concentration in the usual medium) revealed identical behavior with regard to the very low levels of constituitive luminescence and the response of the luminescence system to added arginine. On the other hand, nitrate reduction is involved in some manner with the luminescence system since the addition of 9 x 10-4M nitrate to a culture of wild' type A. fischeri results in an increase in light intensity similar to that found in the nitrate utilizing strain (Figure 12). Since the wild type A. fischeri has a nitrate reductase, but lacks the ability to reduce nitrite (35), this indicates that the effect of nitrate on the luminescence system in wild type is associated with the reduction of that ion. It has been proposed that two separate electron transport chains are involved; one leading to reduction of oxygen, the other to nitrate reduction (35). The significance of these alternate pathways relative to the present problem is not clear at this time, nor is the role of nitrate reduction. A reasonable initial step toward investigating this relationship seems to lie in determining the effect of adding nitrate on the intracellular pool of FMNH2. Effect of Arginine on Growth

McElroy and Farghaly (11) found that arginine must be supplied to the arginine requiring mutants in quantities sufficient to exceed a minimum level of growth in order to develop the bioluminescent system. Although they did not measure growth rate (change in turbidity in time) during the incubation period, it is assumed that in two cultures receiving equal inoculations initially, a difference in turbidity after a fixed incubation period (48 hours) represents a difference in growth rates in the two cultures. In order to determine if the nitrate utilizing mutant is analogous to their arginine requiring mutants, the immediate effect of arginine on growth rate in this strain was examined. The hypothesis





44












2.5


Complete medium
-4
--. Complete medium plus NaNO3 (9 x 10 M) added at time 0

o



1 r
2.0












1 .5 1 1 1 1 1 1.I 1 I
10 20 30 40 50 60 70 80 90 100 110 120 Time (minutes)







Figure 12: Effect of added nitrate in low concentration on luminescence in A. fischeri.





45

for these experiments was that endogenous arginine was present in quantities which would support growth but not luminescence. Plots of turbidity readings taken at 10-minute intervals on parallel samples of a culture, one containing 2.4 x 10 4M arginine and the other serving as an arginineless control, were essentially congruent over a three-hour period following the addition of arginine. This information is shown in Figure 13. It is noted that the periodicity seen in the arginine treated culture is not observed in the control.

The significance of this congruency is doubtful. Kjeldgard et al.(36) found a definite lag in the alteration of growth rate in their work on Salmonella typhimurium. Moreover, McElroy and Farghaly (11) found that the rate of change in bioluminescence was much more rapid than that in growth as the initial arginine concentration was increased above 0.01 mg per ml. Therefore an increase in arginine concentration could be expected to influence luminescence more than growth rate over a period of time.

An experiment was then performed to determine the long-term effect of arginine, and other compounds, on growth rate. This was done by estimating the doubling time of log phase cultures (having effectively the same turbidity) which were growing in liquid media supplemented with the indicated compound in a final concentration of 9 x 10-4M. For each substance listed, media were prepared with and without the standard concentration of nitrate present to determine the growth-supporting ability of the various materials listed. In addition to determining the doubling time, the bioluminescent intensity was measured for each culture at the same turbidity (where growth had occurred) and both were related to their respective values for cultures grown in medium supplemented with

1.0 percent peptone. These results are summarized in Table 3. In order















140 Arginine (2.4 x 104 M) added at time 0

130 Arginine present


120 Arginine absent

110


" 100

90

80

70
70 I I I I I I I

20 40 60 80 100 120 140 160 180








Figure 13: Short-term effect of arginine on growth.










LONG-TERM EFFECT OF VARIOUS NITROGENOUS COMPOUNDS ON LUMINESCENCE AND GROWTH


TABLE 3

NITROGEN NITRATE PRESENT NITRATE ABSENT
SOURCE % Growth Rate % Luminescence % Growth Rate % Luminescence


Peptone 100 100 100 100 Nitrate 54 0.06 60 0.14 Arginine 61 90 44 56

Aspartic acid 56 1.90 69 0.14 Ornithine 29 0.65 10 0.25 Citrulline 28 0.39 22 0.18 Urea 30 7.70 6 1.80

Glutamic acid 22 0.16 31 0.29 Proline 61 26 53 2.50

Homoserine No growth or luminescence measurable Agmatine 36 2.90 14 0.36 Homoarginine
Canavanine 10 0.04 No growth or luminescence detected

NH3 51 3.50 53 0.18 Lysine 43 0.21 12 0.18 Methionine 61 5.40 77 7.20 Histidine 58 4.60 39 0.28 Serine 34 11 17 2.70





48

to compensate for total available nitrogen, the cultures containing nitrate were prepared with nitrogen concentration equivalent to that in the other media in the.same column.

Two observations are important to the problem being discussed. First, growth is more rapid on a medium containing both arginine and nitrate than it is on a medium containing nitrate only. However, a very similar growth rate may be obtained by culturing on media containing L-proline, L-methionine or L-histidine instead of L-arginine. Secondly, whereas arginine plus nitrate supports a level of luminescence approximating that obtained in complete medium (containing peptone), the other amino acid supplements which provide growth rates comparable to that on arginine plus nitrate were, at best, only one-fourth as efficient in supporting luminescence. On the other hand, serine, which did not promote growth as well as L-methionine or L-histidine, supported a luminescent intensity which was more than twice that for the L-methionine or L-histidine. Therefore, it may be said that although arginine does increase the growth rate of a nitrate-grown culture over a long period, other amino acids are equally effective in doing this without the concomitant increase in luminescence. Also the relationship between doubling time and luminescence is not a direct one, in view of the data for serine. Thus, while arginine does enhance the growth rate of these bacteria over an extended period, the degree to which it enhances luminescence appears to be specific and independent of the effect on growth.

One arginine metabolic pathway known to exist in bacteria deserves particular consideration. In Streptococcus faecalis, arginine is hydrolyzed to yield citrulline and ammonia (37), and the citrulline is phosphorolyzed to yield ornithine and carbamyl phosphate (38). The





49

carbamyl phosphate then reacts with ADP to produce ATP in a reaction which favors ATP formation (39). This mechanism for phosphorylation of ADP via arginine metabolism is noteworthy because it bypasses the electron transport system which is intimately involved with nitrate reduction and the bioluminescent reaction. Levels of ADP and ATP are known to serve in a regulatory capacity for other enzyme systems (9,40,41). Indirect evidence from several lines of investigation suggeststhat the carbamyl phosphate pathway for phosphorylation of ADP is not likely to be involved in the action of arginine on the bioluminescence system in this strain of bacteria. The first of these is that citrulline would be expected to be at least as effective as arginine in stimulating luminescence if the mechanism involved generation of carbamyl phosphate via the pathway described above; this was found not to be the case. Citrulline was the most effective of all compounds tested (other than arginine) as stimulators of luminescence, and its uptake was similar to that of arginine, but it was not as potent as arginine. A second argument against ATP (or ADP) involvement in controlling bioluminescence is based on the findings that these organisms are obligate aerobes. Attempts to grow:cultures under a variety of anaerobic conditions were unsuccessful. These efforts included conditions with nitrogen, carbon dioxide or hydrogen atmospheres in the culture flasks containing nitrate medium. Addition of arginine did not alleviate this total inhibition of growth. Two assumptions are necessary to make this argument relevant: first, it is assumed that this absolute requirement for oxygen reflects a mandatory operation of the electron transport coupled oxidative phosphorylation system, and,secondly, that ADP phosphorylation via carbamyl phosphate production from arginine could supplant the normal oxidative process to the extent that some growth, even though very slow, could occur.






50

Therefore, although the activity of specific enzymes was not

measured, this evidence indicates that generation of ATP via carbamyl phosphate does not play a primary role, at least in the stimulation of luminescence in these bacteria following the addition of arginine to the medium of a "nitrate" grown culture. Arginine Incorporation into Luciferase

An early consideration was that luciferase might be very rich in arginine and that synthesis would be limited except in the presence of a large arginine pool.; This was investigated by determining the amount of specific radioactivity of partially purified luciferase as compared to similar measurements of general cell protein 60 minutes after'introducing uniformly labeled 14C-arginine (specific activity 0.4 mC per mM,
-4
final concentration 2.4 x 10 M). Results of this experiment showed that total intracellular protein contained an average of 3,186 dpm per mg protein. The luciferase fraction (purified 30 fold) was found to contain 2,142 dpm per mg protein.

Therefore, luciferase does not preferentially incorporate arginine as compared to the general cellular proteins. These results are in agreement with the estimation of five arginyl residues per luciferase molecule (J.W. Hastings, personal communication). Effect of Inhibitors of Bioluminescence Response

Synthesis of new protein is associated with the increase in bioluminescence. This is suggested in Figure 3, in which the in vitro assay of luciferase activity in aliquots of culture collected at timed intervals following the introduction of arginine into the medium is related to in vivo bioluminescence observed in a parallel culture at the same times.

























Figure 14: Effect of mitomycin on the luminescence response to added arginine. Mitomycin (10 g/ml) added to one culture five minutes before adding arginine. Arginine (2.4 x 10-4M) added to all three cultures at time 0. Mitomycin added to second culture at 40 minutes. Third culture received no mitomycin.












10 ~ Mitomycin added at -5 min.
Arginine added at 0 9 Arginine added at 0
Mitomycin added at 40 min.

8 -- Arginine added at 0 No mitomycin added

7







2 -
6








1 Mitomyin
3





Mitomycin
Arginine



-10 -5 0 10 20 30 40 50 60 70 Time (minutes)





53

In order to clarify this point, inhibitors of protein synthesis

were employed. Addition of puromycin (10 pg per ml) or chloramphenicol (10 pg per ml) 5 minutes prior to adding arginine (2.4 x 10-4M) to a log phase culture prevented any increase in bioluminescent intensity. This strongly suggests that new protein synthesis occurs in response to added arginine. Coffey (1) used, in addition to those inhibitors,amino acid analogues, which proved inhibitory, and found that the inhibition was reversible by the addition of the proper amino acid. He also showed that messenger RNA synthesis is probably involved since 5-fluorouracil serves as an inhibitor of the response to added arginine. In support of this conclusion about messenger RNA, it was found that pretreatment of a culture with mitomycin (10 pg per ml added 5 minutes prior to arginine addition) prevented the expected increase in light production (Figure 14). Cheer and Tchen (42) described similar results for the inhibition of 9-galactosidase induction in E. coli. Since a considerable lag occurs before this antibiotic influences synthesis of RNA or protein (43), Cheer and Tchen suggested that mitomycin acts selectively on.genes which are not involved in synthetic activities at the time of exposure to the inhibitor. Later, Iyer and Szybalski (44) indicated that mitomycin acts by cross-linking complementary strands of DNA. Assuming Cheer and Tchen were correct in their analysis, the effect of mitomycin on this system may be interpreted as indicating that the genes for luciferase are not being transcribed prior to addition of arginine, and are therefore susceptible to mitomycin action. If this were true, then the addition of this antibiotic would not be expected to influence the increase in luminescence, once it was initiated by arginine. This, in fact, was observed to be the case (Figure 14). A temporary interruption (2-3 minutes)





54

in the increase in luminescence resulted when mitomycin was added. A similar pause was noted in /-galactosidase in the work of Cheer and Tchen. They showed that general protein synthesis was not inhibited during this period, however, but offered no explanation for the interruption of .f-galactosidase synthesis.

Effect of Initial Arginine Concentration

In Figure 15, the maximum luminescence achieved is plotted against the initial concentration of added arginine. The sigmoidal shape of this curve suggested another possible relationship between arginine and luciferase. One of the more puzzling aspects of this problem is the apparently unrelated nature between arginine and the luminescent reaction. If arginine functions in the capacity of an allosteric effector, however, no obvious relationship is necessary. The curve in Figure 15 suggests that this is a possibility.

"Allosteric" proteins were initially defined by Monod, et al. (3) as those enzymes in which activity is regulated via changes induced in the protein conformation by a molecule (the allosteric effector) which is not necessarily involved in the reaction mediated by.1the enzyme. In their paper, the authors pointed out that a sigmoid curve is obtained when in vitro reaction velocity is plotted against substrate concentration for certain enzymes. This was interpreted as meaning that more than one ligand could be bound to the enzyme at the same time, and,moreover, that some kind of "cooperative interaction" existed between the binding sites. The sigmoid form of the curve was found to linearize by application of the relationship which Hill (45) had described for the hemoglobin oxygen-saturation curve. Atkinson (41) points out, if one assumes that reaction velocity is proportional to the fraction of substrate binding sites saturated, the Hill equation may be expressed as:





























Figure 15: Effect of concentration of added arginine on in vivo luminescence.












6.0 5.0


Points indicate maximum in vivo luminescence observed with each D4.0 initial arginine concentration




2 3.0




2.0 1.0






50 100 200 300 500 Arginine Hydrochloride Concentration (pg per ml)

0N





57

log v/(V-v) = n log (S) Log K

in which v is reaction velocity, V is maximum velocity, n is the number of the substrate binding sites, S is substrate concentration and K is a constant.

Assuming that the maximum in vivo luminescence obtained represents "reaction velocity" (v) for each concentration (s), .and tYt the maximum luminescence achieved by the highest two concentrations (shown in Figure 15) represents maximum reaction velocity (V), a Hlill plot was made and is illustrated in Figure 16. Although it is not linear at the extremes, the region of the curve near the midpoint (where log v/(V-v) = 0) is straight and has a slope of 2.93. Since this value represents a measure of both the strength of cooperative interaction and the number of binding sites (46) it is assumed that if this is an allosteric effect, three arginine binding sites exist on the luciferase molecule.

An alternative interpretation of Figure 15 is that at lower concentrations, a greater proportion of the added arginine is used for purposes other than stimulating bioluminescence; i.e., there is some minimum quantity of arginine that must be supplied for other uses before it can be applied to the bioluminescence system. This is suggested as a possibility in light of the work by McElroy and Farghaly (11) if it is assumed that growth on nitrate medium represents an arginine-limiting condition. Their plot of luminescence versus arginine concentration for an arginine requiring mutant exhibits a sigmoid shape when luminescence is expressed as a percentage of the maximum obtained. Application of the Hill equation to their data results in the plot shown in Figure 17. Here the slope is 3.2 at the midpoint. Thus the value for the slope of the Kill plot of these data agrees reasonably well with the one for the present work. This suggests that a similar relationship might exist for the two systems.





58













1.6




*-4 0.8



Cn
0.0
u m = 2.93


.0 -0.8




-1.6


0


-7.6 -7.2 -6.8 -6.4 -6.0

Log Arginine







Figure 16: Hill plot of arginine concentration effect.





59












1.0




U
( 0.6-)x 0.2
m = 3.2


U
-0.2




8 -0.6




I I I I I
-4.0 -3.9 -3.8 -3.7 -3.6 -3.5

Log Arginine






Figure 17: Hill plot of data for arginine requiring mutant (data from McElroy and Farghaly, 1948).





60

Another experiment involving the effect of arginine concentration on luciferase activity may be interpreted as an allosteric interaction. This experiment consisted of the addition of a second equal amount of arginine to a culture following the re-establishment of the luminescence level at the control value. Results of this experiment are plotted in Figure 18. The second addition of arginine resulted in two changes with respect to the effect of the first: 1) the intensity of the second luminescence response was approximately twice as great as the first (and of longer duration, although this is not shown); and 2) the lag period between the addition of arginine and the first detectable increase in luminescence was shortened. Assuming arginine does act as an inducer of luciferase on the basis of the information presented above, a finite amount of time is required to accomplish this. Coffey (1) has estimated that this is four minutes. During that time, available arginine is continually being removed from the system into protein synthesis. Assuming that arginine also exerts an allosteric effect on luciferase, the total amount of light produced will be a function of the effective concentration of arginine and the duration of that concentration. Provided luciferase is still present but non-functional, due to low arginine levels (resulting in a decrease in luminescence), a second equal addition of arginine should result in a luminescence response of longer duration and greater magnitude. As shown in Figure 18, this was found to be the case.

Again a growth requirement interpretation may be applied. If arginine acts as a simple inducer of luciferase, but must satisfy a growth requirement before induction is accomplished, then a greater proportion of the first addition of arginine would be expected to go for this purpose than would be expected for the second arginine treatment.















10 10 pg Arginine/ml added at time 0; additional 10 pg Arginine/ml added at time 98 minutes.

9

8

7




u 5




3


2

1



10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time (minutes Figure 18: Effect of second addition of arginine.






62

In Vitro Effects of Arginine

Allosteric effects are usually demonstrable in vitro since the interaction exists between the enzyme molecule and the effector, thus requiring no other cellular constituents (as does the induction process which depends on the operation of the genetic system). Therefore, an investigation was made of the effects of arginine on the in vitro behavior of partially purified luciferase.

One method for demonstrating allosteric effects is based on dilution of the presumed effector in vitro (47). If arginine exerts an allosteric effect by blocking an inhibitory action of the luminescence system due to some other chemical species present in the cytoplasm, then a greater than proportional decrease in luciferase activity would occur in dilution with a crude extract of non-arginine treated cells. If no such material exists then dilution of a luciferase preparation with a crude extract of cells not treated with arginine should result in a proportional decrease in the in vitro luciferase activity. Results of such an "admixture" experiment yielded values of 2,500 my peak flash intensity with an extract of arginine4 -4
treated (2.4 x 10 M arginine for 60 minutes)cells, no measurable luminescence in an extract of untreated cells, and 1,300 my peak flash intensity when the enzyme fraction used was a 1:1 mixture of the extracts from treated and untreated cells. Therefore, the existance of a luminescence inhibitive compound which is antagonistic to the action.of arginine was not demonstrated by these experiments.

A long-chain fatty aldehyde requirement for in vitro luciferase activity has been shown (48). The suggestion has been made that the aldehyde acts by altering the configuration of the luciferase protein (28). This offered the possibility that arginine might exert the same effect,





63

particularly since no aldehyde component has been firmly identified in bacterial extracts. It was found, however, that arginine in three different concentrations did not promote a luminescent flash in an assay system containing no aldehyde. Therefore, if arginine does act as an allosteric effector, it does not serve the same function as aldehyde.

The possibility that arginine, acting independently of aldehyde, might enhance the efficiency (flash intensity or decay rate) of the in vitro system was then examined. Tracings of the light response in an in vitro system in the presence and absence of 100, 200 and 300 pg arginine are shown in Figure 19. As may be seen, there is no apparent difference which may be attributed to arginine.

Bovine serum albumin is included in the assay system to aid

against denaturation of the enzyme as a result of the dilution factor. Results of analyses of in vitro luciferase activity in the absence of albumin show that arginine does not have any apparent effect as a replacement for the added protein. Figure 20 shows tracings of such analyses.

Finally, a noteworthy observation was made on the: behavior of the enzyme on stimulation of a second in vitro flash. The intensity of the flash following addition of a second aliquot of FMNH2 was considerably reduced compared to the first flash. Since the second injection of FMNH2 followed the first by five minutes, the control was made up so that the enzyme was exposed to an equal concentration of FMN for five minutes before initiating the first flash. This control showed a greater activity than observed under experimental conditions with enzyme which had been stimulated with FMNI2 before. The effect of arginine on the reduced intensity of the second flash was investigated. Tracings of the





64






5' 4.0
A B
3.0

2.0

o 1.0



10 20 30 40 50 60 10 20 30 40 50 60
Time (seconds)


.I 1 I 1 1 1 1 4.0
C D N 3.0


2.0

S1.0



10 20 30 40 50 60 10 20 30 40 50 60 Time (seconds)





Figure 19: Effect of arginine on in vitro luciferase activity. A : Arginine absent B : 100 pg arginine added C : 200 pg arginine added D : 300 pg arginine added





65



















o


3.0 11l l

2.0

S1.0


10 20 30 40 50 10 20 30 40 50
Time (seconds) Time (seconds)















Figure 20: Effect of arginine on in vitro luciferase activity in the absence of bovine serum albumin. a: Arginine absent b: Arginine present





66


control, first and second flashes in the presence and absence of 150 pg arginine HCI are shown in Figure 21. There is no apparent improvement in the second response in the presence of arginine. Therefore, in these experiments there does not seem to be any in vitro effect by arginine on luciferase activity. Experiments similar to those described above were performed using citrulline, ornithine, or proline i1atead of arginine with the same results. Urea was also tested in the place of arginine, and was found to be slightly inhibitory, which was expected since, at higher concentrations (8M), urea has been shown to reversibly denature bacterial luciferase (49). It does not appear, then, that an early metabolic product of arginine is involved, at least to an extent greater than arginine, as an allosteric effector on the basis of these in vitro studies.

In Vivo Effector Function of Arginine

In mammalian systems, one means of regulating the activity of

enzymes is by controlling the rate of their degrad-.tion (7). Generally speaking, this mechanism is not found in growing bacteria, but apparently does occur in non-growing bacterial cultures (50). Although only log phase cultures were used for the experiments involving the response of the luminescence system to added arginine, the rapid decline in light production (as shown in Figures 1, 18, 19) once peak luminescence had occurred suggested that luciferase might be under a degradative control, or that, in view of the in vitro decline in activity observed with a second FMNH2 stimulation, perhaps the luminescent activity of the enzyme was itself an inactivating (or degrading ) process.

To investigate this possibility, luciferase synthesis was interrupted by the addition of puromycin to a culture following the introduction of arginine and the resultant increase in luminescence. After





67








4 3.0 -1 li l a

0 a b c

o1.0





Time (seconds)





3.0

d -e -f-- f
2.0 C 1.0



0 10 20 30 0 10 20 30 0 -10. 20 30 Time (seconds)






Figure 21: Effect of arginine on second in vitro flash. a,d: First flash b,e: Second flash, 5 minutes after first c,f: FMN control, first flash a,b, and c: Arginine absent d,e, and f: Arginine present (150 ng)






68

a brief continuation in the rise of luminescence, light production began to decline. Two experiments were carried out; one entailed adding puromycin to parallel cultures treated with different amounts of arginine at a point where the luminescence of the cultures was equivalent; the other involved the addition of puromydin following a fixed period post arginine treatment. In each case, it was felt that the decay rate of luminescence

would be different for cultures containing 4.8 x 10-5M arginine and 4.8 x 10'4M arginine if the amino acid were acting to stabilize the

luciferase against a degradative action. Results of these experiments are shown in Figure 22. The rate of decay of luminescence under these

conditions appears to be independent of the initial arginine concentration irrespective of whether the exposure time was the same or the luminescence

intensity was the same.


Discussion of Mechanistic Interpretations

A characteristic of inducible enzyme systems in bacteria is the

relationship between the inducer and the induced enzyme. Although certain s materials which are not acted upon by the induced enzyme, and which are

not metabolized by the cell have the ability to serve as "gratuitous

inducers", these are related to the reaction in the sense that they bear

some similarity to the substrate which is usually assumed to be the "natural" inducer. The most thoroughly studied inducible system is

the ,e-galactosidase system, in which ,e-thioglactoside acts as a "gratuitous inducer" (51). In the case of arginine and bacterial

luciferase, there does not appear to exist such a relationship. Also, though it is not deemed as important as the exceptions just mentioned,

the early kinetics of the luciferase response to arginine are not as

rapid as might be expected on the basis of the inducer model (51).











(a) (b)
1.0
Puromycin added at time 0 Puromcin added at time 0



7.0 w 0.8




S6.0 0.6
x



5.0 0.4
........ 10 ug arginine -10 ug arginine per ml per ml
. 4.0 100 ug arginine/ml \ 0.2 100 ug arginine/ml




3.0 I I I I I I I
5 10 15 20 10 20 30 40 50 Time (minutes) Time (minutes)

Figure 22: Effect of puromycin addition to cultures containing different concentrations of
arginine. (a) Puromycin added when luminescent intensities were equal. (b) Puromycin added after
equal incubation periods with arginine.

'7N





70

Therefore, to call this an inducible enzyme system, implies a departure from the classical model proposed by Jacob and Monod (2).

On the other hand, consideration of this system as one involving allosteric effects is also at odds with the definitions made in describing the model (4). In the formulation of the proposed mechanism, a specific requirement was that the allosteric protein must consist of at least two identical subunits associated in such a way as to occupy symmetrical positions. Bacterial luciferase has been reported as comprised of two kinds of subunits (52). Very recently, active luciferase was shown to contain two and only two subunits which are non-identical (J. W. Hastings, personal communication). Thus far, no indicationsof large aggregates of enzyme have been found. Therefore, bacterial luciferase does not appear to meet the requirements originally established for an allosteric protein.












SUMMARY AND CONCLUSION


The bioluminescent system in the ND strain of the marine bacterium Achromobacter fischeri is not functional when these bacteria are cultured in a minimal medium containing glycerol and nitrate as the only nutrients. Absence of the luminescent reaction is associated with the virtual absence of the enzyme luciferase in functional form. Addition of L-arginine to a log phase culture results in a dramatic increase in in vivo light production and in luciferase activity as estimated in vitro. Results of experiments testing other compounds as stimulators of luminescence and comparison of the time required for arginine conversion to begin in the cell with the appearance of luminescence increase indicate that the response of the luminescent system is due specifically to arginine.

Arginine probably exerts its effect on the luminescent system, via an enzyme "induction" at the genetic level, in a manner similar to the model proposed by Jacob and Monod (2). Evidence for this is found in the apparent increase in luciferase protein, and the prevention of this increase by inhibitors of protein synthesis. These findings are presented in support of the conclusions drawn by Coffey (1) on the basis of his work with A. fischeri ND.

In his work on this problem Coffey (1) presented kinetic evidence indicating that an "activation" step (of unknown nature) was required before luciferase promoted any light production. Certain aspects of the luciferase response to added arginine (in particular the relationship between arginine concentration and the luminescence intensity) suggest that this activation may be the result of an allosteric interaction between arginine and luciferase. Although much of the evidence may be explained 71







72


in terms of such a hypothesis, no results of in vitro or in vivo experiments indicate the existence of such an interaction. Therefore, interpretation of the results in terms of allosteric effectors does not, at

this time, seem appropriate.

This work is offered in confirmation of Coffey's (1) speculation

that L-arginine specifically acts as an inducer of bacterial luciferase

in Achromobacter fischeri ND.





















S .'













BIBLIOGRAPHY


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BIOGRAPHICAL SKETCH


Thomas Stephen Quarles was born October 1, 1941, in Beaumont, Texas. In June,1960, he was graduated from Beaumont High School. In June, 1964, he received the degree of Bachelor of Science with a major in Biology from Lamar State College of Technology in Beaumont, Texas. In September, 1964, he entered the Graduate School of the University of Florida. He worked as a teaching assistant in the Department of Zoology until August, 1967. From September, 1967, until August, 1968, he was a predoctoral fellow of the National Institutes of Health. Since September, 1968, he has been an employee of the Biology Department, Birmingham Southern College, Birmingham, Alabama.

Thomas Stephen Quarles is married to the former Jeannette Downs and

is the father of two children. ie is a member of the American Association for the Advancement of Science, the American Society of Zoologists, and Phi Sigma Biological Society.

























77









This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Arts and Sciences and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1970




Dean, Colle# Ars and Sciences Dean, Graduate School Supervisory Committee: Chairman




Co-Chairman


a~46w*-z"U/I K I/




Full Text
26
effect on the general physiology of the cell prevents the appearance of
an increase in luminescence. It is possible,for example, that luciferase
is synthesized, but is non-functional due to incorporation of canavanine
rather than arginine. This might be clarified by looking for the forma
tion of protein which can be precipitated by anti-luciferase antibody.
Arginine Uptake
The specificity for exogenously added L-arginine and the delay in
the onset of the increase in _in vivo luminescence suggested a possible
lag in arginine uptake by the cells, as might be occasioned by the re
quirement for induction of a permease or by a slow process of simple
diffusion. Therefore a study of the rate of arginine uptake was attempted.
The rate of disappearance of ninhydrin-positive material from the
medium after adding arginine was used as a measure of the uptake of the
amino acid (Figure 5). Treatment with permutit (0.4 g/ml) to remove
ammonia produced no significant change in values obtained from treated
and untreated parallel samples. Although numerical values did not

correspond exactly from one experiment to another, data plotted on the
basis of triplicate ninhydrin assays of each sample in ten experiments
yielded essentially identical curves. Two additional methods more specific
for arginine were also used to measure the uptake of this amino acid. One
was a quantitative fluorescence procedure reported to be specific for
guanido and ureide groups (19); the other was the disappearance of uniformly
labeled ^C-arginine. Results of three experiments involving each of these
methods demonstrated uptake kinetics different from those determined using
the ninhydrin reaction. Figure 5 illustrates data taken from a single
experiment in which all three procedures were employed. Guanido-group
and 1^C-label assays show close agreement in their variation in time, but
the ninhydrin results are seen to be out of phase.


Luminescence
42
Figure 11: Effect of added nitrate in low concentration on
arginine-stimulated luminescence.


74
14. Hastings, J.W., Q.H. Gibson, J. Friedland, and J. Spudich, 1966.
Molecular Mechanisms in Bacterial Bioluminescence: On Energy
Storage Intermediates and the Role of Aldehyde in the Reaction.
In F. H. Johnson and Y. Haneda (eds.). Bioluminescence in
Progress. pp. 151-186. Princeton University Press, Princeton,
New Jersey.
15. Smith, I. (ed.), 1960. Chromatographic and Electrophoretic Techniques
v. 1. Interscience Publishers, Inc., New York.
16. Fink, K R.E. Cline, and R.M. Fink, 1963. Paper Chromatography of
Several Classes of Compounds: Correlated Rf Values in a Variety
of Solvent Systems. Anat. Chem., 35:389-398.
17. Moore, S., D.H. Spackman, and W.H. Stein, 1958. Chromatography of
Amino Acids on Sulfonated Polystyrene Resins. Anat. Chem., 30:
1185-1190.
¡ f
18. Moore, S., and W.H. Stein, 1954. Procedures for the Chromatographic
Determination of Amino Acids on Four Per Cent Cross-Linked
Sulfonated Polystyrene Resins. ^J. Biol. Chem., 211:893-913.
19. Conn, R.B., and R.B. Davis, 1959. Green Fluorescence of Guanidinium
Compounds with Ninhydrin. Nature, 183:1053-1055.
20. Bray, G.A., 1960. A Simple Efficient Liquid Scintillator for Counting
Aqueous Solutions in a Liquid Scintillation Counter. Anat. Biochem
1:279-285.
21. Wang, C.H., and D.L. Willis, 1965. Radiotracer Methodology in Bio
logical Science. Prentice-Hall, Inc., Englewood Cliffs, New Jersey
? J
22. Davidson, J.D., and P. Feigelson, 1957. Practical Aspects of Internal-
Sample Liquid-Scintillation Counting. Int. J. Appl. Rad. Isotopes,
2:1-18.
23. Breed, R.S., E.D.G. Murray and N.R. Smith (eds.), 1957. Bergey's
Manual of Determinative Bacteriology, 7th edition. The Williams
and Wilkins Company, Baltimore, Maryland.
24. Hastings, J.W. and W.D. McElroy, 1955. Purification and Properties of
Bacterial Luciferase. In F. H. Johnson (ed.), The Luminescence of
Biological Systems, American Association for the Advancement of
Science, Washington, D.C.
25. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, 1951.
Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.,
193:265-275.
26.Hastings, J.W., W.H. Riley, and J. Massa, 1965. The Purification,
Properties, and Chemiluminescent Quantum Yield of Bacterial
Luciferase. J. Biol. Chem., 240:1473-1481.


LONG-TERM EFFECT OF VARIOUS NITROGENOUS COMPOUNDS ON LUMINESCENCE AND GROWTH
TABLE 3
NITROGEN NITRATE PRESENT NITRATE ABSENT
SOURCE % Growth Rate L Luminescence /0 Growth Rate % Luminescence
Peptone
100
100
100
100
Nitrate
54
0.06
60
0.14
Arginine
61
90
44
56
Aspartic acid
56
1.90
69
0.14
Ornithine
29
0.65
10
0.25
Citrulline
28
0.39
22
0.18
Urea
30
y
y
7.70
6
1.80
Glutamic acid
22
0.16
31
0.29
Proline
61
26
53
2.50
Homoserine
Agmatine
36
No growth or
2.90
luminescence measurable
14
0.36
Homoarginine
Canavanine
10
0.04
No growth or luminescence
detec
nh3
51
3.50
53
0.18
Lysine
43
. 0.21
12
0.18
Methionine
61
- 5.40
77
7.20
Histidine
58
4.60
39
0.28
Serine
34
11
17
2.70


ACKNOWLEDGMENTS
I wish to thank Dr. P. R. Elliott in the Department of Zoology and
the Office of Radiation Control for their generosity in providing equip
ment and instrumentation for use in the performance of the research
reported here. Appreciation is also expressed to Drs. James Gregg,
William Carr, L. Berner and A.S. Bleiweis for the many helpful suggestions
they offered and the materials they so frequently supplied during the
course of this work. *
ii


45
for these experiments was that endogenous arginine was present in quanti
ties which would support growth but not luminescence. Plots of turbidity
readings taken at 10-minute intervals on parallel samples of a culture,
-4
one containing 2.4 x 10 M arginine and the other serving as an arginine
less control, were essentially congruent over a three-hour period following
the addition of arginine. This information is shown in Figure 13. It is
noted that the periodicity seen in the arginine treated culture is not
observed in the control.
The significance of this congruency is doubtful. Kjeldgard et al. (36)
i f
found a definite lag in the alteration of growth rate in their work on
Salmonella typhimurium. Moreover, McElroy and Farghaly (11) found that
the rate of change in bioluminescence was much more rapid than that in
growth as the initial arginine concentration was increased above 0.01 mg
per ml. Therefore an increase in arginine concentration could be expected
to influence luminescence more than growth rate over a period of time.
An experiment was then performed to determine the long-term effect
, i
of arginine, and other compounds, on growth rate. This was done by
estimating the doubling time of log phase cultures (having effectively
the same turbidity) which were growing in liquid media supplemented
with the indicated compound in a final concentration of 9 x 10^M. For
each substance listed, media were prepared with and without the standard
concentration of nitrate present to determine the growth-supporting
ability of the various materials listed. In addition to determining
the doubling time, the bioluminescent intensity was measured for each culture
at the same turbidity (where growth had occurred) and both were related to
their respective values for cultures grown in medium supplemented with
1.0 percent peptone. These results are summarized in Table 3. In order


LIST OF FIGURES
Figure Page
1. Relationship between growth of culture, arginine-
stimulated bioluminescence and bioluminescence
in the absence of arginine (constituitive lumin
escence) 13
2. Relationship of luminescence and growth in maximum
stationary phase cultures 16
3. Relationship of iji vivo and In vitro measurement of
luminescence following addition of arginine to
a culture 18
4. Onset of luminescence following addition of arginine ... 20
5. Arginine uptake 27
6. Periodicity in growth, luminescence and arginine uptake 29
7. Puromycin inhibition of ^C-argini.ne incorporation into
acid-insoluble material 36
8. Effect of adding puromycin two minutes after adding
arginine 37
9. Proposed electron transport scheme for nitrate *'
reduction in A. fischeri 39
10. Effect of high nitrate ion concentration on
luminescence 41
11. Effect of added nitrate in low concentration on
arginine-stimulated luminescence 42
12. Effect of added nitrate in low concentration on
luminescence in A. fischeri 44
13. Short-term effect of arginine on growth 46
14. Effect of mitomycin on the luminescence response to
added arginine 52
15. Effect of concentration of added arginine on n vivo
luminescence 56
16. Hill plot of arginine concentration effect 58
vi


8
11 mm by 100 cm and 11 mm by 20 cm. A fraction collecter was used to
collect 2 ml samples for ninhydrin analysis or 10 ml fractions for
isolation and subsequent identification of ^C-labeled compounds.
Samples to be de-salted were applied to a Dowex 50-X-8 resin
column (15 mm x 150 mm) in the acid form, eluted with concentrated
NH^OH and evaporated. For one series of experiments, small organic
molecules were isolated from macromolecules and salts by water elution
of the samples from a 9 mm x 150 mm column of Sephadex G-10.
/Quantitative Procedures
The ninhydrin method of Moore and Stein (18) was employed for
measuring free amino acids in membrane (Millipore Corporation, 0.22 micron
pore size) filtered samples of medium and effluent fractions collected
from column chromatography. A spectrophotofluorimetric procedure specific
I
for guanido groups (19) was used to estimate arginine concentration in
these samples.
i
Uniformly labeled ^C-L-arginine was employed in all experiments
involving isotopes. Radiochemical purity of each new supply was checked
by thin-layer chromatography in two solvents. For mostexperiments,
radioactive arginine was added to a final concentration of approximately
10 microcuries per liter and unlabeled arginine used as a carrier to
provide a sufficient concentration for the stimulation of luminescence.
For each experiment, the specific activity of the radioactive arginine
is indicated.
Radioactivity was measured in a liquid scintillation counter using
Bray's solution as the scintillation fluid (20). All samples were counted
for a minimum of ten minutes, or until a significant number of counts
above background were obtained (21). Absolute quantification was made


Log luminescence (mv)
Time (minutes)
00


19
activity since all excess reduced flavin mononucleotide (FMNH2) not
utilized in the reaction within the first second is removed from the
system by autoxidation (27). In view of the luminescent reaction
kinetics, the time limitations imposed by FMNH2 availability have led
Hastings _et a_l. (28) to conclude that only a single catalytic cycle occurs
in vitro.
Observation of the increase in specific activity of luciferase in
crude extracts shown in Figure 3 indicates an increase in the concentration
of functional luciferase relative to total intracellular protein concen
tration. (Because of the extraction procedure, the unlysed cells
and cellular debris were removed by centrifugation to yield a clear
enzyme supernatant fraction.) This increase implies a differential rate
of apparent synthesis of luciferase; that is, addition of arginine to a
culture appears to promote a greater increase in the rate of luciferase
synthesis specifically than it does in general protein synthesizing
activity of the cell.
The _rn vivo luminescence observed in the culture from which the
0 f
enzyme assays were taken is also indicated in Figure 3. The slope, or
rate of increase, of both curves is essentially parallel, deviating only
at the extremes. Since the chemical environment involved in the esti
mation of enzyme activity under these two conditions is so different,
some variation was expected.
Lag Period
A distinct lag of approximately 12 minutes is evident between the
addition of arginine and the first measurable increase in luminescence.
Although it may be seen in Figure 1, the expanded scale of Figure 4 makes
it more obvious. Increasing the arginine concentration ten-fold does not


66
control, first and second flashes in the presence and absence of 150 jig
arginine HC1 are shown in Figure 21. There is no apparent improvement
in the second response in the presence of arginine. Therefore, in these
experiments there does not seem to be any _in vitro effect by arginine on
luciferase activity. Experiments similar to those described above were
performed using citrulline, ornithine, or proline instead of arginine
with the same results. Urea was also tested in the place of arginine,
and was found to be slightly inhibitory, which was expected since, at
higher concentrations (8M), urea has been shown to reversibly denature
bacterial luciferase (49). It does not appear, then, that an early
metabolic product of arginine is involved, at least to an extent greater
than arginine, as an allosteric effector on the basis of these rn vitro
studies.
In Vivo Effector Function of Arginine
In mammalian systems, one means of regulating the activity of
enzymes is by controlling the rate of their degradation (7). Generally
speaking, this mechanism is not found in growing bacteria, but apparently
does occur in non-growing bacterial cultures (50). Although only log
phase cultures were used for the experiments involving the response
of the luminescence system to added arginine, the rapid decline in light
production (as shown in Figures 1, 18, 19) once peak luminescence had
occurred suggested that luciferase might be under a degradative control,
or that, in view of the in vitro decline in activity observed with a
second FMN^ stimulation, perhaps the luminescent activity of the enzyme
was itself an inactivating (or degrading ) process.
To investigate this possibility, luciferase synthesis was inter
rupted by the addition of puromycin to a culture following the intro
duction of arginine and the resultant increase in luminescence. After


Luminescence (mv) Relative Arginine Concentration
Figure 6: Periodicity in growth, luminescence and arginine uptake. (Arginine[,2.4 x 10"^m] ro
added at time 0).
Growth (KS units)


Luminescence (mv
Ln
ON


62
In Vitro Effects of Arginine
Allosteric effects are usually demonstrable In vitro since the
interaction exists between the enzyme molecule and the effector, thus
requiring no other cellular constituents (as does the induction process
which depends on the operation of the genetic system). Therefore, an
investigation was made of the effects of arginine on the jin vitro behavior
of partially purified luciferase.
One method for demonstrating allosteric effects is based on dilution
of the presumed effector in vitro (47). If arginine exerts an allosteric
effect by blocking an inhibitory action of the luminescence system due to
some other chemical species present in the cytoplasm, then a greater than
proportional decrease in luciferase activity would occur in dilution with
a crude extract of non-arginine treated cells. If no such material exists
then dilution of a luciferase preparation with a crude extract of cells
not treated with arginine should result in a proportional decrease in the
in vitro luciferase activity. Results of such an "admixture" experiment
yielded values of 2,500 mv peak flash intensity with an extract of arginine
treated (2.4 x 10 arginine for 60 minutes)cells, no measurable lumines
cence in an extract of untreated cells, and 1,300 mv peak flash intensity
when the enzyme fraction used was a 1:1 mixture of the extracts from
treated and untreated cells. Therefore, the existance of a luminescence
inhibitive compound which is antagonistic to the action.of arginine was
not demonstrated by these experiments.
A long-chain fatty aldehyde requirement for In vitro luciferase
activity has been shown (48). The suggestion has been made that the
aldehyde acts by altering the configuration of the luciferase protein (28).
This offered the possibility that arginine might exert the same effect,


30
labeled ^C-arginine, specific activity 0.6 mC per mM, final concentra
tion 1 x 10~^M). The procedure is somewhat complicated by the fact that
arginine is utilized in protein synthesis and thus care must be exercised
to measure only free arginine as the internal pool.
Interpretation of the above experiment must be in terms of the
radioactivity per unit volume of cytoplasm and medium. To do this, the
number of cells per ml of culture was determined directly by microscopic
counts. The volume of a single cell was calculated as the volume of a
cylinder having dimensions of the average cell size determined from
f
electron photomicrographs of bacteria taken from log phase cultures. The
dimensions used were found to compare favorably with those listed for
wild type A. fischeri in Bergev's Manual (23).
Several sources of error in this method must be considered. The
**
first is that direct counting of cells, in addition to sampling and dilu
tion errors, does not distinguish viable cells. Since viable counts
correspond very closely to direct microscopic counts of cells in early
log phase cultures, and these experiments were performed on such cultures,
it was assumed that direct counts would accurately represent the number of
viable cells. A second error source is that the cells are not perfect
cylinders; therefore the volume calculations are not exact. Deviation
from cylindrical form in these bacilli occurs primarily as a rounding of
the ends, thus introducing little variation from the volume of a cylinder.
Thirdly, the use of average cell dimensions introduces error since all the
cells are not the same size. By using the mean value, it was assumed that
variation about that measurement would be compensated. Further, it was
assumed that the cells used to determine the average dimensions of these
bacteria represented a random sample. Finally, error in volume calculations
is occasioned by inclusion of the cell wall volume. No estimation


10
mixture was placed in the photometer and 0.2 ml FMNH2, 0.1 mg per ml,
injected by syringe through a rubber cap. The peak intensity of the
resulting light flash was used to express enzyme activity in millivolts (mv).
It should be noted that these units are not directly comparable to
similar units of _in vivo luminescence since the geometry and electronic
components of the two systems are not identical. Protein concentration
was estimated by the method of Lowry, e_t al. (25).
/


48
to compensate for total available nitrogen, the cultures containing
nitrate were prepared with nitrogen concentration equivalent to that in
the other media in the same column.
Two observations are important to the problem being discussed.
First, growth is more rapid on a medium containing both arginine and
nitrate than it is on a medium containing nitrate only. However, a
very similar growth rate may be obtained by culturing on media containing
L-proline, L-methionine or L-histidine instead of L-arginine. Secondly,
whereas arginine plus nitrate supports a level of luminescence approximat-
/ f
ing that obtained in complete medium (containing peptone), the other amino
acid supplements which provide growth rates comparable to that on arginine
plus nitrate were, at best, only one-fourth as efficient in supporting
luminescence. On the other hand, serine, which did not promote growth as
well as L-methionine or L-histidine, supported a luminescent intensity
which was more than twice that for the L-methionine or L-histidine. There
fore, it may be said that although arginine does increase the growth rate
of a nitrate-grown culture over a long period, other amino cids are
equally effective in doing this without the concomitant.increase in
luminescence. Also the relationship between doubling time and lumin
escence is not a direct one, in view of the data for serine. Thus,
while arginine does enhance the growth rate of these bacteria over an
extended period, the degree to which it enhances luminescence appears
to be specific and independent of the effect on growth.
One arginine metabolic pathway known to exist in bacteria deserves
particular consideration. In Streptococcus faecalis, arginine is hydro
lyzed to yield citrulline and ammonia (37), and the citrulline is
phosphorolyzed to yield ornithine and carbamyl phosphate (38). The


Luminescence (mv x 1CP) Luminescence (mv
64
Figure 19: Effect of arginine on jin vitro luciferase activity.
A : Arginine absent
B : 100 jug arginine added
C : 200 jug arginine added
D : 300 jug arginine added


43
nitrogen source (nitrite present in equal molarity to the nitrate concen
tration in the usual medium) revealed identical behavior with regard to
the very low levels of constituitive luminescence and the response of
the luminescence system to added arginine. On the other hand, nitrate
reduction is involved in some manner with the luminescence system since
the addition of 9 x 10^M nitrate to a culture of wild type A. fischeri
results in an increase in light intensity similar to that found in the
nitrate utilizing strain (Figure 12) Since the wild type A_. fischeri
has a nitrate reductase, but lacks the ability to reduce nitrite (35),
this indicates that the effect of nitrate on the luminescence system in
wild type is associated with the reduction of that ion. It has been proposed
that two separate electron transport chains are involved; one leading to
reduction of oxygen, the other to nitrate reduction (35). The significance
of these alternate pathways relative to the present problem is not clear
at this time, nor is the role of nitrate reduction. A reasonable initial
step toward investigating this relationship seems to lie in determining
the effect of adding nitrate on the intracellular pool of FMNH2.
Effect of Arginine on Growth
McElroy and Farghaly (11) found that arginine must be supplied to
the arginine requiring mutants in quantities sufficient to exceed a
minimum level of growth in order to develop the bioluminescent system.
Although they did not measure growth rate (change in turbidity in time)
during the incubation period, it is assumed that in two cultures receiving
equal inoculations initially, a difference in turbidity after a fixed
incubation period (48 hours) represents a difference in growth rates in
the two cultures. In order to determine if the nitrate utilizing mutant
is analogous to their arginine requiring mutants, the immediate effect
of arginine on growth rate in this strain was examined. The hypothesis


Luminescence
Figure 17: Hill plot of data for arginine requiring mutant
(data from McElroy and Farghaly, 1948).


75
27. Gibson, Q.H., and J.W. Hastings, 1962. The Oxidation of Reduced
Flavin Mononucleotide by Molecular Oxygen. Biochem. J., 83:
368-377.
28. Hastings, J.W., Q.H. Gibson, and C. Greenwood, 1965. Evidence for
High Energy Storage Intermediates in Bioluminescence, Photochem.
Photobiol., 4:1227-1241.
29. Middlehove, W.J., 1964. The Pathway of Arginine Breakdown in
Saccharomyces cerevisiae. Biochim. Biophys. Acta, 93:650-652.
30. Wood, W.A., 1956. Symposium on Microbial Amino Acid Metabolism,
Bact. Rev., 20:285-288.
31. Schwartz, J.H., and W.K. Maas, 1960. Analysis of the Inhibition of
Growth Produced by Canavanine in Escherichia coli. J. Bact.,
J79:794-799.
/
32. Nathans, D., 1964. Puromycin Inhibition of Protein Synthesis: Incorpor
ation of Puromycin into Peptide Chains. Proc. Nat. Acad. Sci.,
U.S., 51:585-593.
33. Sells, B.H. 1965. Puromycin: Effect on Messenger RNA Synthesis and
^-Galactosidase Formation in Escherichia coli 15T~ Science,
148:371-373.
34. Sadana, J.C., and W.D. McElroy, 1957. Nitrate Reductase from
Achromobacter fischeri. Purification and Properties: Function
of Flavins and Cytochrome. Arch. Biochem. Biophys., 67:16-34.
35. McElroy, W.D., 1961. Bacterial Luminescence, pp. 479-508. _I
I.C. Gunsalus and R.Y. Stanier (eds.), The Bacteria, vol. 2.
Academic Press, Inc., New York.
36. Kjeldgard, N.O., 0. Maaloe, and M. Schaechter, 1958., The Transition
Between Different Physiological States During Balanced Growth
of Salmonella typhimurium. J. Gen. Microbiol., 19:607-616.
37. Petrack, B., L. Sullivan, and S. Rutner, 1957. Behavior of Purified
Arginine Desiminase from S. faecalis. Arch. Biochem. Biophys.,
6j): 187-197.
38. Jones, M.E., A.D. Anderson, C. Anderson, and S. Hodes, 1961.
Citrulline Synthesis in Rat Tissues. Arch. Biochem. Biophys.,
95:499-507.
39. Jones, M.E., L. Spector, and F. Lipmann, 1955. Carbamyl Phosphate.
The Carbamyl Donor in Enzymatic Citrulline Synthesis. J. Am.
Chem. Soc., 77:819-820.
40.Tomkins, G.M., K.L. Yielding, N. Talal, and J.F. Curran, 1963.
Protein Structure and Biological Regulation. Cold Spr. Harb.
Symp. Quant. Biol., 28:461-471.


Figure 2: Relationship of luminescence and growth in maximum stationary phase cultures.
(Data are plotted as in Figure 1. Stimulated luminescence data are from culture treated with
L-arginine at time indicated in Figure 1.
Growth (KS units)


Therefore, to call this an inducible enzyme system, implies a departure
from the classical model proposed by Jacob and Monod (2).
70
On the other hand, consideration of this system as one involving
allosteric effects is also at odds with the definitions made in des
cribing the model (4). In the formulation of the proposed mechanism,
a specific requirement was that the allosteric protein must consist
of at least two identical subunits associated in such a way as to occupy
symmetrical positions. Bacterial luciferase has been reported as com
prised of two kinds of subunits (52). Very recently, active luciferase
/ f
was shown to contain two and only two subunits which are non-identical
(J. W. Hastings, personal communication). Thus far, no indications of
large aggregates of enzyme have been found. Therefore, bacterial
luciferase does not appear to meet the requirements originally established
for an allosteric protein.


31
is available for this volume. However, the influence of this error is
to decrease the apparent intracellular concentration of ^C-labeled
material. Since the actual volume of cytoplasm in which the ^C-label is
concentrated is less than the estimated volume, the real concentration
gradient is greater than the calculated one. The error introduced by
including any non-viable cells in the calculations would have a similar
influence on the estimated concentration gradient.
Within these limitations, calculations indicated concentration
gradients of 1,180-fold.after 5 minutes and 1,870-fold after 10 minutes.
In light of the above discussion of errors, these estimates are probably
low. According to A.G. DeBusk (personal communication) a 500-fold con
centration gradient estimated by these methods is sufficient to indicate
a concentrating mechanism. Therefore, these values strongly suggest the
existance of a mechanism for taking up arginine against a concentration
gradient.
Arginine Metabolism
Figure 5 shows data for arginine disappearance from the medium in
time. Although the curves for the measurements of guanido groups and
14 \
C-arginine show close agreement at ll stages, the assay for ninhydrin
positive material was out of phase with these two. Since the data for
all three curves were obtained from the same samples, the presence of
ninhydrin-positive materials other than arginine was indicated. There
fore, an examination of the medium by chromatography was carried out.
Proline was identified as the only ninhydrin-positive compound
present in detectable quantities in minimal nitrate medium of a log
phase culture prior to the addition of arginine. Chromatographed sample
concentrations sufficient to yield a readily discernible yellow spot for


Luminescence (mv
(a)
(b)
Figure 22: Effect of puromycin addition to cultures containing different concentrations of
arginine. (a) Puromycin added when luminescent intensities were equal. (b) Puromycin added after
equal incubation periods with arginine.
O'


THE ROLE OF ARGININE AS THE INDUCER
OF BACTERIAL LUCIFERASE IN
ACHROMOBACTER FISCHERI ND
By
THOMAS STEPHEN QUARLES
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1970

ACKNOWLEDGMENTS
I wish to thank Dr. P. R. Elliott in the Department of Zoology and
the Office of Radiation Control for their generosity in providing equip
ment and instrumentation for use in the performance of the research
reported here. Appreciation is also expressed to Drs. James Gregg,
William Carr, L. Berner and A.S. Bleiweis for the many helpful suggestions
they offered and the materials they so frequently supplied during the
course of this work. *
ii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . ii
LIST OF TABLES v
LIST OF FIGURES vi
INTRODUCTION 1
MATERIALS AND METHODS 6
> >
Culture Techniques and Procedure for Arginine Addition ... 6
Light Measurement 6
Chromatographic Procedures 7
Quantitative Procedures 8
Special Procedures 9
RESULTS AND DISCUSSION 11
Luminescent Response to Arginine 11
In vitro estimation of enzyme activity 15
Lag period 19
Arginine Specificity 21
Stimulation by other compounds 21
Arginine Uptake 26
Induced Periodicity 28
Arginine Concentrating Ability 28
Arginine Metabolism 31
Mechanism of Arginine Action 38
Effects of the nitrate ion 38
Effect of arginine on growth 43
iii

Arginine incorporation into luciferase 50
Effect of inhibitors of bioluminescence response .... 50
Effect of initial arginine concentration 54
In vitro effects of arginine 62
In vivo effector function of arginine 66
Discussion of Mechanistic Interpretations 68
SUMMARY AND CONCLUSION 71
BIBLIOGRAPHY 73
BIOGRAPHICAL SKETCH 77
/ !
IV

LIST OF TABLES
Table Page
1. Compounds Tested for Stimulation of In Vivo
i Luminescence 22
2. Arginine Sparing of Canavanine Inhibition of Growth 25
3. Long-term Effect of Various Nitrogenous Compounds on
Luminescence and Growth 47

v

LIST OF FIGURES
Figure Page
1. Relationship between growth of culture, arginine-
stimulated bioluminescence and bioluminescence
in the absence of arginine (constituitive lumin
escence) 13
2. Relationship of luminescence and growth in maximum
stationary phase cultures 16
3. Relationship of iji vivo and In vitro measurement of
luminescence following addition of arginine to
a culture 18
4. Onset of luminescence following addition of arginine ... 20
5. Arginine uptake 27
6. Periodicity in growth, luminescence and arginine uptake 29
7. Puromycin inhibition of ^C-argini.ne incorporation into
acid-insoluble material 36
8. Effect of adding puromycin two minutes after adding
arginine 37
9. Proposed electron transport scheme for nitrate *'
reduction in A. fischeri 39
10. Effect of high nitrate ion concentration on
luminescence 41
11. Effect of added nitrate in low concentration on
arginine-stimulated luminescence 42
12. Effect of added nitrate in low concentration on
luminescence in A. fischeri 44
13. Short-term effect of arginine on growth 46
14. Effect of mitomycin on the luminescence response to
added arginine 52
15. Effect of concentration of added arginine on n vivo
luminescence 56
16. Hill plot of arginine concentration effect 58
vi

Figure Page
17. Hill plot of data for arginine requiring mutant 59
18. Effect of second addition of arginine 61
19.Effect of arginine on _in vitro luciferase activity .... 64
20. Effect of arginine on jin vitro luciferase activity
in the absence of bovine serum albumin 65
21. Effect of arginine on second n vitro flash 67
22. Effect of puromycin addition to cultures containing
different concentrations of arginine 69
vii

INTRODUCTION
An arginine-inducible bacterial luciferase has been described
recently (1). Two aspects of the phenomenon are unusual compared to
previously described inducible bacterial enzyme systems. The first of
these is that no known relationship exists between arginine and the
reaction catalyzed by the induced enzyme. The other unusual feature is
that these bacteria are.capable of _de novo synthesis of the inducer and,
in fact, do produce sufficient amounts of arginine to support growth
under the conditions necessary for induction. This raises the question
as to how the addition of exogenous arginine results in the observed
increase in luciferase activity.
In 1961, Jacob and Monod (2) suggested a mechanism by which protein
biosynthesis could be genetically controlled. Briefly, their model pro
poses a set of three regions on the genome associated with the synthesis
of a protein. These regions are 1) the structural gene, which dictates
the amino acid sequence of the specific enzyme protein, 2) the operator
region, which controls the initiation xof transcription of the structural
\
gene, and 3) the repressor region, which produces a product that, by
combining with the operator region, prevents initiation of protein bio
synthesis. Another property of the repressor product, in an inducible
system, is its ability to combine specifically with a small molecule, the
inducer, such as a sugar or amino acid.
When combined with the inducer, the repressor material loses its
capacity to attach to the operator region of the genome. This frees the
operator, permitting the initiation of enzyme synthesis. Alternately,
1

2
in a repressible system, the small molecule (termed co-repressor) combines
with the repressor product and attaches to the operator to prevent trans
cription of the structural gene. The system under consideration in this
study has been considered to be inducible rather than repressible (1).
In bacterial inducible enzyme systems, the inducer molecule often is
related to the induced enzyme as the substrate or as' a homologue of
the substrate. However, this does not appear to be an absolute require
ment (3) .
An entirely different means for controlling enzyme activity has
been suggested by Monod, Changeux and Jacob (4), involving an "allosteric
effector". The molecule serving as the "effector" is not necessarily a
component in the reaction catalyzed by the affected enzyme, but may
elicit a quantitative change in enzyme activity. According to the
proposed model, combination of the effector with its target enzyme causes
a structural modification of the protein which may result in an increase
(activation) in the rate or a decrease (inactivation) in the rate of the
catalyzed reaction, or in a stabilization of the enzyme protein (5-10).
Since, under certain conditions, data for an enzyme exhibiting allosteric
properties vitro demonstrate an ^Ln vivo behavior very similar to an
inducible system (7) both models will be considered here.
The organism used in the present study is the ND strain of the marine
bacterium Achromobacter fischeri. Originally isolated for its ability to
utilize nitrate as its sole nitrogen source, it was found to be essentially
non-luminescent when cultured in a minimal nitrate medium (nitrate dark).
P. R. Elliott and A. H. Farghaly (personal communication) reported that
the strain luminesced normally when grown in the presence of peptone,
and that the component responsible for this effect was specifically
L-arginine. Early studies of the system indicated that the virtual

3
absence of light production in cultures grown with nitrate as the
sole nitrogen source was due to the lack of functional enzyme required
for luminescence.
Through the extensive use of various inhibitors of protein synthesis,
Coffey (1) has demonstrated that the addition of L-arginine to a log
phase culture growing on minimal nitrate medium results in the synthesis
of new luciferase. He has found that new mRNA synthesis is required to
effect this response. Elliott and Farghaly (personal communication) had
previously shown that added arginine did not affect the level of activity
of six other randomly selected enzymes, thus arguing against a generalized
enzyme induction. These data, combined with those of Coffey, strongly
suggest that exogenously added arginine is an inducer for the specific
synthesis of luciferase.
The investigations of Coffey (1) indicated a very high specificity
of L(+)-arginine as the inducer of luciferase in his strain of bacteria.
Other amino acids, structural analogues of arginine, and ammonium salts
were ineffective as inducers with the exceptions of L-proline, L-aspartic
acid, and the arginine precursors L-ornithine, L-citrulline and L-arginino-
succinic acid. The most efficient of these, L-citrulline, elicited a
response that was only 37.3 percent as strong as that produced by L-arginine.
On the basis of this and ^C-arginine experiments, Coffey concluded that
arginine is the specific inducer molecule, and that the positive response
to the other compounds results from intracellular conversions to or sparing
of arginine. He also stated that the growth rate of the culture is not
altered during the first two hours after treatment with arginine.
The first studies of the influence of arginine on bacterial growth
and luminescent systems were carried out by McElroy and Farghaly (11) in
a series of experiments on A. fischeri mutants isolated after exposure

4
to ultraviolet light. Among these were two strains which exhibited a
specific nutritional requirement for arginine. Both mutants showed a
linear growth response to low arginine concentrations. No luminescence
was evident, however, unless the cell density after 48 hours exceeded
30 percent of that attained by the wild type A. fischeri in arginine-
free medium after the same incubation period. As the arginine concen
tration was increased beyond the level satisfying this condition, the
slope of the growth versus arginine plot diminished and luminescence
developed rapidly. Both growth and luminescence were measured only
{ \
after an incubation period of forty-eight hours. No attempt to determine
the short-term effect of added arginine was reported. The only other
growth-supporting amino acid, ornithine, gave results similar to those
reported for arginine. The workers concluded that the growth and
luminescent systems were competing for th.e common factor arginine or some
component containing arginine. The also pointed out that the wild type
A. fischeri exhibited a distinct lag in the production of light during
the early stages of rapid growth. This suggested that strong competition
*
existed for some component common to both luminescent and growth systems
even in the normal strain.
In a later study, Farghaly (12) determined the influence of various
amino acids on light production and growth in wild type A. fischeri.
Arginine proved no more effective than any other amino acid for stimu
lating luminescence and was less so than either lysine, methionine,
histidine or ammonium salts.
The requirements for _in vitro luminescence by the bacterial system
have been well defined. Reduced flavine mononucleotide (FMNl^),
luciferase, a long-chain saturated aldehyde in a buffer, and oxygen
are sufficient to produce a flash of light (13). Work by Hastings and

5
his group (14) has indicated that FMNH2 reduces the enzyme directly
and that no substrate in which arginine could be a component is
involved in the reaction. It would appear, therefore, that arginine,
as an inducer, does not bear a typical relationship to the induced
enzyme.
The work reported here was carried out in an attempt to answer the
following questions: Is arginine an inducer of luciferase or is some
arginine metabolite responsible for the phenomenon? Does the luminescent
response to arginine occur as a result of an induction process or as a
result of some other mechanism? Finally, does arginine serve a double
function by acting both as an inducer and as a stabilizer of the luciferase
molecule?

MATERIALS AND METHODS
Culture Techniques and Procedure for Arginine Addition
In all experiments involving stimulation of luciferase activity,
the cultures were grown in the minimal liquid medium described by
Farghaly (12) modified to contain 0.76 grams per liter (g/1) NaNO^
(approximately 9 millimolar) to replace the (NH^)H^PO^. Complete
broth contained both (NH^^PO,, 0.5 g/1, and 1.0 percent peptone
(weight per volume) in addition to minimal salts and glycerol (12).
Cultures were incubated 18 to 20 hours at 23C with reciprocal
shaking of 80 cycles per minute. Cell density was determined turbidi-
metrically with a Klett-Summerson colorimeter using a number 42 filter.
Arginine was added to the culture when a reading was obtained of 80 to
100 Klett units. If the culture had overgrown this density, it was
diluted with fresh medium to a reading of about 40 units and allowed
to grow back to the usual cell concentration before introducing arginine
Light Measurement
In. vivo light measurements were carried out in a light-excluded
tank maintained at 23C by a circulating water bath. An RCA 1P21 photo
multiplier tube was positioned below a one-inch diameter window located
in the bottom of the chamber. Mounted inside the tank was a rack for
six 125 ml Erlenmeyer flasks, any one of which could be rotated into a
fixed position over the phototube window. Aeration of the culture was
maintained by reciprocal shaking of the rack at 80 cycles per minute.
Normal operation of the photomultiplier was at a regulated 1000 volts
potential. Cathodal current signal was measured on a strip-chart
6

7
servo-recorder with variable sensitivity by use of precision resistors
for signal attenuation. The instrumentation gave linear response across
the full range of light measurements.
In vitro bioluminescence measurements were carried out in 10 x 75 mm
test tubes placed in a fixed geometry relative to an RCA 1P21 photomultiplier
tube in a light-excluded chamber. Reaction components were injected with
a hypodermic syringe and needle without admission of extraneous light. The
photomultiplier was operated at 1000 volts and the output signal traced by
a recorder with 5 milliseconds response time.
Chromatographic Procedures
Thin-layer chromatography was performed using plastic sheets pre
coated with silica gel G (Eastman Distillation Products Industries) and
a sandwich style developing chamber. Routine detection of amino acids
was obtained with a 0.25 percent ninhydrin in acetone spray followed by
heating at 100C for about five minutes. Ehrlich's Reagent was sprayed
for detection of ureides and Sakaguchi reagent for the identification of
arginine (15) Tricarboxylic acid cycle intermediates were located by
spraying with bromocresol green reagent (16) In addition to identification
by specific color reactions, each unknown was required to co-chromatograph
with a known standard in at least three solvent systems before being
considered identified (15, 16). A similar procedure was employed to
N.
ascertain the chemical purity of compounds used to stimulate luminescence
in the cultures.
Ion exchange chromatography columns were prepared and operated
according to the procedure of Moore, Spackman and Stein (17) with the
exceptions that Dowex 50-X-8 resin, 200-400 mesh, was used without
additional particle sizing, and the dimensions of the columns were

8
11 mm by 100 cm and 11 mm by 20 cm. A fraction collecter was used to
collect 2 ml samples for ninhydrin analysis or 10 ml fractions for
isolation and subsequent identification of ^C-labeled compounds.
Samples to be de-salted were applied to a Dowex 50-X-8 resin
column (15 mm x 150 mm) in the acid form, eluted with concentrated
NH^OH and evaporated. For one series of experiments, small organic
molecules were isolated from macromolecules and salts by water elution
of the samples from a 9 mm x 150 mm column of Sephadex G-10.
/Quantitative Procedures
The ninhydrin method of Moore and Stein (18) was employed for
measuring free amino acids in membrane (Millipore Corporation, 0.22 micron
pore size) filtered samples of medium and effluent fractions collected
from column chromatography. A spectrophotofluorimetric procedure specific
I
for guanido groups (19) was used to estimate arginine concentration in
these samples.
i
Uniformly labeled ^C-L-arginine was employed in all experiments
involving isotopes. Radiochemical purity of each new supply was checked
by thin-layer chromatography in two solvents. For mostexperiments,
radioactive arginine was added to a final concentration of approximately
10 microcuries per liter and unlabeled arginine used as a carrier to
provide a sufficient concentration for the stimulation of luminescence.
For each experiment, the specific activity of the radioactive arginine
is indicated.
Radioactivity was measured in a liquid scintillation counter using
Bray's solution as the scintillation fluid (20). All samples were counted
for a minimum of ten minutes, or until a significant number of counts
above background were obtained (21). Absolute quantification was made

9
using the internal standard method (22). The following calculations
were made for each vial:
cPmu
(cpmu + cpms) cpmu = dPmu
dpms
where cpmu and cpmg are counts per minute due to the unknown and standard
respectively and dpmu and dpmg are similar expressions for disintegrations
per minute.
i Special Procedures
The ability of the bacteria to concentrate arginine was determined
by measuring the difference in radioactivity per unit volume in samples
of membrane-filtered medium and simultaneously collected samples of
membrane-filtered culture containing trichloroacetic acid to lyse the
bacteria and precipitate the proteins. The number of bacteria per ml of
culture was determined by direct microscopic counts. Using the average
dimensions of a cell of this species of bacteria as indicated in Bergey1s
# 9
Manual (23) and confirmed by measurements taken from electron photomicro
graphs, the volume of cells (including the cell walls) per ml of culture
was calculated. By appropriate corrections for dilutions, the disintegrations
per minute per unit volume of bacterial cells and per unit volume of extra
cellular medium were determined.

Cell-free extracts of the bacteria were prepared by osmotic lysis
in distilled water. Luciferase was partially purified by the method of
Hastings and McElroy (24). Enzyme activity was determined in a 10 mm x 75 mm
test tube containing 1.5 ml 0.1 M phosphate buffer, pH 7.4; 0.5 ml 1
percent bovine serum albumin in water; 0.1 ml partially purified enzyme;
and 0.5 ml dodecanal-saturated water solution. This tube with the assay

10
mixture was placed in the photometer and 0.2 ml FMNH2, 0.1 mg per ml,
injected by syringe through a rubber cap. The peak intensity of the
resulting light flash was used to express enzyme activity in millivolts (mv).
It should be noted that these units are not directly comparable to
similar units of _in vivo luminescence since the geometry and electronic
components of the two systems are not identical. Protein concentration
was estimated by the method of Lowry, e_t al. (25).
/

RESULTS AND DISCUSSION
Luminescent Response to Arginine
The primary question concerns the role of arginine in the bio-
luminescent response evoked by the addition of this amino acid to a
log phase culture of Achromobacter fischeri ND grown on minimal nitrate
medium. Figure 1 shows the relationship of growth, the small amount of
measurable luminescence of an untreated culture (constituitive luminescence),
and the luminescence observed after adding arginine (final concentration
2.4 x 10 4 Molar) to early log phase cells. In Figure 1, and all succeeding
figures containing data on light production, luminescence is plotted as
readings taken from continuous recordings at one minute intervals in those
regions of particular concern and at five minute intervals over the remainder
of the curve.
As may be seen in Figure 1, the addition of arginine results in a
2,000-fold increase in _in vivo luminescence compared to an untreated
culture. The constituitive luminescence is measured at 12 to 15 mv
compared to approximately thirty volts following treatment with arginine.
Maximum luminescence was observed to vary by as much as a factor of five,
ranging from about ten volts to fifty volts in repeated experiments.
Attempts to grow and treat cultures under uniform conditions did not
improve the reproducibility of the measured maximum luminescence after
adding arginine. Careful examination revealed that results from parallel
samples of the same culture were comparable and findings from repeated
experiments were consistent relative to controls, irrespective of the
variation in maximum luminescence intensity.
11

Figure 1: Relationship between growth of culture, arginine-stimulated bioluminescence and
bioluminescence in the absence of arginine (constituitive luminescence). L-arginine in final
concentration of 2.4 x 10^M is added at arrow. Growth is determined turbidimetrically and
expressed in Klett-Summerson (K-S) units. Luminescence curves are plotted from continuous record
ings and expressed in millivolts (mv).

Luminescence (mv
-- 600
500
400
300
-- 200
-- 100
u>
Growth (KS units)

14
The cause for this variation in susceptibility to arginine stimu
lation of light production is not known at this time. Several factors
thought to be potentially responsible were investigated. Cultures
grown on successive days in aliquots of medium taken from the same
large batch preparation did not respond equally to added arginine. Addi
tion of arginine from the same stock solution did not insure a reproducible
maximum luminescence of the treated culture, independent of whether the
solution had been stored at room temperature, 4C or frozen at -15C
between use. The age of the culture from which subsequent inoculations
were taken did not appear to be a factor since variations were noted in
cultures inoculated with log phase, stationary phase, or death phase
cells. Although culture age does influence the sensitivity to arginine
stimulation, as will be discussed later, the maximum response varied
whether the amino acid was added after the same period of incubation
or at the same cell density (as estimated turbidimetrically). Other
factors controlled prior to the addition of arginine included the adjust
ment of pH of the medium to 7.0, adding a fresh supply of glycerol and
supplementing the nitrate concentration. Only one variable showed any
correlation to the problem of reproducibility; with increasing serial
transfers in minimal nitrate medium, there was a tendency toward a
decrease in maximal luminescence when all other factors were held constant.
Sub-culturing in complete medium usually resulted in an increase of
sensitivity to arginine stimulation in subsequent minimal nitrate cultures.
In his work on arginine stimulation of bioluminescence in similar cultures
grown on minimal nitrate medium, Coffey (1) reported this same difficulty
with peak response to added arginine but offered no suggestions as to the
cause of the problem.

15
By the time a culture has reached the late log phase or early
stationary phase of its growth cycle, the constituitive luminescence
increases. The onset of this increase may be seen in Figure 1 and,
in Figure 2, it is shown in relation to culture age and luminescence
resulting from added arginine. Although not shown in Figure 2, the
constituitive luminescence maintains a relatively constant level for
about seven hours and then begins to decline before the culture enters
the death phase (indicated by viable cell counts performed every four
hours). This is observed independent of previous arginine treatment.
/ f
The addition of arginine to a culture during this period results in
an increase in light production; however, the response is not as ex
tensive as that elicited by an equal concentration of the amino acid
added in early log phase.
In Vitro Estimation of Enzyme Activity
The increased luminescence of a culture following addition of
arginine to the medium reflects an increase in the intracellular concen
tration of functional luciferase, as measured by jin vitro assays of enzyme
activity. Cell-free crude extracts of aliquots removed from a culture
at timed intervals after the introduction of arginine into the medium
were analyzed for luciferase activity and protein concentration. In
vitro light production per mg of protein (specific activity) in these ex
tracts is plottedas a logarithmic function against the time at which
the aliquots were removed from the culture (Figure 3).
Use of the peak flash intensity as a measurement of enzyme activity,
as described in the Methods section, is a standard procedure in such
assays. Flash height has been shown to be directly proportional to
the total light output measured as the area under the response curve (12, 26).
Moreover, this area (rather than a rate) is believed to represent enzyme

Figure 2: Relationship of luminescence and growth in maximum stationary phase cultures.
(Data are plotted as in Figure 1. Stimulated luminescence data are from culture treated with
L-arginine at time indicated in Figure 1.
Growth (KS units)

Figure 3: Relationship of in vivo and _in vitro measurement of luminescence following addition
of arginine (2.4 x 104M) to a culture at time zero. _In vitro luminescence is expressed in millivolts
per milligram protein in crude cell-free extract. _In vivo luminescence is expressed in millivolts per
ml of culture. i 1.-

Log luminescence (mv)
Time (minutes)
00

19
activity since all excess reduced flavin mononucleotide (FMNH2) not
utilized in the reaction within the first second is removed from the
system by autoxidation (27). In view of the luminescent reaction
kinetics, the time limitations imposed by FMNH2 availability have led
Hastings _et a_l. (28) to conclude that only a single catalytic cycle occurs
in vitro.
Observation of the increase in specific activity of luciferase in
crude extracts shown in Figure 3 indicates an increase in the concentration
of functional luciferase relative to total intracellular protein concen
tration. (Because of the extraction procedure, the unlysed cells
and cellular debris were removed by centrifugation to yield a clear
enzyme supernatant fraction.) This increase implies a differential rate
of apparent synthesis of luciferase; that is, addition of arginine to a
culture appears to promote a greater increase in the rate of luciferase
synthesis specifically than it does in general protein synthesizing
activity of the cell.
The _rn vivo luminescence observed in the culture from which the
0 f
enzyme assays were taken is also indicated in Figure 3. The slope, or
rate of increase, of both curves is essentially parallel, deviating only
at the extremes. Since the chemical environment involved in the esti
mation of enzyme activity under these two conditions is so different,
some variation was expected.
Lag Period
A distinct lag of approximately 12 minutes is evident between the
addition of arginine and the first measurable increase in luminescence.
Although it may be seen in Figure 1, the expanded scale of Figure 4 makes
it more obvious. Increasing the arginine concentration ten-fold does not

Luminescence (mv)
20
Figure 4: Onset of luminescence following addition of arginine.

21
reduce the length of this lag; however, the lag begins to increase as
-4
the concentration is lowered below 1 x 10 M arginine. Coffey (1) has
dealt rather extensively with this aspect of the problem and has con
cluded that approximately eight of the twelve minutes represent a period
of "activation" of the enzyme, the primary structure of the protein being
completed in the first four minutes after adding arginine to the medium.
This lag period and Coffey's interpretation will be given further con
sideration later.
Arginine Specificity
t
Stimulation by Other Compounds
The stimulation of bioluminescence in A. fischeri ND shows a high
degree of specificity for L-arginine. A number of compounds were tested
for their ability to serve as stimulators; these are listed in Table 1.
With the exceptions of L-citrulline and L-proline, as discussed below,
they were uniformly ineffective in stimulation of _in vivo bioluminescence
over control levels.
The groups of compounds in Table 1 were selected to provide some
insight into the mechanism by which arginine exerts its stimulating effect
on the bioluminescence system. Because of the participation of arginine
in the cycle leading to urea biosynthesis, the various intermediates in
that pathway (Table 1, Group 1) were tested for their ability to stimulate
luminescence in this strain of bacteria. In view of the lag period dis
cussed above, which might represent the time required to effect a conversion
of arginine to another compound, this approach seemed particularly promising.
Also included in this group are L-glutamic acid and L-proline which are
related to the urea cycle, and are derivable from arginine in Sacchromyces
cerevisiae by the following initial steps:

TABLE 1
Compounds Tested for Stimulation of _In Vivo Luminescence
GROUP 1: Urea Precursors
L-Glutamic acid
Carbamyl Phosphate
L-Proline
Urea
L-Ornithine
L-Citrulline
L-Aspartic acid
GROUP 2: Other
Glycine
L-Alanine
L-Valine
L-Leucine
L-Isoleucine
L-Serine
L-Tyrosine
L-Tryptophan
L-Threonine
L-Phenylalanine
"Amino" Compounds
L-Cysteine
L-Methionine
L-Lysine
L-Histidine
L-Proline
Ammonium Chloride
Ammonium Phosphate
Ammonium Sulphate
Adenine
Cytosine
GROUP 3: Non-amino Compounds
D-Glucose Citrate (Na+ salt)
Pyruvate (Na+ salt) Dodecanal
Nitrite
GROUP 4: Arginine Analogues
Agmatine
L-Homoserine
Guanidoacetic acid
L-Canavanine
L-Homoarginine
D-Arginine

23
arginine *. ornithine glutamic V semialdehyde
+
urea
(29). The glutamic-X -semialdehyde may then be oxidized to glutamic
acid or cyclized (via dehydration) to lead to proline synthesis (30).
Of the compounds listed in Group 1, Table 1, two resulted in increased
light production by the log phase cultures to which they had been added:
L-citrulline and L-proline produced 400-fold and 20-fold increases re
spectively in _in vivo luminescence compared to the luminescence level
of an untreated culture. L-ornithine failed to show any stimulatory
effect; however, within the limits of sensitivity of the ninhydrin pro
cedure, no ornithine uptake could be detected. The response to added
citrulline and proline is believed to result from intracellular conversion
of these amino acids to arginine or as arginine sparing intermediates.
This is acceptable as a working hypothesis, since citrulline and proline
are involved in the general pathway leading to arginine synthesis and
their effectiveness is directly related to their remoteness from arginine
in that pathway. -
In view of the apparent specificity for arginine, knowledge of the
effectiveness of arginine analogues (Group 4, Table 1) should yield some
insight into the specific functional group requirements for stimulation
of bioluminescence in this organism. The importance of the guanido
group was examined by supplying it in. the form of guanidoacetic acid, by
replacing it with an hydroxyl group in the arginine structure (homoserine),
and by adding both guanidoacetic acid and homoserine simultaneously to the
culture. Agmatine was tested to determine the effectiveness of the
guanido and amino groups in combination. None of these compounds, either
singly or in combination, demonstrated any stimulatory effect on the
luminescence reaction.

24
Specificity for the three-dimensional structure of arginine was
also investigated (Group 4, Table 1). An increase of one carbon atom
in the chain length (L-homoarginine) and the substitution of an oxygen
for a carbon atom in the L-arginine chain (L-canavanine) were tested.
Each of these changes alters the fundamental structure of L-arginine
sufficiently to make it ineffective as a stimulator of the luminescence
reaction.
D-arginine was tested to determine the influence of optical iso
merism on arginine specificity. Addition of this compound to a culture
produced no change in luminescence intensity relative to controls. However,
ninhydrin analysis of the medium indicated that D-arginine does not
enter the cells. For this reason, no conclusion may be drawn in terms
of the effect of optical isomerism on the intracellular molecular specifi
city of the bioluminescence response.
It should be noted that the addition of L-canavanine to one of two
-4
cultures containing equal amounts of L-arginine (2.4 x 10 M) resulted
in approximately 36 percent inhibition of the luminescence response com
pared to the arginine control. Canavanine was also found to inhibit
growth of the organism. This inhibition could not be completely reversed
by the addition of arginine to the medium in 10 times the canavanine
concentration. The arginine sparing of growth inhibition by canavanine
is shown in Table 2. Because of this'general inhibition of growth, and
indications in other bacteria that canavanine interferes with the utiliza
tion of arginine for protein biosynthesis (31), the significance of the
failure of canavanine to stimulate luminescence is somewhat difficult to
assess. It is not known whether the specificity requirements are such
that canavanine is not suitable as an arginine substitute or if its

TABLE 2
ARGININE SPARING OF CANAVANINE INHIBITION OF GROWTH
Flask Number'
Arginine Concentration
x 10"^ m
7o Control Growth
Rate
Control*
-
100
1
-
17.4
2
1.0
22.9
3
2.5
26.7
4
5.0
32.1
5
25
57.2
Each experimental
flask contains 2.5 x 10"^M
Canavanine
* Standard nitrate medium with no additions

26
effect on the general physiology of the cell prevents the appearance of
an increase in luminescence. It is possible,for example, that luciferase
is synthesized, but is non-functional due to incorporation of canavanine
rather than arginine. This might be clarified by looking for the forma
tion of protein which can be precipitated by anti-luciferase antibody.
Arginine Uptake
The specificity for exogenously added L-arginine and the delay in
the onset of the increase in _in vivo luminescence suggested a possible
lag in arginine uptake by the cells, as might be occasioned by the re
quirement for induction of a permease or by a slow process of simple
diffusion. Therefore a study of the rate of arginine uptake was attempted.
The rate of disappearance of ninhydrin-positive material from the
medium after adding arginine was used as a measure of the uptake of the
amino acid (Figure 5). Treatment with permutit (0.4 g/ml) to remove
ammonia produced no significant change in values obtained from treated
and untreated parallel samples. Although numerical values did not

correspond exactly from one experiment to another, data plotted on the
basis of triplicate ninhydrin assays of each sample in ten experiments
yielded essentially identical curves. Two additional methods more specific
for arginine were also used to measure the uptake of this amino acid. One
was a quantitative fluorescence procedure reported to be specific for
guanido and ureide groups (19); the other was the disappearance of uniformly
labeled ^C-arginine. Results of three experiments involving each of these
methods demonstrated uptake kinetics different from those determined using
the ninhydrin reaction. Figure 5 illustrates data taken from a single
experiment in which all three procedures were employed. Guanido-group
and 1^C-label assays show close agreement in their variation in time, but
the ninhydrin results are seen to be out of phase.

Relative Value
1.0
0.9
0.8
0.7
0.6
0.5
0.4
H 1 I 1 1 1 1 1
10 20 30 40 50 60 70 80 90
Time (minutes)
Figure 5: Arginine uptake. (For each of the three methods plotted, the values were obtained from
membrane-filtered samples of medium removed from a culture at the indicated times.
ho

28
Induced Periodicity
The regular fluctuation in the arginine uptake studies, shown in
Figure 5, suggests a rhythmic behavior of the culture. Examination of
other factors revealed a similar periodicity in both growth and in in
vivo luminescence following the addition of arginine to a culture (Figure 6).
Arginine uptake is taken from the "^C-label information presented in
Figure 5. Growth is indicated in Klett-Summerson turbidity units measured
at ten-minute intervals, and luminescence is plotted as points read from
a recording of in vivo light production. A similar periodicity exists
i ?
in all three factors measured.
There does not appear to be an inherent periodicity in either
bacterial luminescence or cell division: continuous recordings of light
intensity produced by the wild type A. fischeri or by the strain being
studied here (when grown in luminescence-supporting medium) reveal only
a uniform increase as the culture develops. In addition, studies of
cultures in complete or minimal nitrate medium (prior to adding arginine)
t i'
indicate no periodicity in growth rate. Therefore, the rhythmicity observed
in the factors shown in Figure 6 is believed to be due to the addition of
arginine. Further work will be required to define more carefully the
nature of the periodicity and to clarify the relationship shown in Figure 6.
Arginine Concentrating Ability
The rapid initial rate of arginine removal from the medium sug
gested that some system was operating to take up the amino acid at a rate
greater than that expected for simple diffusion. Experiments were conducted
to determine if this strain of bacteria was capable of accumulating intra
cellular arginine against a concentration gradient based on the relative
intra- and extra-cellular concentration of ^C-label (introduced as uniformly

Luminescence (mv) Relative Arginine Concentration
Figure 6: Periodicity in growth, luminescence and arginine uptake. (Arginine[,2.4 x 10"^m] ro
added at time 0).
Growth (KS units)

30
labeled ^C-arginine, specific activity 0.6 mC per mM, final concentra
tion 1 x 10~^M). The procedure is somewhat complicated by the fact that
arginine is utilized in protein synthesis and thus care must be exercised
to measure only free arginine as the internal pool.
Interpretation of the above experiment must be in terms of the
radioactivity per unit volume of cytoplasm and medium. To do this, the
number of cells per ml of culture was determined directly by microscopic
counts. The volume of a single cell was calculated as the volume of a
cylinder having dimensions of the average cell size determined from
f
electron photomicrographs of bacteria taken from log phase cultures. The
dimensions used were found to compare favorably with those listed for
wild type A. fischeri in Bergev's Manual (23).
Several sources of error in this method must be considered. The
**
first is that direct counting of cells, in addition to sampling and dilu
tion errors, does not distinguish viable cells. Since viable counts
correspond very closely to direct microscopic counts of cells in early
log phase cultures, and these experiments were performed on such cultures,
it was assumed that direct counts would accurately represent the number of
viable cells. A second error source is that the cells are not perfect
cylinders; therefore the volume calculations are not exact. Deviation
from cylindrical form in these bacilli occurs primarily as a rounding of
the ends, thus introducing little variation from the volume of a cylinder.
Thirdly, the use of average cell dimensions introduces error since all the
cells are not the same size. By using the mean value, it was assumed that
variation about that measurement would be compensated. Further, it was
assumed that the cells used to determine the average dimensions of these
bacteria represented a random sample. Finally, error in volume calculations
is occasioned by inclusion of the cell wall volume. No estimation

31
is available for this volume. However, the influence of this error is
to decrease the apparent intracellular concentration of ^C-labeled
material. Since the actual volume of cytoplasm in which the ^C-label is
concentrated is less than the estimated volume, the real concentration
gradient is greater than the calculated one. The error introduced by
including any non-viable cells in the calculations would have a similar
influence on the estimated concentration gradient.
Within these limitations, calculations indicated concentration
gradients of 1,180-fold.after 5 minutes and 1,870-fold after 10 minutes.
In light of the above discussion of errors, these estimates are probably
low. According to A.G. DeBusk (personal communication) a 500-fold con
centration gradient estimated by these methods is sufficient to indicate
a concentrating mechanism. Therefore, these values strongly suggest the
existance of a mechanism for taking up arginine against a concentration
gradient.
Arginine Metabolism
Figure 5 shows data for arginine disappearance from the medium in
time. Although the curves for the measurements of guanido groups and
14 \
C-arginine show close agreement at ll stages, the assay for ninhydrin
positive material was out of phase with these two. Since the data for
all three curves were obtained from the same samples, the presence of
ninhydrin-positive materials other than arginine was indicated. There
fore, an examination of the medium by chromatography was carried out.
Proline was identified as the only ninhydrin-positive compound
present in detectable quantities in minimal nitrate medium of a log
phase culture prior to the addition of arginine. Chromatographed sample
concentrations sufficient to yield a readily discernible yellow spot for

32
proline after spraying with ninhydrin revealed no other distinct area
having a positive reaction. Thirty minutes after adding uniformly labeled
^C-arginine (specific activity 0.4 mC per mM; final concentration of
-4
2.4 x 10 M) to a culture, proline was chromatographically isolated from the
medium, but the eluted material contained no radioactivity. However
14
C-label was isolated in the forms of citrulline and urea as well as
arginine. The findings implicated these materials in the discrepancy be-
14
tween the ninhydrin and C-label assays depicted in Figure 5.
A series of experiments were designed to elucidate the intracellular
/ f
fate of arginine. For this work, an early log phase culture was exposed
to uniformly labeled ^c-arginine (specific activity 6 mC per mM; final
-4
concentration 2.4 x 10 M) for 90 minutes. A trichloracetic acid (5 percent)
extract of washed cells was chromatographed on a heated ion exchange column
using a series of buffers for elution. Ten ml fractions were collected
and assayed for radioactivity. Those fractions containing ^c-label were
compared to an elution pattern achieved with known amino acids. Having
thus made a tentative identification of most of the ^C-labeled compounds,
confirmation was obtained by thin-layer co-chromatography with standards
in two dimensions with two solvent pairs. The radioactive material was
required to chromatograph with and only with the standard in all instances.
Compounds identified as bearing ^C-label (which had been introduced
14
as uniformly labeled C-arginine) were as follows: arginine, urea, ornithine,
citrulline, arginino-succinic acid, proline, glutamic acid, <-keto-glutaric
acid, succinic acid, fumaric acid and malic acid. The latter four compounds
demonstrated very low specific activity and do not appear to constitute
a pathway of great importance in the early stages of arginine metabolism
in this organism. Compounds showing the highest specific activity (other
than arginine) were ornithine and citrulline.

33
Evidence from additional experiments does not indicate the presence
of urease in this strain of bacteria. Aliquots of A. fischeri N D
cultures were incubated in sealed vials in the presence of uniformly
labeled ^C-arginine and hyamine hydroxide (Packard) for 20 minutes.
At the end of this period, concentrated sulfuric acid was injected into
the vial to kill the cells and to drive dissolved C0£ out of solution.
Subsequent analysis of the hyamine hydroxide for ^(X>2 did not demonstrate
any of this gas.
From the above results, it is clear that early arginine metabolism
in this strain of bacteria does not lead to intermediates which were not
also tried as stimulators of luminescence (Table 1). This argues against
the hypothesis that arginine is converted to some other compound which
then acts to stimulate the bioluminescence reaction. If such a hypothesis
were correct, one of the arginine metabolites would be expected to be at
least as effective as arginine for stimulating the apparent synthesis of
luciferase. It can be argued that the preceding statement does not apply
to ornithine, since there appears to be virtually no uptake of this amino
acid.
A final series of experiments was carried out in an effort to confirm
arginine specificity. It was essential to determine the time at which
intracellular arginine conversion to other compounds begins. This was
done by collecting one ml samples of -the culture at one minute intervals
following the addition of uniformly labeled ^C-arginine. Each sample was
washed with cold minimal salts and then each filter was placed in 5 ml
of cold distilled water with gentle shaking for 20 minutes. Microscopic
examination showed very few whole cells remaining after this treatment.
The extracts were then concentrated under vacuum at room temperature

34
(about 27C) to a volume less than 1 ml. These concentrated samples were
applied to a Sephadex G-10 column (1 cm x 12 cm) to separate the small
organic molecules from macromolecules and inorganic salts by elution with
\
distilled water. The isolated compounds were then co-chromatographed with
10 /ig arginine in two dimensions, and in two solvent pairs (n-butanol:
acetic acid:water, 3:1:1; ethanol:ammonia:water, 8:1:1; t-butanol:2-butanone:
formic acid: water, 8:6:3:3; and n-butanol:pyridine: water, 1:1:1). Ninhydrin
was used to locate the arginine, and the adsorbant in that area was scraped
into vials for quantification of radioactivity. The remaining adsorbant
was also analyzed for radioactivity. Results of these experiments indicated
14
C-label associated only with arginine for the first eight minutes. The
nine-minute sample showed 20 counts per minute above background which were
located in areas other than that associated with arginine. Based on the
products of arginine catabolism indicated above, and on published Rf values
for these compounds in the solvents used, the area containing arginine was
believed to be isolated from areas containing other potentially labeled
compounds. Therefore, within the sensitivity limits of the present methods,
- no_conversion of arginine occurs for eight minutes after introducing it
into a culture.
Puromycin acts by interrupting the growth of polypeptides (32).
Preliminary work on the amino acid composition of luciferase has indicated
the presence of at least five arginyl residues per molecule (J.W. Hastings,
14
personal communication). Therefore, the cessation of C-arginine incorp
oration into TCA insoluable material following treatment with puromycin
is felt to reflect the blockage of luciferase synthesis. This represents
a maximum limit; biosynthesis of some proteins appears to be more sus
ceptible than others to puromycin inhibition (33). The degree to which
luciferase synthesis is sensitive to puromycin is not known, but the

35
apparent cessation to total protein synthesis implies the halt of luci-
ferase synthesis. The time required for puromycin inhibition of total
protein synthesis was estimated to be six minutes (Figure 7).
The factors to be considered in the final experiment in this series
are three-fold: 1) there is an apparent lag of eight minutes between the
addition of arginine to a log phase culture and the occurance of any arginine
metabolites in the cytoplasm of the bacteria; 2) evidence indicates that
puromycin completely inhibits protein synthesis within six minutes after
its addition to a log phase culture; and 3) in vivo luminescence may be
considered as a measure of intracellular luciferase concentration. If the
first two considerations are valid, then it should be possible to add
arginine to a log phase culture, and after a period of two minutes, add
puromycin with the result that protein synthesis is completely stopped
at about the same time that arginine metabolites become available to
the cells. The appearance of any increase in luciferase concentration
(implying de novo synthesis of the enzyme) in a culture treated in this
manner would most probably be attributable to the presence of arginine
rather than an arginine metabolite. Results of such an experiment were
interpreted in this manner (Figure 8).
Although the time element is critical to the interpretation of this
experiment, three considerations suggest that this approach is valid with
respect to time. First, independent of the mechanism by which an arginine
metabolite could affect luciferase synthesis, a finite time interval should
be required to exert this influence. Second, and also independent of the
mechanism, there must be a sufficient concentration of the molecular
species responsible for the effect: this may reflect an affinity factor,
or the element of probability that the proper interaction will occur.
Lastly, there must be enough time for the synthesis of complete luciferase

Time (minutes)
Figure 7: Puromycin inhibition of ^C-arginine incorporation into acid-insoluble material.
(Arginine added ten minutes before adding puromycin. Puromycin added at arrow.)
O'

Luminescence (mv)
30
5 --
0 1 1 1 1 1 : 1 1 1
-5 0 5 10 15 20 25 30 35
Time (minutes)
Figure 8: Effect of adding puromycin two minutes after adding arginine.
u>

38
molecules: since puromycin interrupts growing polypeptide chains (32),
any luciferase molecules synthesized in response to the added arginine,
must be completed prior to the inhibition resulting from the presence of
puromycin. Collectively, the hypothesis that a metabolite of arginine
is responsible for the bioluminescence response following the addition
of arginine to the medium of log phase cultures requires that the meta
bolite be present in sufficient concentration to exert its effect early
enough to permit the synthesis of complete luciferase molecules, but
still be below the limit of detection by the procedure employed. This
seems rather unlikely.
Using a somewhat similar approach to the problem of arginine
specificity, Coffey (1) concluded that the stimulation of bioluminescence
in a nitrate-utilizing strain of A. fischeri results from the presence of
t
arginine specifically rather than a metabolite of that amino acid. The
evidence presented here is offered in support of that conclusion.
Mechanism of Arginine Action *
m *
Effects of the Nitrate Ion
Studies of the enzyme nitrate reductase from wild type A. fischeri
led to the proposal of the electron transport scheme shown in Figure 9 (34).
Knowledge that FMNH2 serves as the reducing agent in the bacterial
luminescence system suggested to McElroy (35) that the addition of
nitrate ions to a culture would inhibit light production. The rationale
for this depends on the assumption that the supply of electrons to the
cytochrome system is constant. If this is true, addition of nitrate
should add a pathway for electron removal, thus diminishing the FMNH2
available to the light reaction. Therefore, one would expect luminescence
to be reduced in a culture containing nitrate as compared to one without

DPNH(TPNH)
*> FMN(FAD)
Fe
Bacterial __ n
"^Cytochrome ^ 2
Nitrate Reductase
NO,
Abbreviations used are:
DPNH(TPNH) Reduced di-(tri) phosphopyridine nucleotide
FAD Flavin adenine dinucleotide
j | j
Fe Ferric ion .
(>2 Molecular oxygen
NO^ Nitrate ion
Figure 9: Proposed electron transport scheme for nitrate
reduction in A. fischeri (34).

40
that ion. In fact the simultaneous addition of large quantities of
-4
nitrate and arginine (2.4 x 10 M final concentration) to a log phase
culture growing on nitrate medium does reduce the intensity of the light
produced when measured 60 minutes after the time of the addition (Figure 10).
Although a pronounced effect on growth (estimated turbidimetrically) is
detected only after several hours, the reduction in luminescence intensity
is probably related to growth inhibition rather than nitrate ions,
since the addition of NaCl in high concentration produces a similar
dimming of luminescence.
The addition of a much lower concentration of nitrate ions (9 x 10"^M)
results in an enhancement of luminescence (Figure 11). This suggests that
the presumed drain on FMNH2 supplies which is imposed by nitrate is not
solely responsible for the greatly reduced light production. If it had
been found otherwise, i.e., that the presence of nitrate in the concen
trations used in the medium did inhibit luminescence, then the addition
of amino-nitrogen rich arginine could be interpreted as relieving the
nitrate reduction pathway so that a normal flow of electrons to the
bioluminescence reaction would be restored. This interpretation is,
however, not consistent with the results presented.
Additional evidence against the argument that nitrate reduction
depletes the FMNH2 pool at the expense of bioluminescence is found in
several other observations. If growth on minimal nitrate medium results
in amino-nitrogen limiting conditions, as discussed above, then other
amino acids, and especially ammonium salts, would be expected to be at
least as effective as arginine in stimulating light production; they were
not.
The role of the step in which nitrate is reduced to nitrite is also
questionable. Observations of cultures grown with nitrite as the sole

41
NaN03 (M)
Figure
(Data taken
nitrate ions
10: Effect of high nitrate ion concentration on luminescence.
60 minutes after simultaneous addition of large concentration of
and 2.4 x 10^M arginine.)

Luminescence
42
Figure 11: Effect of added nitrate in low concentration on
arginine-stimulated luminescence.

43
nitrogen source (nitrite present in equal molarity to the nitrate concen
tration in the usual medium) revealed identical behavior with regard to
the very low levels of constituitive luminescence and the response of
the luminescence system to added arginine. On the other hand, nitrate
reduction is involved in some manner with the luminescence system since
the addition of 9 x 10^M nitrate to a culture of wild type A. fischeri
results in an increase in light intensity similar to that found in the
nitrate utilizing strain (Figure 12) Since the wild type A_. fischeri
has a nitrate reductase, but lacks the ability to reduce nitrite (35),
this indicates that the effect of nitrate on the luminescence system in
wild type is associated with the reduction of that ion. It has been proposed
that two separate electron transport chains are involved; one leading to
reduction of oxygen, the other to nitrate reduction (35). The significance
of these alternate pathways relative to the present problem is not clear
at this time, nor is the role of nitrate reduction. A reasonable initial
step toward investigating this relationship seems to lie in determining
the effect of adding nitrate on the intracellular pool of FMNH2.
Effect of Arginine on Growth
McElroy and Farghaly (11) found that arginine must be supplied to
the arginine requiring mutants in quantities sufficient to exceed a
minimum level of growth in order to develop the bioluminescent system.
Although they did not measure growth rate (change in turbidity in time)
during the incubation period, it is assumed that in two cultures receiving
equal inoculations initially, a difference in turbidity after a fixed
incubation period (48 hours) represents a difference in growth rates in
the two cultures. In order to determine if the nitrate utilizing mutant
is analogous to their arginine requiring mutants, the immediate effect
of arginine on growth rate in this strain was examined. The hypothesis

Luminescence (mv
44
Figure 12: Effect of added nitrate in low concentration on
luminescence in A. fischeri.

45
for these experiments was that endogenous arginine was present in quanti
ties which would support growth but not luminescence. Plots of turbidity
readings taken at 10-minute intervals on parallel samples of a culture,
-4
one containing 2.4 x 10 M arginine and the other serving as an arginine
less control, were essentially congruent over a three-hour period following
the addition of arginine. This information is shown in Figure 13. It is
noted that the periodicity seen in the arginine treated culture is not
observed in the control.
The significance of this congruency is doubtful. Kjeldgard et al. (36)
i f
found a definite lag in the alteration of growth rate in their work on
Salmonella typhimurium. Moreover, McElroy and Farghaly (11) found that
the rate of change in bioluminescence was much more rapid than that in
growth as the initial arginine concentration was increased above 0.01 mg
per ml. Therefore an increase in arginine concentration could be expected
to influence luminescence more than growth rate over a period of time.
An experiment was then performed to determine the long-term effect
, i
of arginine, and other compounds, on growth rate. This was done by
estimating the doubling time of log phase cultures (having effectively
the same turbidity) which were growing in liquid media supplemented
with the indicated compound in a final concentration of 9 x 10^M. For
each substance listed, media were prepared with and without the standard
concentration of nitrate present to determine the growth-supporting
ability of the various materials listed. In addition to determining
the doubling time, the bioluminescent intensity was measured for each culture
at the same turbidity (where growth had occurred) and both were related to
their respective values for cultures grown in medium supplemented with
1.0 percent peptone. These results are summarized in Table 3. In order

Growth (KS units)
Figure 13: Short-term effect of arginine on growth.

LONG-TERM EFFECT OF VARIOUS NITROGENOUS COMPOUNDS ON LUMINESCENCE AND GROWTH
TABLE 3
NITROGEN NITRATE PRESENT NITRATE ABSENT
SOURCE % Growth Rate L Luminescence /0 Growth Rate % Luminescence
Peptone
100
100
100
100
Nitrate
54
0.06
60
0.14
Arginine
61
90
44
56
Aspartic acid
56
1.90
69
0.14
Ornithine
29
0.65
10
0.25
Citrulline
28
0.39
22
0.18
Urea
30
y
y
7.70
6
1.80
Glutamic acid
22
0.16
31
0.29
Proline
61
26
53
2.50
Homoserine
Agmatine
36
No growth or
2.90
luminescence measurable
14
0.36
Homoarginine
Canavanine
10
0.04
No growth or luminescence
detec
nh3
51
3.50
53
0.18
Lysine
43
. 0.21
12
0.18
Methionine
61
- 5.40
77
7.20
Histidine
58
4.60
39
0.28
Serine
34
11
17
2.70

48
to compensate for total available nitrogen, the cultures containing
nitrate were prepared with nitrogen concentration equivalent to that in
the other media in the same column.
Two observations are important to the problem being discussed.
First, growth is more rapid on a medium containing both arginine and
nitrate than it is on a medium containing nitrate only. However, a
very similar growth rate may be obtained by culturing on media containing
L-proline, L-methionine or L-histidine instead of L-arginine. Secondly,
whereas arginine plus nitrate supports a level of luminescence approximat-
/ f
ing that obtained in complete medium (containing peptone), the other amino
acid supplements which provide growth rates comparable to that on arginine
plus nitrate were, at best, only one-fourth as efficient in supporting
luminescence. On the other hand, serine, which did not promote growth as
well as L-methionine or L-histidine, supported a luminescent intensity
which was more than twice that for the L-methionine or L-histidine. There
fore, it may be said that although arginine does increase the growth rate
of a nitrate-grown culture over a long period, other amino cids are
equally effective in doing this without the concomitant.increase in
luminescence. Also the relationship between doubling time and lumin
escence is not a direct one, in view of the data for serine. Thus,
while arginine does enhance the growth rate of these bacteria over an
extended period, the degree to which it enhances luminescence appears
to be specific and independent of the effect on growth.
One arginine metabolic pathway known to exist in bacteria deserves
particular consideration. In Streptococcus faecalis, arginine is hydro
lyzed to yield citrulline and ammonia (37), and the citrulline is
phosphorolyzed to yield ornithine and carbamyl phosphate (38). The

49
carbamyl phosphate then reacts with ADP to produce ATP in a reaction which
favors ATP formation (39). This mechanism for phosphorylation of ADP
via arginine metabolism is noteworthy because it bypasses the electron
transport system which is intimately involved with nitrate reduction
and the bioluminescent reaction. Levels of ADP and ATP are known to
serve in a regulatory capacity for other enzyme systems (9,40,41).
Indirect evidence from several lines of investigation suggests that the
carbamyl phosphate pathway for phosphorylation of ADP is not likely to
be involved in the action of arginine on the bioluminescence system in
i
this strain of bacteria. The first of these is that citrulline would
be expected to be at least as effective as arginine in stimulating lumin-
V
escence if the mechanism involved generation of carbamyl phosphate via the
pathway described above; this was found not to be the case. Citrulline
was the most effective of all compounds tested (other than arginine) as
stimulators of luminescence, and its uptake was similar to that of arginine,
but it was not as potent as arginine. A second argument against ATP (or ADP)
involvement in controlling bioluminescence is based on the findings that
these organisms are obligate aerobes. Attempts to grow', cultures under
a variety of anaerobic conditions were unsuccessful. These efforts
. \
\
included conditions with nitrogen, carbon dioxide or hydrogen atmospheres
in the culture flasks containing nitrate medium. Addition of arginine
did not alleviate this total inhibition of growth. Two assumptions are
necessary to make this argument relevant: first, it is assumed that this
absolute requirement for oxygen reflects a mandatory operation of the
electron transport coupled oxidative phosphorylation system, and,secondly,
that ADP phosphorylation via carbamyl phosphate production from arginine
could supplant the normal oxidative process to the extent that some growth,
even though very slow, could occur.

50
Therefore, although the activity of specific enzymes was not
measured, this evidence indicates that generation of ATP via carbamyl
phosphate does not play a primary role, at least in the stimulation of
luminescence in these bacteria following the addition of arginine to the
medium of a "nitrate" grown culture.
Arginine Incorporation into Luciferase
An early consideration was that luciferase might be very rich in
arginine and that synthesis would be limited except in the presence of
a large arginine pool./ This was investigated by determining the amount
of specific radioactivity of partially purified luciferase as compared
to similar measurements of general cell protein 60 minutes after' intro
ducing uniformly labeled ^C-arginine (specific activity 0.4 mC per mM,
-4
final concentration 2.4 x 10 M). Results of this experiment showed
that total intracellular protein contained an average of 3,186 dpm per
mg protein. The luciferase fraction (purified 30 fold) was found to
contain 2,142 dpm per mg protein.
Therefore, luciferase does not preferentially incorporate arginine
as compared to the general cellular proteins. These results are in
agreement with the estimation of fiv^ arginyl residues per luciferase
molecule (J.W. Hastings, personal communication).
Effect of Inhibitors of Bioluminescence Response
Synthesis of new protein is associated with the increase in bio
luminescence. This is suggested in Figure 3, in which the _in vitro
assay of luciferase activity in aliquots of culture collected at timed
intervals following the introduction of arginine into the medium is
related to _in vivo bioluminescence observed in a parallel culture at
the same times.

Figure 14: Effect of mitomycin on the luminescence response to added arginine. Mitomycin
(10 jug/ml) added to one culture five minutes before adding arginine. Arginine (2.4 x 10"^M)
added to all three cultures at time 0. Mitomycin added to second culture at 40 minutes. Third
culture received no mitomycin.

11
10
9
8
7
6
5
4
3
2
1
0
Mitomycin added at -5 min.
Arginine added at 0

53
In order to clarify this point, inhibitors of protein synthesis
were employed. Addition of puromycin (10 pg per ml) or chloramphenicol
(10 pg per ml) 5 minutes prior to adding arginine (2.4 x 10^M) to a
log phase culture prevented any increase in bioluminescent intensity.
This strongly suggests that new protein synthesis occurs in response to
added arginine. Coffey (1) used, in addition to those inhibitors, amino
acid analogues, which proved inhibitory, and found that the inhibition
was reversible by the addition of the proper amino acid. He also showed
that messenger RNA synthesis is probably involved since 5-fluorouracil
i f
serves as an inhibitor of the response to added arginine. In support
of this conclusion about messenger RNA, it was found that pretreatment
of a culture with mitomycin (10 jig Per ml added 5 minutes prior to arginine
addition) prevented the expected increase in light production (Figure 14).
Cheer and Tchen (42) described similar results for the inhibition of
y^-gal'actosidase induction in E. coli. Since a considerable lag occurs
before this antibiotic influences synthesis of RNA or protein (43), Cheer
and Tchen suggested that mitomycin acts selectively on. genes which are
not involved in synthetic activities at the time of exposure to the
inhibitor. Later, Iyer and Szybalski (44) indicated that mitomycin acts
by cross-linking complementary strands of DNA. Assuming Cheer and Tchen
were correct in their analysis, the effect of mitomycin on this system
may be interpreted as indicating that the genes for luciferase are not
being transcribed prior to addition of arginine, and are therefore sus
ceptible to mitomycin action. If this were true, then the addition of
this antibiotic would not be expected to influence the increase in
luminescence, once it was initiated by arginine. This, in fact, was
observed to be the case (Figure 14). A temporary interruption (2-3 minutes)

54
in the increase in luminescence resulted when mitomycin was added. A
similar pause was noted in p -galactosidase in the work of Cheer and Tchen.
They showed that general protein synthesis was not inhibited during this
period, however, but offered no explanation for the interruption of
^-galactosidase synthesis.
Effect of Initial Arginine Concentration
In Figure 15, the maximum luminescence achieved is plotted against
the initial concentration of added arginine. The sigmoidal shape of
this curve suggested another possible relationship between arginine and
/
luciferase. One of the more puzzling aspects of this problem is the
apparently unrelated nature between arginine and the luminescent reaction.
If arginine functions in the capacity of an allosteric effector, however,
no obvious relationship is necessary. The curve in Figure 15 suggests
that this is a possibility.
"Allosteric" proteins were initially defined by Monod, j2t _al. (3)
as those enzymes in which activity is regulated via changes induced in
the protein conformation by a molecule (the allosteric, effector) which
is not necessarily involved in the reaction mediated by.-the enzyme.
In their paper, the authors pointed out that a sigmoid curve is obtained
when In vitro reaction velocity is plotted against substrate concentration
for certain enzymes. This was interpreted as meaning that more than
one ligand could be bound to the enzyme at the same time, and, moreover,
that some kind of "cooperative interaction" existed between the binding
sites. The sigmoid form of the curve was found to linearize by appli
cation of the relationship which Hill (45) had described for the hemo
globin oxygen-saturation curve. Atkinson (41) points out, if one
assumes that reaction velocity is proportional to the fraction of sub
strate binding sites saturated, the Hill equation may be expressed as:

Figure 15: Effect of concentration of added arginine on _in vivo luminescence.
I

Luminescence (mv
Ln
ON

57
log v/(V-v) = n log (S) Log K
in which v is reaction velocity, V is maximum velocity, n is the number
of the substrate binding sites, S is substrate concentration and K is a
constant.
Assuming that the maximum _in vivo luminescence obtained represents
"reaction velocity" (v) for each concentration (s), and that the maximum
luminescence achieved by the highest two concentrations (shorn in Figure 15)
represents maximum reaction velocity (V), a Hill plot was made and is illus
trated in Figure 16. Although it is not linear at the extremes, the region
> f
of the curve near the midpoint (where log v/(V-v) = 0) is straight and has
a slope of 2.93. Since this value represents a measure of both the strength
of cooperative interaction and the number of binding sites (46) it is assumed
that if this is an allosteric effect, three arginine binding sites exist on
the luciferase molecule.
An alternative interpretation of Figure 15 is that at lower concentra
tions, a greater proportion of the added arginine is used for purposes
other than stimulating bioluminescence; i,.j;. there is some minimum quantity
of arginine that must be supplied for other uses before,it can be applied to
the bioluminescence system. This is suggested as a possibility in light
of the work by McElroy and Farghaly (11) if it is assumed that growth on
nitrate medium represents an arginine-limiting condition. Their plot of
luminescence versus arginine concentration for an arginine requiring
mutant exhibits a sigmoid shape when luminescence is expressed as a per
centage of the maximum obtained. Application of the Hill equation to
their data results in the plot shown in Figure 17. Here the slope is 3.2
at the midpoint. Thus the value for the slope of the Hill plot of these
data agrees reasonably well with the one for the present work. This
suggests that a similar relationship might exist for the two systems.

Luminescence
Luminescence max luminescence
58
Figure 16: Hill plot of arginine concentration effect.

Luminescence
Figure 17: Hill plot of data for arginine requiring mutant
(data from McElroy and Farghaly, 1948).

60
Another experiment involving the effect of arginine concentration
on luciferase activity may be interpreted as an allosteric interaction.
This experiment consisted of the addition of a second equal amount of
arginine to a culture following the re-establishment of the luminescence
level at the control value. Results of this experiment are plotted in
Figure 18. The second addition of arginine resulted in two changes with
respect to the effect of the first: 1) the intensity of the second
luminescence response was approximately twice as great as the first (and of
longer duration, although this is not shown); and 2) the lag period between
> f
the addition of arginine and the first detectable increase in luminescence
was shortened. Assuming arginine does act as an inducer of luciferase on
the basis of the information presented above, a finite amount of time is
required to accomplish this. Coffey (1) has estimated that this is four
minutes. During that time, available arginine is continually being removed
from the system into protein synthesis. Assuming that arginine also
exerts an allosteric effect on luciferase, the total amount of light pro
duced will be a function of the effective concentration of arginine and
the duration of that concentration. Provided luciferase is still present
but non-functional, due to low arginine levels (resulting in a decrease in
\
luminescence), a second equal addition of arginine should result in a
luminescence response of longer duration and greater magnitude. As shown
in Figure 18, this was found to be the case.
Again a growth requirement interpretation may be applied. If
arginine acts as a simple inducer of luciferase, but must satisfy a
growth requirement before induction is accomplished, then a greater pro
portion of the first addition of arginine xrould be expected to go for this
purpose than would be expected for the second arginine treatment.

Figure 18: Effect of second addition of arginine

62
In Vitro Effects of Arginine
Allosteric effects are usually demonstrable In vitro since the
interaction exists between the enzyme molecule and the effector, thus
requiring no other cellular constituents (as does the induction process
which depends on the operation of the genetic system). Therefore, an
investigation was made of the effects of arginine on the jin vitro behavior
of partially purified luciferase.
One method for demonstrating allosteric effects is based on dilution
of the presumed effector in vitro (47). If arginine exerts an allosteric
effect by blocking an inhibitory action of the luminescence system due to
some other chemical species present in the cytoplasm, then a greater than
proportional decrease in luciferase activity would occur in dilution with
a crude extract of non-arginine treated cells. If no such material exists
then dilution of a luciferase preparation with a crude extract of cells
not treated with arginine should result in a proportional decrease in the
in vitro luciferase activity. Results of such an "admixture" experiment
yielded values of 2,500 mv peak flash intensity with an extract of arginine
treated (2.4 x 10 arginine for 60 minutes)cells, no measurable lumines
cence in an extract of untreated cells, and 1,300 mv peak flash intensity
when the enzyme fraction used was a 1:1 mixture of the extracts from
treated and untreated cells. Therefore, the existance of a luminescence
inhibitive compound which is antagonistic to the action.of arginine was
not demonstrated by these experiments.
A long-chain fatty aldehyde requirement for In vitro luciferase
activity has been shown (48). The suggestion has been made that the
aldehyde acts by altering the configuration of the luciferase protein (28).
This offered the possibility that arginine might exert the same effect,

63
particularly since no aldehyde component has been firmly identified in
bacterial extracts. It was found, however, that arginine in three
different concentrations did not promote a luminescent flash in an assay
system containing no aldehyde. Therefore, if arginine does act as an
allosteric effector, it does not serve the same function as aldehyde.
The possibility that arginine, acting independently of aldehyde,
might enhance the efficiency (flash intensity or decay rate) of the
in vitro system was then examined. Tracings of the light response in an
in vitro system in the presence and absence of 100, 200 and 300 jig arginine
> f
are shown in Figure 19. As may be seen, there is no apparent difference
which may be attributed to arginine.
Bovine serum albumin is included in the assay system to aid
against denaturation of the enzyme as a result of the dilution factor.
Results of analyses of jiji vitro luciferase activity in the absence of
albumin show that arginine does not have any apparent effect as a
replacement for the added protein. Figure 20 shows tracings of such
, X
analyses. .
Finally, a noteworthy observation was made on the-, behavior of the
enzyme on stimulation of a second _in vitro flash. The intensity of the
flash following addition of a second aliquot of FMNH^ was considerably
reduced compared to the first flash. Since the second injection of
FMNH^ followed the first by five minutes, the control was made up so
that the enzyme was exposed to an equal concentration of FMN for five
minutes before initiating the first flash. This control showed a greater
activity than observed under experimental conditions with enzyme which
had been stimulated with FMNH2 before. The effect of arginine on the
reduced intensity of the second flash was investigated. Tracings of the

Luminescence (mv x 1CP) Luminescence (mv
64
Figure 19: Effect of arginine on jin vitro luciferase activity.
A : Arginine absent
B : 100 jug arginine added
C : 200 jug arginine added
D : 300 jug arginine added

m
o
Figure 20: Effect of arginine on ini vitro luciferase activity in
the absence of bovine serum albumin.
a:
b;
Arginine absent
Arginine present

66
control, first and second flashes in the presence and absence of 150 jig
arginine HC1 are shown in Figure 21. There is no apparent improvement
in the second response in the presence of arginine. Therefore, in these
experiments there does not seem to be any _in vitro effect by arginine on
luciferase activity. Experiments similar to those described above were
performed using citrulline, ornithine, or proline instead of arginine
with the same results. Urea was also tested in the place of arginine,
and was found to be slightly inhibitory, which was expected since, at
higher concentrations (8M), urea has been shown to reversibly denature
bacterial luciferase (49). It does not appear, then, that an early
metabolic product of arginine is involved, at least to an extent greater
than arginine, as an allosteric effector on the basis of these rn vitro
studies.
In Vivo Effector Function of Arginine
In mammalian systems, one means of regulating the activity of
enzymes is by controlling the rate of their degradation (7). Generally
speaking, this mechanism is not found in growing bacteria, but apparently
does occur in non-growing bacterial cultures (50). Although only log
phase cultures were used for the experiments involving the response
of the luminescence system to added arginine, the rapid decline in light
production (as shown in Figures 1, 18, 19) once peak luminescence had
occurred suggested that luciferase might be under a degradative control,
or that, in view of the in vitro decline in activity observed with a
second FMN^ stimulation, perhaps the luminescent activity of the enzyme
was itself an inactivating (or degrading ) process.
To investigate this possibility, luciferase synthesis was inter
rupted by the addition of puromycin to a culture following the intro
duction of arginine and the resultant increase in luminescence. After

67
CO
o
CO
o
0 .. 10 20 30
Figure 21: Effect of arginine on second ijn vitro flash.
a ,d:
First flash
b,e:
Second flash, 5 minutes after first
c,f:
FMN control, first flash
a, b, and c:
Arginine absent
d ,e, and f:
Arginine present (150 ig)

68
a brief continuation in the rise of luminescence, light production began
to decline. Two experiments were carried out; one entailed adding puro-
mycin to parallel cultures treated with different amounts of arginine at
a point where the luminescence of the cultures was equivalent; the other
involved the addition of puromydin following a fixed period post arginine
treatment. In each case, it was felt that the decay rate of luminescence
would be different for cultures containing 4.8 x 10"arginine and
4.8 x 10'4M arginine if the amino acid were acting to stabilize the
luciferase against a degradative action. Results of these experiments
are shown in Figure 22. The rate of decay of luminescence under these
conditions appears to be independent of the initial arginine concentration
irrespective of whether the exposure time was the same or the luminescence
intensity was the same.
Discussion of Mechanistic Interpretations
A characteristic of inducible enzyme systems in bacteria is the
relationship between the inducer and the induced enzyme. Although certain
* materials which are not acted upon by the induced enzyme, and which are
not metabolized by the cell have the ability to serve as "gratuitous
inducers", these are related to the reaction in the sense that they bear
some similarity to the substrate which is usually assumed to be the
"natural" inducer. The most thoroughly studied inducible system is
the /^-galactosidase system, in which /^-thioglactoside acts as a
"gratuitous inducer" (51). In the case of arginine and bacterial
luciferase, there does not appear to exist such a relationship. Also,
though it is not deemed as important as the exceptions just mentioned,
the early kinetics of the luciferase response to arginine are not as
rapid as might be expected on the basis of the inducer model (51).

Luminescence (mv
(a)
(b)
Figure 22: Effect of puromycin addition to cultures containing different concentrations of
arginine. (a) Puromycin added when luminescent intensities were equal. (b) Puromycin added after
equal incubation periods with arginine.
O'

Therefore, to call this an inducible enzyme system, implies a departure
from the classical model proposed by Jacob and Monod (2).
70
On the other hand, consideration of this system as one involving
allosteric effects is also at odds with the definitions made in des
cribing the model (4). In the formulation of the proposed mechanism,
a specific requirement was that the allosteric protein must consist
of at least two identical subunits associated in such a way as to occupy
symmetrical positions. Bacterial luciferase has been reported as com
prised of two kinds of subunits (52). Very recently, active luciferase
/ f
was shown to contain two and only two subunits which are non-identical
(J. W. Hastings, personal communication). Thus far, no indications of
large aggregates of enzyme have been found. Therefore, bacterial
luciferase does not appear to meet the requirements originally established
for an allosteric protein.

SUMMARY AND CONCLUSION
The bioluminescent system in the ND strain of the marine bacterium
Achromobacter fischeri is not functional when these bacteria are cultured
in a minimal medium containing glycerol and nitrate as the only nutrients.
Absence of the luminescent reaction is associated with the virtual absence
of the enzyme luciferase in functional form. Addition of L-arginlne to
a log phase culture results in a dramatic increase in _in vivo light pro
duction and in luciferase activity as estimated JLn vitro. Results of
experiments testing other compounds as stimulators of luminescence and
comparison of the time required for arginine conversion to begin in the
cell with the appearance of luminescence increase indicate that the response
of the luminescent system is due specifically to arginine.
Arginine probably exerts its effect on the luminescent system,
via an enzyme "induction" at the genetic level, in a manner similar to
the model proposed by Jacob and Monod (2). Evidence for this is found
-in the apparent increase in luciferase protein, and the prevention of
this increase by inhibitors of protein synthesis. These findings are
presented in support of the conclusions drawn by Coffey (1) on the basis
of his work with A. fischeri ND. '
In his work on this problem Coffey (1) presented kinetic evidence
indicating that an "activation" step (of unknown nature) was required
before luciferase promoted any light production. Certain aspects of the
luciferase response to added arginine (in particular the relationship
between arginine concentration and the luminescence intensity) suggest
that this activation may be the result of an allosteric interaction between
arginine and luciferase. Although much of the evidence may be explained
71

72
in terms of such a hypothesis, no results of iii vitro or in vivo experi
ments indicate the existence of such an interaction. Therefore, inter
pretation of the results in terms of allosteric effectors does not, at
this time, seem appropriate.
This work is offered in confirmation of Coffey's (1) speculation
that L-arginine specifically acts as an inducer of bacterial luciferase
in Achromobacter fischeri ND.

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3. Hartman, R.E., and L.N. Zimmerman, 1960. Effect of the Arginine
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6. Freundlich, M., and H.E. Umbarger, 1963. The Effects of Analogues
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14. Hastings, J.W., Q.H. Gibson, J. Friedland, and J. Spudich, 1966.
Molecular Mechanisms in Bacterial Bioluminescence: On Energy
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15. Smith, I. (ed.), 1960. Chromatographic and Electrophoretic Techniques
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18. Moore, S., and W.H. Stein, 1954. Procedures for the Chromatographic
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22. Davidson, J.D., and P. Feigelson, 1957. Practical Aspects of Internal-
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25. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, 1951.
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26.Hastings, J.W., W.H. Riley, and J. Massa, 1965. The Purification,
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27. Gibson, Q.H., and J.W. Hastings, 1962. The Oxidation of Reduced
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29. Middlehove, W.J., 1964. The Pathway of Arginine Breakdown in
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30. Wood, W.A., 1956. Symposium on Microbial Amino Acid Metabolism,
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31. Schwartz, J.H., and W.K. Maas, 1960. Analysis of the Inhibition of
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32. Nathans, D., 1964. Puromycin Inhibition of Protein Synthesis: Incorpor
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U.S., 51:585-593.
33. Sells, B.H. 1965. Puromycin: Effect on Messenger RNA Synthesis and
^-Galactosidase Formation in Escherichia coli 15T~ Science,
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34. Sadana, J.C., and W.D. McElroy, 1957. Nitrate Reductase from
Achromobacter fischeri. Purification and Properties: Function
of Flavins and Cytochrome. Arch. Biochem. Biophys., 67:16-34.
35. McElroy, W.D., 1961. Bacterial Luminescence, pp. 479-508. _I
I.C. Gunsalus and R.Y. Stanier (eds.), The Bacteria, vol. 2.
Academic Press, Inc., New York.
36. Kjeldgard, N.O., 0. Maaloe, and M. Schaechter, 1958., The Transition
Between Different Physiological States During Balanced Growth
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37. Petrack, B., L. Sullivan, and S. Rutner, 1957. Behavior of Purified
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Citrulline Synthesis in Rat Tissues. Arch. Biochem. Biophys.,
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39. Jones, M.E., L. Spector, and F. Lipmann, 1955. Carbamyl Phosphate.
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40.Tomkins, G.M., K.L. Yielding, N. Talal, and J.F. Curran, 1963.
Protein Structure and Biological Regulation. Cold Spr. Harb.
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41. Atkinson, D.E., 1966. Regulation of Enzyme Activity. Ann. Rev.
Biochem., 35:85-124.
42. Cheer, S., and T.T.Tchen, 1962. Effect of Mitomycin C on the
Synthesis of Induced /-Galactosidase in EL coli. Biochem.
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Inhibition of Formation of Deoxyribonucleic Acid in E. coli by
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44. Iyer, V.N., and W. Szybalski, 1963. A Molecular Mechanism of
Mitomycin Action: Linking of Complementary DNA Strands. Proc.
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46. Atkinson, D.E., J.A. Hathway, and E.C. Smith, 1965. Kinetics of Reg
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Bacterial Luciferin-Luciferase Reaction _In Vitro. Arch. Biochem.
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Denaturation of Bacterial Luciferase. Biochem., 6:2893-2900.
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Populations of Escherichia coli. Biochem. J., 69:40-119.
51. Kepes, A., 1963. Kinetic Analysis of the Early Events in Induced
Enzyme Synthesis. Cold Spr. Harb. Symp. Quant. Biol., 28:325-327.
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52.

BIOGRAPHICAL SKETCH
Thomas Stephen Quarles was born October 1, 1941, in Beaumont, Texas.
In June,1960, he was graduated from Beaumont High School. In June, 1964,
he received the degree of Bachelor of Science with a major in Biology
from Lamar State College of Technology in Beaumont, Texas. In September,
1964, he entered the Graduate School of the University of Florida. He
worked as a teaching assistant in the Department of Zoology until August,
1967. From September, 1967, until August, 1968, he was a predoctoral
fellow of the National Institutes of Health. Since September, 1968, he
has been an employee of the Biology Department, Birmingham Southern
College, Birmingham, Alabama.
Thomas Stephen Quarles is married to the former Jeannette Downs and
is the father of two children. He is a member of the American Association
for the Advancement of Science, the American Society of Zoologists, and
0 *
Phi Sigma Biological Society.
\
77

This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to
the Dean of the College of Arts and Sciences and to the Graduate
Council, and was approved as partial fulfillment of the require
ments for the degree of Doctor of Philosophy.
March, 1970
Dean, Graduate School
Supervisory Committee:



4
to ultraviolet light. Among these were two strains which exhibited a
specific nutritional requirement for arginine. Both mutants showed a
linear growth response to low arginine concentrations. No luminescence
was evident, however, unless the cell density after 48 hours exceeded
30 percent of that attained by the wild type A. fischeri in arginine-
free medium after the same incubation period. As the arginine concen
tration was increased beyond the level satisfying this condition, the
slope of the growth versus arginine plot diminished and luminescence
developed rapidly. Both growth and luminescence were measured only
{ \
after an incubation period of forty-eight hours. No attempt to determine
the short-term effect of added arginine was reported. The only other
growth-supporting amino acid, ornithine, gave results similar to those
reported for arginine. The workers concluded that the growth and
luminescent systems were competing for th.e common factor arginine or some
component containing arginine. The also pointed out that the wild type
A. fischeri exhibited a distinct lag in the production of light during
the early stages of rapid growth. This suggested that strong competition
*
existed for some component common to both luminescent and growth systems
even in the normal strain.
In a later study, Farghaly (12) determined the influence of various
amino acids on light production and growth in wild type A. fischeri.
Arginine proved no more effective than any other amino acid for stimu
lating luminescence and was less so than either lysine, methionine,
histidine or ammonium salts.
The requirements for _in vitro luminescence by the bacterial system
have been well defined. Reduced flavine mononucleotide (FMNl^),
luciferase, a long-chain saturated aldehyde in a buffer, and oxygen
are sufficient to produce a flash of light (13). Work by Hastings and


49
carbamyl phosphate then reacts with ADP to produce ATP in a reaction which
favors ATP formation (39). This mechanism for phosphorylation of ADP
via arginine metabolism is noteworthy because it bypasses the electron
transport system which is intimately involved with nitrate reduction
and the bioluminescent reaction. Levels of ADP and ATP are known to
serve in a regulatory capacity for other enzyme systems (9,40,41).
Indirect evidence from several lines of investigation suggests that the
carbamyl phosphate pathway for phosphorylation of ADP is not likely to
be involved in the action of arginine on the bioluminescence system in
i
this strain of bacteria. The first of these is that citrulline would
be expected to be at least as effective as arginine in stimulating lumin-
V
escence if the mechanism involved generation of carbamyl phosphate via the
pathway described above; this was found not to be the case. Citrulline
was the most effective of all compounds tested (other than arginine) as
stimulators of luminescence, and its uptake was similar to that of arginine,
but it was not as potent as arginine. A second argument against ATP (or ADP)
involvement in controlling bioluminescence is based on the findings that
these organisms are obligate aerobes. Attempts to grow', cultures under
a variety of anaerobic conditions were unsuccessful. These efforts
. \
\
included conditions with nitrogen, carbon dioxide or hydrogen atmospheres
in the culture flasks containing nitrate medium. Addition of arginine
did not alleviate this total inhibition of growth. Two assumptions are
necessary to make this argument relevant: first, it is assumed that this
absolute requirement for oxygen reflects a mandatory operation of the
electron transport coupled oxidative phosphorylation system, and,secondly,
that ADP phosphorylation via carbamyl phosphate production from arginine
could supplant the normal oxidative process to the extent that some growth,
even though very slow, could occur.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . ii
LIST OF TABLES v
LIST OF FIGURES vi
INTRODUCTION 1
MATERIALS AND METHODS 6
> >
Culture Techniques and Procedure for Arginine Addition ... 6
Light Measurement 6
Chromatographic Procedures 7
Quantitative Procedures 8
Special Procedures 9
RESULTS AND DISCUSSION 11
Luminescent Response to Arginine 11
In vitro estimation of enzyme activity 15
Lag period 19
Arginine Specificity 21
Stimulation by other compounds 21
Arginine Uptake 26
Induced Periodicity 28
Arginine Concentrating Ability 28
Arginine Metabolism 31
Mechanism of Arginine Action 38
Effects of the nitrate ion 38
Effect of arginine on growth 43
iii


35
apparent cessation to total protein synthesis implies the halt of luci-
ferase synthesis. The time required for puromycin inhibition of total
protein synthesis was estimated to be six minutes (Figure 7).
The factors to be considered in the final experiment in this series
are three-fold: 1) there is an apparent lag of eight minutes between the
addition of arginine to a log phase culture and the occurance of any arginine
metabolites in the cytoplasm of the bacteria; 2) evidence indicates that
puromycin completely inhibits protein synthesis within six minutes after
its addition to a log phase culture; and 3) in vivo luminescence may be
considered as a measure of intracellular luciferase concentration. If the
first two considerations are valid, then it should be possible to add
arginine to a log phase culture, and after a period of two minutes, add
puromycin with the result that protein synthesis is completely stopped
at about the same time that arginine metabolites become available to
the cells. The appearance of any increase in luciferase concentration
(implying de novo synthesis of the enzyme) in a culture treated in this
manner would most probably be attributable to the presence of arginine
rather than an arginine metabolite. Results of such an experiment were
interpreted in this manner (Figure 8).
Although the time element is critical to the interpretation of this
experiment, three considerations suggest that this approach is valid with
respect to time. First, independent of the mechanism by which an arginine
metabolite could affect luciferase synthesis, a finite time interval should
be required to exert this influence. Second, and also independent of the
mechanism, there must be a sufficient concentration of the molecular
species responsible for the effect: this may reflect an affinity factor,
or the element of probability that the proper interaction will occur.
Lastly, there must be enough time for the synthesis of complete luciferase


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38
molecules: since puromycin interrupts growing polypeptide chains (32),
any luciferase molecules synthesized in response to the added arginine,
must be completed prior to the inhibition resulting from the presence of
puromycin. Collectively, the hypothesis that a metabolite of arginine
is responsible for the bioluminescence response following the addition
of arginine to the medium of log phase cultures requires that the meta
bolite be present in sufficient concentration to exert its effect early
enough to permit the synthesis of complete luciferase molecules, but
still be below the limit of detection by the procedure employed. This
seems rather unlikely.
Using a somewhat similar approach to the problem of arginine
specificity, Coffey (1) concluded that the stimulation of bioluminescence
in a nitrate-utilizing strain of A. fischeri results from the presence of
t
arginine specifically rather than a metabolite of that amino acid. The
evidence presented here is offered in support of that conclusion.
Mechanism of Arginine Action *
m *
Effects of the Nitrate Ion
Studies of the enzyme nitrate reductase from wild type A. fischeri
led to the proposal of the electron transport scheme shown in Figure 9 (34).
Knowledge that FMNH2 serves as the reducing agent in the bacterial
luminescence system suggested to McElroy (35) that the addition of
nitrate ions to a culture would inhibit light production. The rationale
for this depends on the assumption that the supply of electrons to the
cytochrome system is constant. If this is true, addition of nitrate
should add a pathway for electron removal, thus diminishing the FMNH2
available to the light reaction. Therefore, one would expect luminescence
to be reduced in a culture containing nitrate as compared to one without


76
41. Atkinson, D.E., 1966. Regulation of Enzyme Activity. Ann. Rev.
Biochem., 35:85-124.
42. Cheer, S., and T.T.Tchen, 1962. Effect of Mitomycin C on the
Synthesis of Induced /-Galactosidase in EL coli. Biochem.
Biophys. Res. Comm., 271-274.
43. Shiba, S., A. Terawaki, T. Taguchi, and J. Kawamata, 1959. Selective
Inhibition of Formation of Deoxyribonucleic Acid in E. coli by
Mitomycin C. Nature, 183:1056-1057.
44. Iyer, V.N., and W. Szybalski, 1963. A Molecular Mechanism of
Mitomycin Action: Linking of Complementary DNA Strands. Proc.
Nat. Acad. Sci. U._S., 50: 355-367.
45. Hill, A.V., 1913. XLVII The Combinations of Haemoglobin with Oxygen
and with Carbon Monoxide I. Biochem. J., l_\kl 1-480.
46. Atkinson, D.E., J.A. Hathway, and E.C. Smith, 1965. Kinetics of Reg
ulatory Enzymes: Effectors for Yeast Phosphofructokinase Do
Not Alter the Apparent Kinetic Order of Reaction. Biochem.
Biophys. Res. Comm., 18:1-5.
47. Greengard, 0., 1963. The Role of Coenzymes, Cortisone and RNA in
the Control of Liver Enzyme Levels. Advances Enzyme Regulation, 1
61-76.
48. Strehler, B.L., and M.J. Cormier, 1954. Kinetic Aspects of the
Bacterial Luciferin-Luciferase Reaction _In Vitro. Arch. Biochem.
Biophys., 53:138-156.
49. Friedland, J., and J. W. Hastings, 1967. The Reversibility of the
Denaturation of Bacterial Luciferase. Biochem., 6:2893-2900.
50. Mandelstam, J., 1958. Turnover of Protein in Growing and Non-Growing
Populations of Escherichia coli. Biochem. J., 69:40-119.
51. Kepes, A., 1963. Kinetic Analysis of the Early Events in Induced
Enzyme Synthesis. Cold Spr. Harb. Symp. Quant. Biol., 28:325-327.
Friedland, J., and J. W. Hastings, 1967. Nonidentical Subunits of
Bacterial Luciferase: Their Isolation and Recombinations to
Form Active Enzyme. Proc. Nat. Acad. Sci., U.S., 58:2336-2342.
52.


m
o
Figure 20: Effect of arginine on ini vitro luciferase activity in
the absence of bovine serum albumin.
a:
b;
Arginine absent
Arginine present


2
in a repressible system, the small molecule (termed co-repressor) combines
with the repressor product and attaches to the operator to prevent trans
cription of the structural gene. The system under consideration in this
study has been considered to be inducible rather than repressible (1).
In bacterial inducible enzyme systems, the inducer molecule often is
related to the induced enzyme as the substrate or as' a homologue of
the substrate. However, this does not appear to be an absolute require
ment (3) .
An entirely different means for controlling enzyme activity has
been suggested by Monod, Changeux and Jacob (4), involving an "allosteric
effector". The molecule serving as the "effector" is not necessarily a
component in the reaction catalyzed by the affected enzyme, but may
elicit a quantitative change in enzyme activity. According to the
proposed model, combination of the effector with its target enzyme causes
a structural modification of the protein which may result in an increase
(activation) in the rate or a decrease (inactivation) in the rate of the
catalyzed reaction, or in a stabilization of the enzyme protein (5-10).
Since, under certain conditions, data for an enzyme exhibiting allosteric
properties vitro demonstrate an ^Ln vivo behavior very similar to an
inducible system (7) both models will be considered here.
The organism used in the present study is the ND strain of the marine
bacterium Achromobacter fischeri. Originally isolated for its ability to
utilize nitrate as its sole nitrogen source, it was found to be essentially
non-luminescent when cultured in a minimal nitrate medium (nitrate dark).
P. R. Elliott and A. H. Farghaly (personal communication) reported that
the strain luminesced normally when grown in the presence of peptone,
and that the component responsible for this effect was specifically
L-arginine. Early studies of the system indicated that the virtual


Figure 3: Relationship of in vivo and _in vitro measurement of luminescence following addition
of arginine (2.4 x 104M) to a culture at time zero. _In vitro luminescence is expressed in millivolts
per milligram protein in crude cell-free extract. _In vivo luminescence is expressed in millivolts per
ml of culture. i 1.-


Figure 14: Effect of mitomycin on the luminescence response to added arginine. Mitomycin
(10 jug/ml) added to one culture five minutes before adding arginine. Arginine (2.4 x 10"^M)
added to all three cultures at time 0. Mitomycin added to second culture at 40 minutes. Third
culture received no mitomycin.


7
servo-recorder with variable sensitivity by use of precision resistors
for signal attenuation. The instrumentation gave linear response across
the full range of light measurements.
In vitro bioluminescence measurements were carried out in 10 x 75 mm
test tubes placed in a fixed geometry relative to an RCA 1P21 photomultiplier
tube in a light-excluded chamber. Reaction components were injected with
a hypodermic syringe and needle without admission of extraneous light. The
photomultiplier was operated at 1000 volts and the output signal traced by
a recorder with 5 milliseconds response time.
Chromatographic Procedures
Thin-layer chromatography was performed using plastic sheets pre
coated with silica gel G (Eastman Distillation Products Industries) and
a sandwich style developing chamber. Routine detection of amino acids
was obtained with a 0.25 percent ninhydrin in acetone spray followed by
heating at 100C for about five minutes. Ehrlich's Reagent was sprayed
for detection of ureides and Sakaguchi reagent for the identification of
arginine (15) Tricarboxylic acid cycle intermediates were located by
spraying with bromocresol green reagent (16) In addition to identification
by specific color reactions, each unknown was required to co-chromatograph
with a known standard in at least three solvent systems before being
considered identified (15, 16). A similar procedure was employed to
N.
ascertain the chemical purity of compounds used to stimulate luminescence
in the cultures.
Ion exchange chromatography columns were prepared and operated
according to the procedure of Moore, Spackman and Stein (17) with the
exceptions that Dowex 50-X-8 resin, 200-400 mesh, was used without
additional particle sizing, and the dimensions of the columns were


BIOGRAPHICAL SKETCH
Thomas Stephen Quarles was born October 1, 1941, in Beaumont, Texas.
In June,1960, he was graduated from Beaumont High School. In June, 1964,
he received the degree of Bachelor of Science with a major in Biology
from Lamar State College of Technology in Beaumont, Texas. In September,
1964, he entered the Graduate School of the University of Florida. He
worked as a teaching assistant in the Department of Zoology until August,
1967. From September, 1967, until August, 1968, he was a predoctoral
fellow of the National Institutes of Health. Since September, 1968, he
has been an employee of the Biology Department, Birmingham Southern
College, Birmingham, Alabama.
Thomas Stephen Quarles is married to the former Jeannette Downs and
is the father of two children. He is a member of the American Association
for the Advancement of Science, the American Society of Zoologists, and
0 *
Phi Sigma Biological Society.
\
77


Figure Page
17. Hill plot of data for arginine requiring mutant 59
18. Effect of second addition of arginine 61
19.Effect of arginine on _in vitro luciferase activity .... 64
20. Effect of arginine on jin vitro luciferase activity
in the absence of bovine serum albumin 65
21. Effect of arginine on second n vitro flash 67
22. Effect of puromycin addition to cultures containing
different concentrations of arginine 69
vii


67
CO
o
CO
o
0 .. 10 20 30
Figure 21: Effect of arginine on second ijn vitro flash.
a ,d:
First flash
b,e:
Second flash, 5 minutes after first
c,f:
FMN control, first flash
a, b, and c:
Arginine absent
d ,e, and f:
Arginine present (150 ig)


TABLE 1
Compounds Tested for Stimulation of _In Vivo Luminescence
GROUP 1: Urea Precursors
L-Glutamic acid
Carbamyl Phosphate
L-Proline
Urea
L-Ornithine
L-Citrulline
L-Aspartic acid
GROUP 2: Other
Glycine
L-Alanine
L-Valine
L-Leucine
L-Isoleucine
L-Serine
L-Tyrosine
L-Tryptophan
L-Threonine
L-Phenylalanine
"Amino" Compounds
L-Cysteine
L-Methionine
L-Lysine
L-Histidine
L-Proline
Ammonium Chloride
Ammonium Phosphate
Ammonium Sulphate
Adenine
Cytosine
GROUP 3: Non-amino Compounds
D-Glucose Citrate (Na+ salt)
Pyruvate (Na+ salt) Dodecanal
Nitrite
GROUP 4: Arginine Analogues
Agmatine
L-Homoserine
Guanidoacetic acid
L-Canavanine
L-Homoarginine
D-Arginine


54
in the increase in luminescence resulted when mitomycin was added. A
similar pause was noted in p -galactosidase in the work of Cheer and Tchen.
They showed that general protein synthesis was not inhibited during this
period, however, but offered no explanation for the interruption of
^-galactosidase synthesis.
Effect of Initial Arginine Concentration
In Figure 15, the maximum luminescence achieved is plotted against
the initial concentration of added arginine. The sigmoidal shape of
this curve suggested another possible relationship between arginine and
/
luciferase. One of the more puzzling aspects of this problem is the
apparently unrelated nature between arginine and the luminescent reaction.
If arginine functions in the capacity of an allosteric effector, however,
no obvious relationship is necessary. The curve in Figure 15 suggests
that this is a possibility.
"Allosteric" proteins were initially defined by Monod, j2t _al. (3)
as those enzymes in which activity is regulated via changes induced in
the protein conformation by a molecule (the allosteric, effector) which
is not necessarily involved in the reaction mediated by.-the enzyme.
In their paper, the authors pointed out that a sigmoid curve is obtained
when In vitro reaction velocity is plotted against substrate concentration
for certain enzymes. This was interpreted as meaning that more than
one ligand could be bound to the enzyme at the same time, and, moreover,
that some kind of "cooperative interaction" existed between the binding
sites. The sigmoid form of the curve was found to linearize by appli
cation of the relationship which Hill (45) had described for the hemo
globin oxygen-saturation curve. Atkinson (41) points out, if one
assumes that reaction velocity is proportional to the fraction of sub
strate binding sites saturated, the Hill equation may be expressed as:


Growth (KS units)
Figure 13: Short-term effect of arginine on growth.


63
particularly since no aldehyde component has been firmly identified in
bacterial extracts. It was found, however, that arginine in three
different concentrations did not promote a luminescent flash in an assay
system containing no aldehyde. Therefore, if arginine does act as an
allosteric effector, it does not serve the same function as aldehyde.
The possibility that arginine, acting independently of aldehyde,
might enhance the efficiency (flash intensity or decay rate) of the
in vitro system was then examined. Tracings of the light response in an
in vitro system in the presence and absence of 100, 200 and 300 jig arginine
> f
are shown in Figure 19. As may be seen, there is no apparent difference
which may be attributed to arginine.
Bovine serum albumin is included in the assay system to aid
against denaturation of the enzyme as a result of the dilution factor.
Results of analyses of jiji vitro luciferase activity in the absence of
albumin show that arginine does not have any apparent effect as a
replacement for the added protein. Figure 20 shows tracings of such
, X
analyses. .
Finally, a noteworthy observation was made on the-, behavior of the
enzyme on stimulation of a second _in vitro flash. The intensity of the
flash following addition of a second aliquot of FMNH^ was considerably
reduced compared to the first flash. Since the second injection of
FMNH^ followed the first by five minutes, the control was made up so
that the enzyme was exposed to an equal concentration of FMN for five
minutes before initiating the first flash. This control showed a greater
activity than observed under experimental conditions with enzyme which
had been stimulated with FMNH2 before. The effect of arginine on the
reduced intensity of the second flash was investigated. Tracings of the


24
Specificity for the three-dimensional structure of arginine was
also investigated (Group 4, Table 1). An increase of one carbon atom
in the chain length (L-homoarginine) and the substitution of an oxygen
for a carbon atom in the L-arginine chain (L-canavanine) were tested.
Each of these changes alters the fundamental structure of L-arginine
sufficiently to make it ineffective as a stimulator of the luminescence
reaction.
D-arginine was tested to determine the influence of optical iso
merism on arginine specificity. Addition of this compound to a culture
produced no change in luminescence intensity relative to controls. However,
ninhydrin analysis of the medium indicated that D-arginine does not
enter the cells. For this reason, no conclusion may be drawn in terms
of the effect of optical isomerism on the intracellular molecular specifi
city of the bioluminescence response.
It should be noted that the addition of L-canavanine to one of two
-4
cultures containing equal amounts of L-arginine (2.4 x 10 M) resulted
in approximately 36 percent inhibition of the luminescence response com
pared to the arginine control. Canavanine was also found to inhibit
growth of the organism. This inhibition could not be completely reversed
by the addition of arginine to the medium in 10 times the canavanine
concentration. The arginine sparing of growth inhibition by canavanine
is shown in Table 2. Because of this'general inhibition of growth, and
indications in other bacteria that canavanine interferes with the utiliza
tion of arginine for protein biosynthesis (31), the significance of the
failure of canavanine to stimulate luminescence is somewhat difficult to
assess. It is not known whether the specificity requirements are such
that canavanine is not suitable as an arginine substitute or if its


11
10
9
8
7
6
5
4
3
2
1
0
Mitomycin added at -5 min.
Arginine added at 0


Relative Value
1.0
0.9
0.8
0.7
0.6
0.5
0.4
H 1 I 1 1 1 1 1
10 20 30 40 50 60 70 80 90
Time (minutes)
Figure 5: Arginine uptake. (For each of the three methods plotted, the values were obtained from
membrane-filtered samples of medium removed from a culture at the indicated times.
ho


DPNH(TPNH)
*> FMN(FAD)
Fe
Bacterial __ n
"^Cytochrome ^ 2
Nitrate Reductase
NO,
Abbreviations used are:
DPNH(TPNH) Reduced di-(tri) phosphopyridine nucleotide
FAD Flavin adenine dinucleotide
j | j
Fe Ferric ion .
(>2 Molecular oxygen
NO^ Nitrate ion
Figure 9: Proposed electron transport scheme for nitrate
reduction in A. fischeri (34).


MATERIALS AND METHODS
Culture Techniques and Procedure for Arginine Addition
In all experiments involving stimulation of luciferase activity,
the cultures were grown in the minimal liquid medium described by
Farghaly (12) modified to contain 0.76 grams per liter (g/1) NaNO^
(approximately 9 millimolar) to replace the (NH^)H^PO^. Complete
broth contained both (NH^^PO,, 0.5 g/1, and 1.0 percent peptone
(weight per volume) in addition to minimal salts and glycerol (12).
Cultures were incubated 18 to 20 hours at 23C with reciprocal
shaking of 80 cycles per minute. Cell density was determined turbidi-
metrically with a Klett-Summerson colorimeter using a number 42 filter.
Arginine was added to the culture when a reading was obtained of 80 to
100 Klett units. If the culture had overgrown this density, it was
diluted with fresh medium to a reading of about 40 units and allowed
to grow back to the usual cell concentration before introducing arginine
Light Measurement
In. vivo light measurements were carried out in a light-excluded
tank maintained at 23C by a circulating water bath. An RCA 1P21 photo
multiplier tube was positioned below a one-inch diameter window located
in the bottom of the chamber. Mounted inside the tank was a rack for
six 125 ml Erlenmeyer flasks, any one of which could be rotated into a
fixed position over the phototube window. Aeration of the culture was
maintained by reciprocal shaking of the rack at 80 cycles per minute.
Normal operation of the photomultiplier was at a regulated 1000 volts
potential. Cathodal current signal was measured on a strip-chart
6


Luminescence
Luminescence max luminescence
58
Figure 16: Hill plot of arginine concentration effect.


32
proline after spraying with ninhydrin revealed no other distinct area
having a positive reaction. Thirty minutes after adding uniformly labeled
^C-arginine (specific activity 0.4 mC per mM; final concentration of
-4
2.4 x 10 M) to a culture, proline was chromatographically isolated from the
medium, but the eluted material contained no radioactivity. However
14
C-label was isolated in the forms of citrulline and urea as well as
arginine. The findings implicated these materials in the discrepancy be-
14
tween the ninhydrin and C-label assays depicted in Figure 5.
A series of experiments were designed to elucidate the intracellular
/ f
fate of arginine. For this work, an early log phase culture was exposed
to uniformly labeled ^c-arginine (specific activity 6 mC per mM; final
-4
concentration 2.4 x 10 M) for 90 minutes. A trichloracetic acid (5 percent)
extract of washed cells was chromatographed on a heated ion exchange column
using a series of buffers for elution. Ten ml fractions were collected
and assayed for radioactivity. Those fractions containing ^c-label were
compared to an elution pattern achieved with known amino acids. Having
thus made a tentative identification of most of the ^C-labeled compounds,
confirmation was obtained by thin-layer co-chromatography with standards
in two dimensions with two solvent pairs. The radioactive material was
required to chromatograph with and only with the standard in all instances.
Compounds identified as bearing ^C-label (which had been introduced
14
as uniformly labeled C-arginine) were as follows: arginine, urea, ornithine,
citrulline, arginino-succinic acid, proline, glutamic acid, <-keto-glutaric
acid, succinic acid, fumaric acid and malic acid. The latter four compounds
demonstrated very low specific activity and do not appear to constitute
a pathway of great importance in the early stages of arginine metabolism
in this organism. Compounds showing the highest specific activity (other
than arginine) were ornithine and citrulline.


SUMMARY AND CONCLUSION
The bioluminescent system in the ND strain of the marine bacterium
Achromobacter fischeri is not functional when these bacteria are cultured
in a minimal medium containing glycerol and nitrate as the only nutrients.
Absence of the luminescent reaction is associated with the virtual absence
of the enzyme luciferase in functional form. Addition of L-arginlne to
a log phase culture results in a dramatic increase in _in vivo light pro
duction and in luciferase activity as estimated JLn vitro. Results of
experiments testing other compounds as stimulators of luminescence and
comparison of the time required for arginine conversion to begin in the
cell with the appearance of luminescence increase indicate that the response
of the luminescent system is due specifically to arginine.
Arginine probably exerts its effect on the luminescent system,
via an enzyme "induction" at the genetic level, in a manner similar to
the model proposed by Jacob and Monod (2). Evidence for this is found
-in the apparent increase in luciferase protein, and the prevention of
this increase by inhibitors of protein synthesis. These findings are
presented in support of the conclusions drawn by Coffey (1) on the basis
of his work with A. fischeri ND. '
In his work on this problem Coffey (1) presented kinetic evidence
indicating that an "activation" step (of unknown nature) was required
before luciferase promoted any light production. Certain aspects of the
luciferase response to added arginine (in particular the relationship
between arginine concentration and the luminescence intensity) suggest
that this activation may be the result of an allosteric interaction between
arginine and luciferase. Although much of the evidence may be explained
71


Time (minutes)
Figure 7: Puromycin inhibition of ^C-arginine incorporation into acid-insoluble material.
(Arginine added ten minutes before adding puromycin. Puromycin added at arrow.)
O'


BIBLIOGRAPHY
1. Coffey, J.J., 1967. Inducible Synthesis of Bacterial Luciferase:
Specificity and Kinetics of Induction. J. Bacteriol.. 94:1638-
1647.
2. Jacob, F., and J. Monod, 1961. Genetic Regulatory Mechanisms in the
Synthesis of Proteins. J. Mol. Biol., _3:318-356.
3. Hartman, R.E., and L.N. Zimmerman, 1960. Effect of the Arginine
Dihydrolase Enzyme System on Proteinase Biosynthesis by
Streptococcus faecalis Var. Liquefaciens. J. Bacteriol., 80:
753-761.
4. Monod, J., J.P. Changeux, and F. Jacob, 1963. Allosteric Proteins
and Cellular Control Systems. J. Mol. Biol., _6:306-329.
5. Monod, J., J. Wyman, and J.P. Changeux, 1965. On the Nature of
Allosteric Transitions: A Plausible Model. J. Mol. Biol.. 12:
88-118.
6. Freundlich, M., and H.E. Umbarger, 1963. The Effects of Analogues
of Threonine and of Isoleucine on the Properties of Threonine
Deaminase. Cold Spr. Harb. Symp. Quant. Biol., 28: 505-511.
7. Schimke, R.T., 1966. Studies on the Roles of Synthesis and Degrada
tion in the Control of Enzyme Levels in Animal Tissues. Bui.
Soc. Chim. Biol.. 48:1009-1030.
8. Datta, P., H. Gest, and H. L. Segal, 1964. Effects of Feedback
Modifiers on the State of Aggregation of Homoserine Dehydro
genase of Rhodospirillum rubrum. Proc. Nat. Acad. Sci., U._S.,
51:125-130.
9. Marks, P.A., 1961. Glucose -6-P Dehydrogenase Stability, Activation,
and Inactivation. Cold Spr. Harb. Symp. Quant. Biol., 26:343-345.
10. Gerhart, J.C., and A.B. Pardee, 1963. The Effect of Feedback Inhibition,
CTP, on Subunit Interactions in Aspartate Transcarbamylase. Cold
Spr. Harb. Symp. Quant. Biol., 28:491-496.
11. McElroy, W.D., and A.H. Farghaly, 1948. Biochemical Mutants Affecting
the Growth and Light Production in Luminous Bacteria. Arch. Biochem.
_17: 379-390.
12. Farghaly, A.H., 1950. Factors Influencing the Growth and Light Product
ion of Luminous Bacteria. J. Cell Comp. Physiol., 36:165-183.
13. McElroy, W.D., and A.A. Green, 1956. Enzymatic Properties of Bacterial
Luciferase. Arch. Biochem. Biophys., 56:240-255.
73


23
arginine *. ornithine glutamic V semialdehyde
+
urea
(29). The glutamic-X -semialdehyde may then be oxidized to glutamic
acid or cyclized (via dehydration) to lead to proline synthesis (30).
Of the compounds listed in Group 1, Table 1, two resulted in increased
light production by the log phase cultures to which they had been added:
L-citrulline and L-proline produced 400-fold and 20-fold increases re
spectively in _in vivo luminescence compared to the luminescence level
of an untreated culture. L-ornithine failed to show any stimulatory
effect; however, within the limits of sensitivity of the ninhydrin pro
cedure, no ornithine uptake could be detected. The response to added
citrulline and proline is believed to result from intracellular conversion
of these amino acids to arginine or as arginine sparing intermediates.
This is acceptable as a working hypothesis, since citrulline and proline
are involved in the general pathway leading to arginine synthesis and
their effectiveness is directly related to their remoteness from arginine
in that pathway. -
In view of the apparent specificity for arginine, knowledge of the
effectiveness of arginine analogues (Group 4, Table 1) should yield some
insight into the specific functional group requirements for stimulation
of bioluminescence in this organism. The importance of the guanido
group was examined by supplying it in. the form of guanidoacetic acid, by
replacing it with an hydroxyl group in the arginine structure (homoserine),
and by adding both guanidoacetic acid and homoserine simultaneously to the
culture. Agmatine was tested to determine the effectiveness of the
guanido and amino groups in combination. None of these compounds, either
singly or in combination, demonstrated any stimulatory effect on the
luminescence reaction.


THE ROLE OF ARGININE AS THE INDUCER
OF BACTERIAL LUCIFERASE IN
ACHROMOBACTER FISCHERI ND
By
THOMAS STEPHEN QUARLES
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1970


Luminescence (mv
44
Figure 12: Effect of added nitrate in low concentration on
luminescence in A. fischeri.


53
In order to clarify this point, inhibitors of protein synthesis
were employed. Addition of puromycin (10 pg per ml) or chloramphenicol
(10 pg per ml) 5 minutes prior to adding arginine (2.4 x 10^M) to a
log phase culture prevented any increase in bioluminescent intensity.
This strongly suggests that new protein synthesis occurs in response to
added arginine. Coffey (1) used, in addition to those inhibitors, amino
acid analogues, which proved inhibitory, and found that the inhibition
was reversible by the addition of the proper amino acid. He also showed
that messenger RNA synthesis is probably involved since 5-fluorouracil
i f
serves as an inhibitor of the response to added arginine. In support
of this conclusion about messenger RNA, it was found that pretreatment
of a culture with mitomycin (10 jig Per ml added 5 minutes prior to arginine
addition) prevented the expected increase in light production (Figure 14).
Cheer and Tchen (42) described similar results for the inhibition of
y^-gal'actosidase induction in E. coli. Since a considerable lag occurs
before this antibiotic influences synthesis of RNA or protein (43), Cheer
and Tchen suggested that mitomycin acts selectively on. genes which are
not involved in synthetic activities at the time of exposure to the
inhibitor. Later, Iyer and Szybalski (44) indicated that mitomycin acts
by cross-linking complementary strands of DNA. Assuming Cheer and Tchen
were correct in their analysis, the effect of mitomycin on this system
may be interpreted as indicating that the genes for luciferase are not
being transcribed prior to addition of arginine, and are therefore sus
ceptible to mitomycin action. If this were true, then the addition of
this antibiotic would not be expected to influence the increase in
luminescence, once it was initiated by arginine. This, in fact, was
observed to be the case (Figure 14). A temporary interruption (2-3 minutes)


3
absence of light production in cultures grown with nitrate as the
sole nitrogen source was due to the lack of functional enzyme required
for luminescence.
Through the extensive use of various inhibitors of protein synthesis,
Coffey (1) has demonstrated that the addition of L-arginine to a log
phase culture growing on minimal nitrate medium results in the synthesis
of new luciferase. He has found that new mRNA synthesis is required to
effect this response. Elliott and Farghaly (personal communication) had
previously shown that added arginine did not affect the level of activity
of six other randomly selected enzymes, thus arguing against a generalized
enzyme induction. These data, combined with those of Coffey, strongly
suggest that exogenously added arginine is an inducer for the specific
synthesis of luciferase.
The investigations of Coffey (1) indicated a very high specificity
of L(+)-arginine as the inducer of luciferase in his strain of bacteria.
Other amino acids, structural analogues of arginine, and ammonium salts
were ineffective as inducers with the exceptions of L-proline, L-aspartic
acid, and the arginine precursors L-ornithine, L-citrulline and L-arginino-
succinic acid. The most efficient of these, L-citrulline, elicited a
response that was only 37.3 percent as strong as that produced by L-arginine.
On the basis of this and ^C-arginine experiments, Coffey concluded that
arginine is the specific inducer molecule, and that the positive response
to the other compounds results from intracellular conversions to or sparing
of arginine. He also stated that the growth rate of the culture is not
altered during the first two hours after treatment with arginine.
The first studies of the influence of arginine on bacterial growth
and luminescent systems were carried out by McElroy and Farghaly (11) in
a series of experiments on A. fischeri mutants isolated after exposure


15
By the time a culture has reached the late log phase or early
stationary phase of its growth cycle, the constituitive luminescence
increases. The onset of this increase may be seen in Figure 1 and,
in Figure 2, it is shown in relation to culture age and luminescence
resulting from added arginine. Although not shown in Figure 2, the
constituitive luminescence maintains a relatively constant level for
about seven hours and then begins to decline before the culture enters
the death phase (indicated by viable cell counts performed every four
hours). This is observed independent of previous arginine treatment.
/ f
The addition of arginine to a culture during this period results in
an increase in light production; however, the response is not as ex
tensive as that elicited by an equal concentration of the amino acid
added in early log phase.
In Vitro Estimation of Enzyme Activity
The increased luminescence of a culture following addition of
arginine to the medium reflects an increase in the intracellular concen
tration of functional luciferase, as measured by jin vitro assays of enzyme
activity. Cell-free crude extracts of aliquots removed from a culture
at timed intervals after the introduction of arginine into the medium
were analyzed for luciferase activity and protein concentration. In
vitro light production per mg of protein (specific activity) in these ex
tracts is plottedas a logarithmic function against the time at which
the aliquots were removed from the culture (Figure 3).
Use of the peak flash intensity as a measurement of enzyme activity,
as described in the Methods section, is a standard procedure in such
assays. Flash height has been shown to be directly proportional to
the total light output measured as the area under the response curve (12, 26).
Moreover, this area (rather than a rate) is believed to represent enzyme


72
in terms of such a hypothesis, no results of iii vitro or in vivo experi
ments indicate the existence of such an interaction. Therefore, inter
pretation of the results in terms of allosteric effectors does not, at
this time, seem appropriate.
This work is offered in confirmation of Coffey's (1) speculation
that L-arginine specifically acts as an inducer of bacterial luciferase
in Achromobacter fischeri ND.


50
Therefore, although the activity of specific enzymes was not
measured, this evidence indicates that generation of ATP via carbamyl
phosphate does not play a primary role, at least in the stimulation of
luminescence in these bacteria following the addition of arginine to the
medium of a "nitrate" grown culture.
Arginine Incorporation into Luciferase
An early consideration was that luciferase might be very rich in
arginine and that synthesis would be limited except in the presence of
a large arginine pool./ This was investigated by determining the amount
of specific radioactivity of partially purified luciferase as compared
to similar measurements of general cell protein 60 minutes after' intro
ducing uniformly labeled ^C-arginine (specific activity 0.4 mC per mM,
-4
final concentration 2.4 x 10 M). Results of this experiment showed
that total intracellular protein contained an average of 3,186 dpm per
mg protein. The luciferase fraction (purified 30 fold) was found to
contain 2,142 dpm per mg protein.
Therefore, luciferase does not preferentially incorporate arginine
as compared to the general cellular proteins. These results are in
agreement with the estimation of fiv^ arginyl residues per luciferase
molecule (J.W. Hastings, personal communication).
Effect of Inhibitors of Bioluminescence Response
Synthesis of new protein is associated with the increase in bio
luminescence. This is suggested in Figure 3, in which the _in vitro
assay of luciferase activity in aliquots of culture collected at timed
intervals following the introduction of arginine into the medium is
related to _in vivo bioluminescence observed in a parallel culture at
the same times.


41
NaN03 (M)
Figure
(Data taken
nitrate ions
10: Effect of high nitrate ion concentration on luminescence.
60 minutes after simultaneous addition of large concentration of
and 2.4 x 10^M arginine.)


LIST OF TABLES
Table Page
1. Compounds Tested for Stimulation of In Vivo
i Luminescence 22
2. Arginine Sparing of Canavanine Inhibition of Growth 25
3. Long-term Effect of Various Nitrogenous Compounds on
Luminescence and Growth 47

v


28
Induced Periodicity
The regular fluctuation in the arginine uptake studies, shown in
Figure 5, suggests a rhythmic behavior of the culture. Examination of
other factors revealed a similar periodicity in both growth and in in
vivo luminescence following the addition of arginine to a culture (Figure 6).
Arginine uptake is taken from the "^C-label information presented in
Figure 5. Growth is indicated in Klett-Summerson turbidity units measured
at ten-minute intervals, and luminescence is plotted as points read from
a recording of in vivo light production. A similar periodicity exists
i ?
in all three factors measured.
There does not appear to be an inherent periodicity in either
bacterial luminescence or cell division: continuous recordings of light
intensity produced by the wild type A. fischeri or by the strain being
studied here (when grown in luminescence-supporting medium) reveal only
a uniform increase as the culture develops. In addition, studies of
cultures in complete or minimal nitrate medium (prior to adding arginine)
t i'
indicate no periodicity in growth rate. Therefore, the rhythmicity observed
in the factors shown in Figure 6 is believed to be due to the addition of
arginine. Further work will be required to define more carefully the
nature of the periodicity and to clarify the relationship shown in Figure 6.
Arginine Concentrating Ability
The rapid initial rate of arginine removal from the medium sug
gested that some system was operating to take up the amino acid at a rate
greater than that expected for simple diffusion. Experiments were conducted
to determine if this strain of bacteria was capable of accumulating intra
cellular arginine against a concentration gradient based on the relative
intra- and extra-cellular concentration of ^C-label (introduced as uniformly


Figure 1: Relationship between growth of culture, arginine-stimulated bioluminescence and
bioluminescence in the absence of arginine (constituitive luminescence). L-arginine in final
concentration of 2.4 x 10^M is added at arrow. Growth is determined turbidimetrically and
expressed in Klett-Summerson (K-S) units. Luminescence curves are plotted from continuous record
ings and expressed in millivolts (mv).


RESULTS AND DISCUSSION
Luminescent Response to Arginine
The primary question concerns the role of arginine in the bio-
luminescent response evoked by the addition of this amino acid to a
log phase culture of Achromobacter fischeri ND grown on minimal nitrate
medium. Figure 1 shows the relationship of growth, the small amount of
measurable luminescence of an untreated culture (constituitive luminescence),
and the luminescence observed after adding arginine (final concentration
2.4 x 10 4 Molar) to early log phase cells. In Figure 1, and all succeeding
figures containing data on light production, luminescence is plotted as
readings taken from continuous recordings at one minute intervals in those
regions of particular concern and at five minute intervals over the remainder
of the curve.
As may be seen in Figure 1, the addition of arginine results in a
2,000-fold increase in _in vivo luminescence compared to an untreated
culture. The constituitive luminescence is measured at 12 to 15 mv
compared to approximately thirty volts following treatment with arginine.
Maximum luminescence was observed to vary by as much as a factor of five,
ranging from about ten volts to fifty volts in repeated experiments.
Attempts to grow and treat cultures under uniform conditions did not
improve the reproducibility of the measured maximum luminescence after
adding arginine. Careful examination revealed that results from parallel
samples of the same culture were comparable and findings from repeated
experiments were consistent relative to controls, irrespective of the
variation in maximum luminescence intensity.
11


TABLE 2
ARGININE SPARING OF CANAVANINE INHIBITION OF GROWTH
Flask Number'
Arginine Concentration
x 10"^ m
7o Control Growth
Rate
Control*
-
100
1
-
17.4
2
1.0
22.9
3
2.5
26.7
4
5.0
32.1
5
25
57.2
Each experimental
flask contains 2.5 x 10"^M
Canavanine
* Standard nitrate medium with no additions


This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to
the Dean of the College of Arts and Sciences and to the Graduate
Council, and was approved as partial fulfillment of the require
ments for the degree of Doctor of Philosophy.
March, 1970
Dean, Graduate School
Supervisory Committee:


34
(about 27C) to a volume less than 1 ml. These concentrated samples were
applied to a Sephadex G-10 column (1 cm x 12 cm) to separate the small
organic molecules from macromolecules and inorganic salts by elution with
\
distilled water. The isolated compounds were then co-chromatographed with
10 /ig arginine in two dimensions, and in two solvent pairs (n-butanol:
acetic acid:water, 3:1:1; ethanol:ammonia:water, 8:1:1; t-butanol:2-butanone:
formic acid: water, 8:6:3:3; and n-butanol:pyridine: water, 1:1:1). Ninhydrin
was used to locate the arginine, and the adsorbant in that area was scraped
into vials for quantification of radioactivity. The remaining adsorbant
was also analyzed for radioactivity. Results of these experiments indicated
14
C-label associated only with arginine for the first eight minutes. The
nine-minute sample showed 20 counts per minute above background which were
located in areas other than that associated with arginine. Based on the
products of arginine catabolism indicated above, and on published Rf values
for these compounds in the solvents used, the area containing arginine was
believed to be isolated from areas containing other potentially labeled
compounds. Therefore, within the sensitivity limits of the present methods,
- no_conversion of arginine occurs for eight minutes after introducing it
into a culture.
Puromycin acts by interrupting the growth of polypeptides (32).
Preliminary work on the amino acid composition of luciferase has indicated
the presence of at least five arginyl residues per molecule (J.W. Hastings,
14
personal communication). Therefore, the cessation of C-arginine incorp
oration into TCA insoluable material following treatment with puromycin
is felt to reflect the blockage of luciferase synthesis. This represents
a maximum limit; biosynthesis of some proteins appears to be more sus
ceptible than others to puromycin inhibition (33). The degree to which
luciferase synthesis is sensitive to puromycin is not known, but the


Luminescence (mv)
30
5 --
0 1 1 1 1 1 : 1 1 1
-5 0 5 10 15 20 25 30 35
Time (minutes)
Figure 8: Effect of adding puromycin two minutes after adding arginine.
u>


68
a brief continuation in the rise of luminescence, light production began
to decline. Two experiments were carried out; one entailed adding puro-
mycin to parallel cultures treated with different amounts of arginine at
a point where the luminescence of the cultures was equivalent; the other
involved the addition of puromydin following a fixed period post arginine
treatment. In each case, it was felt that the decay rate of luminescence
would be different for cultures containing 4.8 x 10"arginine and
4.8 x 10'4M arginine if the amino acid were acting to stabilize the
luciferase against a degradative action. Results of these experiments
are shown in Figure 22. The rate of decay of luminescence under these
conditions appears to be independent of the initial arginine concentration
irrespective of whether the exposure time was the same or the luminescence
intensity was the same.
Discussion of Mechanistic Interpretations
A characteristic of inducible enzyme systems in bacteria is the
relationship between the inducer and the induced enzyme. Although certain
* materials which are not acted upon by the induced enzyme, and which are
not metabolized by the cell have the ability to serve as "gratuitous
inducers", these are related to the reaction in the sense that they bear
some similarity to the substrate which is usually assumed to be the
"natural" inducer. The most thoroughly studied inducible system is
the /^-galactosidase system, in which /^-thioglactoside acts as a
"gratuitous inducer" (51). In the case of arginine and bacterial
luciferase, there does not appear to exist such a relationship. Also,
though it is not deemed as important as the exceptions just mentioned,
the early kinetics of the luciferase response to arginine are not as
rapid as might be expected on the basis of the inducer model (51).


40
that ion. In fact the simultaneous addition of large quantities of
-4
nitrate and arginine (2.4 x 10 M final concentration) to a log phase
culture growing on nitrate medium does reduce the intensity of the light
produced when measured 60 minutes after the time of the addition (Figure 10).
Although a pronounced effect on growth (estimated turbidimetrically) is
detected only after several hours, the reduction in luminescence intensity
is probably related to growth inhibition rather than nitrate ions,
since the addition of NaCl in high concentration produces a similar
dimming of luminescence.
The addition of a much lower concentration of nitrate ions (9 x 10"^M)
results in an enhancement of luminescence (Figure 11). This suggests that
the presumed drain on FMNH2 supplies which is imposed by nitrate is not
solely responsible for the greatly reduced light production. If it had
been found otherwise, i.e., that the presence of nitrate in the concen
trations used in the medium did inhibit luminescence, then the addition
of amino-nitrogen rich arginine could be interpreted as relieving the
nitrate reduction pathway so that a normal flow of electrons to the
bioluminescence reaction would be restored. This interpretation is,
however, not consistent with the results presented.
Additional evidence against the argument that nitrate reduction
depletes the FMNH2 pool at the expense of bioluminescence is found in
several other observations. If growth on minimal nitrate medium results
in amino-nitrogen limiting conditions, as discussed above, then other
amino acids, and especially ammonium salts, would be expected to be at
least as effective as arginine in stimulating light production; they were
not.
The role of the step in which nitrate is reduced to nitrite is also
questionable. Observations of cultures grown with nitrite as the sole


9
using the internal standard method (22). The following calculations
were made for each vial:
cPmu
(cpmu + cpms) cpmu = dPmu
dpms
where cpmu and cpmg are counts per minute due to the unknown and standard
respectively and dpmu and dpmg are similar expressions for disintegrations
per minute.
i Special Procedures
The ability of the bacteria to concentrate arginine was determined
by measuring the difference in radioactivity per unit volume in samples
of membrane-filtered medium and simultaneously collected samples of
membrane-filtered culture containing trichloroacetic acid to lyse the
bacteria and precipitate the proteins. The number of bacteria per ml of
culture was determined by direct microscopic counts. Using the average
dimensions of a cell of this species of bacteria as indicated in Bergey1s
# 9
Manual (23) and confirmed by measurements taken from electron photomicro
graphs, the volume of cells (including the cell walls) per ml of culture
was calculated. By appropriate corrections for dilutions, the disintegrations
per minute per unit volume of bacterial cells and per unit volume of extra
cellular medium were determined.

Cell-free extracts of the bacteria were prepared by osmotic lysis
in distilled water. Luciferase was partially purified by the method of
Hastings and McElroy (24). Enzyme activity was determined in a 10 mm x 75 mm
test tube containing 1.5 ml 0.1 M phosphate buffer, pH 7.4; 0.5 ml 1
percent bovine serum albumin in water; 0.1 ml partially purified enzyme;
and 0.5 ml dodecanal-saturated water solution. This tube with the assay


60
Another experiment involving the effect of arginine concentration
on luciferase activity may be interpreted as an allosteric interaction.
This experiment consisted of the addition of a second equal amount of
arginine to a culture following the re-establishment of the luminescence
level at the control value. Results of this experiment are plotted in
Figure 18. The second addition of arginine resulted in two changes with
respect to the effect of the first: 1) the intensity of the second
luminescence response was approximately twice as great as the first (and of
longer duration, although this is not shown); and 2) the lag period between
> f
the addition of arginine and the first detectable increase in luminescence
was shortened. Assuming arginine does act as an inducer of luciferase on
the basis of the information presented above, a finite amount of time is
required to accomplish this. Coffey (1) has estimated that this is four
minutes. During that time, available arginine is continually being removed
from the system into protein synthesis. Assuming that arginine also
exerts an allosteric effect on luciferase, the total amount of light pro
duced will be a function of the effective concentration of arginine and
the duration of that concentration. Provided luciferase is still present
but non-functional, due to low arginine levels (resulting in a decrease in
\
luminescence), a second equal addition of arginine should result in a
luminescence response of longer duration and greater magnitude. As shown
in Figure 18, this was found to be the case.
Again a growth requirement interpretation may be applied. If
arginine acts as a simple inducer of luciferase, but must satisfy a
growth requirement before induction is accomplished, then a greater pro
portion of the first addition of arginine xrould be expected to go for this
purpose than would be expected for the second arginine treatment.


Arginine incorporation into luciferase 50
Effect of inhibitors of bioluminescence response .... 50
Effect of initial arginine concentration 54
In vitro effects of arginine 62
In vivo effector function of arginine 66
Discussion of Mechanistic Interpretations 68
SUMMARY AND CONCLUSION 71
BIBLIOGRAPHY 73
BIOGRAPHICAL SKETCH 77
/ !
IV


Figure 18: Effect of second addition of arginine


Figure 15: Effect of concentration of added arginine on _in vivo luminescence.
I


33
Evidence from additional experiments does not indicate the presence
of urease in this strain of bacteria. Aliquots of A. fischeri N D
cultures were incubated in sealed vials in the presence of uniformly
labeled ^C-arginine and hyamine hydroxide (Packard) for 20 minutes.
At the end of this period, concentrated sulfuric acid was injected into
the vial to kill the cells and to drive dissolved C0£ out of solution.
Subsequent analysis of the hyamine hydroxide for ^(X>2 did not demonstrate
any of this gas.
From the above results, it is clear that early arginine metabolism
in this strain of bacteria does not lead to intermediates which were not
also tried as stimulators of luminescence (Table 1). This argues against
the hypothesis that arginine is converted to some other compound which
then acts to stimulate the bioluminescence reaction. If such a hypothesis
were correct, one of the arginine metabolites would be expected to be at
least as effective as arginine for stimulating the apparent synthesis of
luciferase. It can be argued that the preceding statement does not apply
to ornithine, since there appears to be virtually no uptake of this amino
acid.
A final series of experiments was carried out in an effort to confirm
arginine specificity. It was essential to determine the time at which
intracellular arginine conversion to other compounds begins. This was
done by collecting one ml samples of -the culture at one minute intervals
following the addition of uniformly labeled ^C-arginine. Each sample was
washed with cold minimal salts and then each filter was placed in 5 ml
of cold distilled water with gentle shaking for 20 minutes. Microscopic
examination showed very few whole cells remaining after this treatment.
The extracts were then concentrated under vacuum at room temperature


21
reduce the length of this lag; however, the lag begins to increase as
-4
the concentration is lowered below 1 x 10 M arginine. Coffey (1) has
dealt rather extensively with this aspect of the problem and has con
cluded that approximately eight of the twelve minutes represent a period
of "activation" of the enzyme, the primary structure of the protein being
completed in the first four minutes after adding arginine to the medium.
This lag period and Coffey's interpretation will be given further con
sideration later.
Arginine Specificity
t
Stimulation by Other Compounds
The stimulation of bioluminescence in A. fischeri ND shows a high
degree of specificity for L-arginine. A number of compounds were tested
for their ability to serve as stimulators; these are listed in Table 1.
With the exceptions of L-citrulline and L-proline, as discussed below,
they were uniformly ineffective in stimulation of _in vivo bioluminescence
over control levels.
The groups of compounds in Table 1 were selected to provide some
insight into the mechanism by which arginine exerts its stimulating effect
on the bioluminescence system. Because of the participation of arginine
in the cycle leading to urea biosynthesis, the various intermediates in
that pathway (Table 1, Group 1) were tested for their ability to stimulate
luminescence in this strain of bacteria. In view of the lag period dis
cussed above, which might represent the time required to effect a conversion
of arginine to another compound, this approach seemed particularly promising.
Also included in this group are L-glutamic acid and L-proline which are
related to the urea cycle, and are derivable from arginine in Sacchromyces
cerevisiae by the following initial steps:


Luminescence (mv
-- 600
500
400
300
-- 200
-- 100
u>
Growth (KS units)


57
log v/(V-v) = n log (S) Log K
in which v is reaction velocity, V is maximum velocity, n is the number
of the substrate binding sites, S is substrate concentration and K is a
constant.
Assuming that the maximum _in vivo luminescence obtained represents
"reaction velocity" (v) for each concentration (s), and that the maximum
luminescence achieved by the highest two concentrations (shorn in Figure 15)
represents maximum reaction velocity (V), a Hill plot was made and is illus
trated in Figure 16. Although it is not linear at the extremes, the region
> f
of the curve near the midpoint (where log v/(V-v) = 0) is straight and has
a slope of 2.93. Since this value represents a measure of both the strength
of cooperative interaction and the number of binding sites (46) it is assumed
that if this is an allosteric effect, three arginine binding sites exist on
the luciferase molecule.
An alternative interpretation of Figure 15 is that at lower concentra
tions, a greater proportion of the added arginine is used for purposes
other than stimulating bioluminescence; i,.j;. there is some minimum quantity
of arginine that must be supplied for other uses before,it can be applied to
the bioluminescence system. This is suggested as a possibility in light
of the work by McElroy and Farghaly (11) if it is assumed that growth on
nitrate medium represents an arginine-limiting condition. Their plot of
luminescence versus arginine concentration for an arginine requiring
mutant exhibits a sigmoid shape when luminescence is expressed as a per
centage of the maximum obtained. Application of the Hill equation to
their data results in the plot shown in Figure 17. Here the slope is 3.2
at the midpoint. Thus the value for the slope of the Hill plot of these
data agrees reasonably well with the one for the present work. This
suggests that a similar relationship might exist for the two systems.


INTRODUCTION
An arginine-inducible bacterial luciferase has been described
recently (1). Two aspects of the phenomenon are unusual compared to
previously described inducible bacterial enzyme systems. The first of
these is that no known relationship exists between arginine and the
reaction catalyzed by the induced enzyme. The other unusual feature is
that these bacteria are.capable of _de novo synthesis of the inducer and,
in fact, do produce sufficient amounts of arginine to support growth
under the conditions necessary for induction. This raises the question
as to how the addition of exogenous arginine results in the observed
increase in luciferase activity.
In 1961, Jacob and Monod (2) suggested a mechanism by which protein
biosynthesis could be genetically controlled. Briefly, their model pro
poses a set of three regions on the genome associated with the synthesis
of a protein. These regions are 1) the structural gene, which dictates
the amino acid sequence of the specific enzyme protein, 2) the operator
region, which controls the initiation xof transcription of the structural
\
gene, and 3) the repressor region, which produces a product that, by
combining with the operator region, prevents initiation of protein bio
synthesis. Another property of the repressor product, in an inducible
system, is its ability to combine specifically with a small molecule, the
inducer, such as a sugar or amino acid.
When combined with the inducer, the repressor material loses its
capacity to attach to the operator region of the genome. This frees the
operator, permitting the initiation of enzyme synthesis. Alternately,
1


Luminescence (mv)
20
Figure 4: Onset of luminescence following addition of arginine.


14
The cause for this variation in susceptibility to arginine stimu
lation of light production is not known at this time. Several factors
thought to be potentially responsible were investigated. Cultures
grown on successive days in aliquots of medium taken from the same
large batch preparation did not respond equally to added arginine. Addi
tion of arginine from the same stock solution did not insure a reproducible
maximum luminescence of the treated culture, independent of whether the
solution had been stored at room temperature, 4C or frozen at -15C
between use. The age of the culture from which subsequent inoculations
were taken did not appear to be a factor since variations were noted in
cultures inoculated with log phase, stationary phase, or death phase
cells. Although culture age does influence the sensitivity to arginine
stimulation, as will be discussed later, the maximum response varied
whether the amino acid was added after the same period of incubation
or at the same cell density (as estimated turbidimetrically). Other
factors controlled prior to the addition of arginine included the adjust
ment of pH of the medium to 7.0, adding a fresh supply of glycerol and
supplementing the nitrate concentration. Only one variable showed any
correlation to the problem of reproducibility; with increasing serial
transfers in minimal nitrate medium, there was a tendency toward a
decrease in maximal luminescence when all other factors were held constant.
Sub-culturing in complete medium usually resulted in an increase of
sensitivity to arginine stimulation in subsequent minimal nitrate cultures.
In his work on arginine stimulation of bioluminescence in similar cultures
grown on minimal nitrate medium, Coffey (1) reported this same difficulty
with peak response to added arginine but offered no suggestions as to the
cause of the problem.


5
his group (14) has indicated that FMNH2 reduces the enzyme directly
and that no substrate in which arginine could be a component is
involved in the reaction. It would appear, therefore, that arginine,
as an inducer, does not bear a typical relationship to the induced
enzyme.
The work reported here was carried out in an attempt to answer the
following questions: Is arginine an inducer of luciferase or is some
arginine metabolite responsible for the phenomenon? Does the luminescent
response to arginine occur as a result of an induction process or as a
result of some other mechanism? Finally, does arginine serve a double
function by acting both as an inducer and as a stabilizer of the luciferase
molecule?