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Low-temperature conditional cell division mutants of Escherichia coli

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Low-temperature conditional cell division mutants of Escherichia coli
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Cell division mutants of Escherichia coli
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Sturgeon, Joyce Anne, 1951-
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English
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ix, 94 leaves : ill. ; 28 cm.

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Antibiotics ( jstor )
Cell division ( jstor )
DNA ( jstor )
Escherichia coli ( jstor )
Genetics ( jstor )
Lesions ( jstor )
Lipoproteins ( jstor )
Membrane proteins ( jstor )
Phospholipids ( jstor )
RNA ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Escherichia coli ( lcsh )
Microbiology and Cell Science thesis Ph. D
Mutation (Biology) ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis--University of Florida.
Bibliography:
Bibliography: leaves 84-93.
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Typescript.
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Vita.
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by Joyce Anne Sturgeon.

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Full Text
LOi-T ZLRATLTE CONDITIONAL CELL DIVISION ,lUTA L.,TS OF ESCHERICHIA COLI
By
JOYCE ANNE SUjEGEON
A DISSERTATION PPESERTED TO THE GRADUATE COUNCIL OF THE JE iIVERSITY OF FLORIDA IN PAL.ETIAL FULFILIXalKNT OF THE REQUIRENET"S FOR TIE, DEGRF.
OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA 1977




ACKNOWLEDGMENTS
The time and effort that I have invested in my work and the writing of this dissertation are really the products of several people that are close to me. Wy parents helped to shape my attitudes and study habits from an early age by never appearing bored whenever I asked questions. My husband helped to develop my confidence and provided advice and encouragement when problems seemed unsurmountable. I dedicate my love and this dissertation to these three people.
I want to acknowledge and thank the entire faculty, staff,and graduate students of the Department of Microbiology and Cell Sjience for their friendship. I will sorely miss the conversations and companionship inherit in this department.
I have worked in the laboratories of Drs. Achey, Duckworth, Previc, Preston, Duggan, Smith,and Bleiweis. I would like to specifically thank all of these people for the use of their facilities and space. In addition, special thanks goes to Glenda Dunn for her excellent instructions on several techniques, most notably disc gel electrophoresis. Steve Hurst was most helpful in his explanations of the amino acid analyzer. Ed Hendrickson enlightened me with his experience of E. coli genetics. Both Dr.Aldrich and Bill Doherty have provided electron micrographs cf my cell division mutant. Tom Buttke was particularly kind to do the lipopolysaccharide analysis of strains X462 and 615A. Pam Spoutz and Dixie Donovan always provided me with the essentials of bacterial life-media and culture tubes. Most recently, Tom Hallam and John Silber have ii




introduced me to the technique of sucrose gradient centrifugation.and some special equipment in Dr. Achey's laboratory.
My commit-Lee members,Drs. Aldrich, Bleiveis, Duckworth, Duggan, and Hoffmann, have provided many helpful comments on my work and the writing of my dissertation. M/y warmest thanks, however, goes to Dr. ingram, the chairman of my comittee, for his enthusiasm, patience ,and continual support.
Finally, I want to thank three special friends for their advice
and encouragement throughout my graduate career--Nola Masterson, Carolyn
Napoli ,and Ruth Emyanitoff.
lii




TABLE OF CONTENTS
Page
AC12OWLEDGIMENTS ............................................... ii
LIST OF TABLES ................................................ v
LIST OF FIGURES ............................................... vi
ABSTRACT ....................................................... viii
GENERAL INTRODUCTION ................................................. 1
PHYSIOLOGIC 2D GENETIC CHARACTERIZATION OF LOW-TEMPERATURE
CONDITIONAL DIVISION MUTANTS OF ESCHERICIIlA COLI ........... 3
Introduction ............................................. 3
Materials and Methods ..................................... 4
Results .................................................. 11
Discussion ............................................... 19
BIOCHEMICAL CHARACTERIZATION OF THE ENVELOPE IMPAIRMENT OF
STRAIN 615A ................................................ 26
Introduction ............................................. 26
Materials and Methods .................................... 27
Results .................................................. 32
Discussion ............................................... 35
LITERATURE CITED .............................................. 84
BIOGRAPHICAL SKETCH ........................................... 94
iv




LIST OF TABLES
Table Page
1 Bacterial strains ........................................ 38
2 Antibiotic disc assays .................................. 39
3 DNA per mass ratios of strain X462 and strain 615A ...... 40
Results of Hfr and 615A uninterrupted matings ........... 41
5 Transductional analyses ................................. 42
6 Phospholipid comoosition in strains X462, 615A and 71, 43 7 Fatty acid composition in strains X462 and 615A ......... 44 8 Analysis of peptidoglycan in strains x462 and 615A ...... h5
9 In vivo cross--linking analysis of peptidoglycan in strains x462 and 615A .................................... 46
10 Analysis of bound lipoprotein in strains X462 and
615A .................................................... 47
V




LIST OF FIGURES
Figure Page
1 Linkage map of Eseherichia coli K-12 showing the
origins and directions of transfer of Hfr strains
(arrows) .................................................49
2 Micrographs of strain 615A, a representative lowtemperature conditional mutant and its parent, strain
x462 ....................................................51
3 Cell division following a shift from 39C to 3C.... 53
14 Viability of strain 615A following a temperature
shift from 39C to 3C....................................55
5 Cell division following a shift from 30C to 39C .... 57
6 Growth of strains 615A and X462 in the presence and
absence of deoxycholate ..................................59
7 Effects of a shift from 39C to 30C on growth and DNA
synthesis in strains 615A (A) and Q462 (B)...............61,62
8 Pulse-label measurements of DNA synthesis in strains
615A (A) and x1462 QB following a shift from 39C to
3C.....................................................64
9 Pulse-label measurements of RNA synthesis in strains
615A (A) and x462 (B following a shift from 39C to
3C......................................................66
10 Effect of chloramphenicol on induced division of filaments of strain 615A .................................68
11. Summry of the effects of antibiotic additions on induced cell division of strain 615A .....................70
12 Measurements of growth and DNA synthesis in strains 615A (A) and x1462 (B) following a shift from 30C to
39C .....................................................72,73
13 Phospholipid turnover during induced division in strain 615A ..............................................75
Ai




LIST OF FIGURES--CONTINUED
Figure Page
14 Pulse-label measurements of phospholipid synthesis
in strains 615A (A) and X462 (B) following a shift
from 30C to 39C ...................................... 77
15 Thin-layer chromatography of LPS isolated from strain
X462 and strain 615A ................................. 79
A6 Difference plots of membrane protein profiles from strains X462, 615A and 71 ............................ 81
17 Comparison of membrane proteins from cultures of strain Qh62 (dashed line) and strain 615A (solid
line) grown at 30C ......................... ......... 83
vii




Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial FuLIfillnent of the Requirements
for the Degree of Doctor of Philosophy
LOW-TE1PERATURE CONDITIONAL CELL DIVISION MUTANTS OF ESCIFHRCHIA COLI
By
Joyce Anne Sturgeon
June 1977
Chairman: Lonnie 0. Ingram
Major Department: Microbiology and Cell Science
Fifteen low-temperature conditional division mutants of Escherichia coli K-12 have been isolated. These grow normally at 390C and form filaments at 30*C. All exhibit a coordinated burst of cell division upon exposing filaments to the permissive temperature. None of the various agents which stimulated cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability or stimulating cell division in broth. One of these mutants, strain 615A, appears to have an altered cell envelope as evidenced by its increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonuclease I, a periplasmic enzyme. However, no significant differences between the parent and mutant strains are observed in lipopolysaccharide structure, phospholipid composition, fatty acid composition, peptidoglycan composition, or in the percentage of cross-linking. In addition, no differences are detected between the two strains in the inner and outer membrane protein compositions. This mutant has normal rates of deoxyribonucleic acid synthesis, ribonucleic viii




acid synthesis, and phospholipid synthesis for division of filaments at the permissive temperature. However, strain 615A requires new protein synthesis in the apparent absence of new ribonucleic acid synthesis for division of filaments at the permissive temperature. The division lesion in strain 615A is cotransducible with malA, aroB, and QD, and maps within minutes 72-75 on the E. coli chromosome.
ix




GEIRAL INTRODUCTION
Cell division in bacteria is a complex process involving the coordination and regulation of deoxyribonucleic acid synthesis, nuclear segregation, septation,and the physical separation of newly formed daughter cells. One method of studying bacterial cell division is the isolation of mutants blocked in different recognizable aspects of the cell cycle. The majority of division mutants isolated and characterized 'in Escherichia coli have been high-temperature conditional cell division mutants (2,4,20,35-37,44,67,69,85,108,115,121). A number of genetic loci involved in cross-wall formation has been identified using these mutants (3,30,36,37,41,60,88,95,108,109,120). In an effort to discover new and different loci as well as learn more about the roles of existing loci in the division process, we have isolated low-temperature conditional division mutants in E. coli K-12. The main thrust of our efforts has been the physiologic and genetic characterization of one of these mutants, strain 615A. Section I contains these results as well as a comparison of the phenotypic and genotypic properties of our mutants with previously isolated division mutants.
The isolation and characterization of strain 615A has possibly revealed another locus involved in cell division in E. coli (Section I). This division lesion appears to involve an impairment in the integrity of the envelope as evidenced by the increased sensitivity of strain 615A to deoxycholate and antibiotics as well as leakage of ribonuclease I, a periplasmic enzyme. It seemed logical to investigate the nature of
1




2
this envelope lesion by systematically analyzing the major components of the envelope. Section II is the results and discussion of our efforts.
The regulation of cell division is a fundamental process of all
living organisms. Currently very little is understood about the reulation of any step in the cell division process of any organism. Thus, the elucidation of the division process remains one of the most important areas of investigation in molecular biology today.




SECTION I
PHYSIOLOGIC AND GENETIC CHARACTERIZATION OF LOW-TMPERATURE CONDITIONAL DIVISION MUTANTS OF ESCIERICHIA COLT
Introduction
Cell division in bacteria is a complex process involving the coordination and regulation of DNA replication, nuclear segregation, septation,and the physical separation of newly formed daughter cells. Jones and Donachie (47) have envisioned the cell cycle of Escherichia coli as involving two parallel series of events: chromosome replication and the synthesis of division proteins. Both series are seen as capable of proceeding independently to produce products which must act in a coordinate fashion leading to cell division. DNA synthesis, nuclear segregation, RNA synthesis,and protein synthesis are capable of proceeding normally in the absence of cell division (4,16,20,35,36,41,60,67,85,88, 109,121). However, a strict requirement for an event related to DNA synthesis has been observed for division except under very unusual conditions where DNA-less cells are produced (1,35-37,39,44,45).
One method of studying bacterial cell division is the isolation of mutants blocked in different recognizable aspects of the cell cycle. Nmethyl-N'-nitro-N-nitrosoguanidine has been frequently employed by many investigators (2,4,20,67,69,85,92,103,108,115,121). It suffers from the disadvantage of inducing multiple, closely linked mutations (62). Using this mutagen, a variety of high-temperature conditional cell division mutants (2,4,20,35-37,44,67,69,85,108,115,121) have been isolated. Such
3




4
mutants are generally accepted as resulting from missense mutations which lead to the production of less stable proteins unable to function properly at the elevated temperature (32,87). However, this interpretation is not as readily applicable to "cold-sensitive mutants"(ll0). Thus, it seemed possible that the isolation and characterization of low-temperature conditional cell division mutants might reveal new and different loci involved in the cell division process. This paper describes the physiology and genetic lesion in a UV-induced "cold-sensitive" mutant of Escherichia coli K-12.
Materials and Methods
Bacterial Strains
The bacterial strains and their genotypes are listed in Table 1. In addition, the approximate origins and direction of transfer of the Hfr strains are shown in relation to the genetic markers of the parent strain (Fig. 1). Genetic designations are according to Taylor and Trotter (106). Map positions of amino acids, sugars ,and antibiotics are in accordance with the revised linkage map of Bachmann, Low, and Taylor (8). Whenever possible, the revised map positions will be used. However, in discussing the genetic lesions in division previously described, we have used the former linkage map. Bacteriophage Plvir was part of the strain kit obtained from Cold Spring Harbor Laboratory (Cold Spring Harbor, NY).
Strains X462mg, 615Am,and LW3m are malA- derivatives of strains
X462, 615A,and LW3, respectively. The malA- lesion was introduced into these three strains by 2-aminopurine mutagenesis (62) and subsequent selection of mal- colonies on MacConkey agar supplemented with 0.2% maltose. The glpD lesion was introduced into strain Yh62malA- in an




5
analagous fashion.
Media
Dix and Heimstetter minimal medium (P4M) (21) and Luria (L) broth
(52) were used for isolation, enrichment,and subsequent experiments involving the low-temperature conditional cell division mutants. Both were supplemented iith glucose (2 g/l) and thiamine (10 mg/!). L-amino aids were added to minimal media at a final concentration of 40 ig/ml.
-Streptomycin sulfate (100 pg/ml) was used as a donor counterselective agent in mating experiments.
Solid minimal medium was prepared by mixing equal volumes of 2X
Dix and Helmstetter minimal medium and 2X agar (1.5% final concentration) after autoclaving. L-agar was prepared by adding agar to L-broth at a final concentration of 1.5% before autoclaving. L-NaC! or 13-NaCl redia were prepared by adding 15 g of NaCl per liter to the above recipes prior to autoclaving. L-sucrose and MM-sucrose media were prepared by ad-ing sucrose at a final concentration of 10% to the agar solutions after autoclaving separately. L-deoxycholate and MM-deoxycholate media ccntained 0.25% sodium deoxycholate. Selective plates for all genetic experiments were prepared as described by Miller (62). Growth Conditions
Liquid cultures were grown in 22 X 175 mm tubes with constant,forced aeration. Overnight cultures grown at 39C were diluted into fresh, prewarmed medium. Incubation was continued at 39C until cultures were growing exponentially. Studies involving temperature shifts plus or minus various agents were generally conducted as follows: filaments were induced by growth at 30C for 1-1/2 to 2 hours in L-broth or 3 to 4 hours in MM. These cultures along with appropriate controls were then split,




6
treated with various agents ,and incubated at 30C and 39C.
Bacterial cell numbers were determined with a Celloscope electronic particle counter (orifice, 24 in; amplification, 1; electrozone, 24;
lower threshold, 18). Optical density of bacterial cultures was measured at 550 ran using either a Spectronic 70 spectrophotometer or a Beckman Model 25 spectrophotometer. Isolation and Enrichment
The low-temperature conditional cell division mutants were originally derived from Escherichia coli K-12, strain X462 (Table 1), a generous gift from A. C. Frazer and R. Curtiss III. A log-phase culture was
mutagenized with ultraviolet light (1.247 survival) and allowed to grow overnight in the dark at 37C. Thermosensitive cell division mutants were enriched using a procedure modified from van de Putte et al. (108). The
mutagenized culture was initially diluted 1/20 and allowed to reach an OD550 between 0.4 and 0.6 (log phase) at 37C. This culture was then filtered through a 3 p pore size nuclepore filter (Nuclepore, General Electric). The filtrate, containing small cells, was used as the inoculum into fresh, prewarmed broth at 30C (1/20). After this culture had reached an OD550 between 0.4 and 0.6, 10 ml was filtered and washed with an equal volume of fresh medium through a 5 p pore size nuclepore filter. The filter, containing filamentous cells, was used to inoculate a fresh culture tube at 37C. This description of the filtration procedure constituted one cycle; five such cycles of filament enrichment followed
before dilutions of the !ast culture at 37C were plated onto L-agar for isolated colonies. At no time throughout the enrichment procedure were the cultures allowed to reach late log to stationary phase. This was done to ensure that a physiologically uniform period was employed during




7
both filament formation and stimulated cell division. Enzyme and Antibiotic Disc AssaLys
Release of ribonuclease I was determined by the method of Lopes et
al. (56). L-agar plates were overlaid with 2.5 ml of top agar containing
1.5% PA (pH adjusted to 7.0). The parent or mutant strain was spotted onto the hardened overlay and after incubation at 37C or 25C for 2 hcurs, the plates were flooded with 0.1 N hydrochloric acid. Areas of RNA digestion were clearly visible in the opaque background.
Alkaline phosphatase activity was assayed by the p-nitrcphenol
overlay method and spectrophotometrically as described by Willsky et al. (116).
Sensitivity to antibiotics was determined using disc assays. T6
agar was seeded (4 X 10 cells/ml) with an overnight culture (parent, mutant, revertant, or transductant strain). Portions of 15 rl each -were poured into sterile petri plates and allowed to harden. Antibiotic impregnated discs (Difco Laboratories, Inc., Detroit, MI) were placed in the center of individual plates. After 18 hours of incubation at either 39C or 30C, the diameter of the zone of clearing was measure,. Measurement of DNA Synthesis
DNA synthesis was measured in two ways in the parent and mutant
strains. The first method was to sample cultures that had been uniformly labeled with 3H-thymidine. To measure DNA synthesis in filaments induced to divide by a temperature shift to 39C, an exponential culture at 39C was inoculated into prewarmed medium to which thymidine (1 pCi/ml,
5 jg/ml) and deoxyadenosine (250 jg/ml) had been added. The cultures were allowed to grow in the presence of the label for 4-5 generations by incubating the culture at 39C for 2 generations and then shifting to




8
30C for 3 generations. After this period, cultures of strain 615A had formed filaments. Cultures were split and one portion was incubated at
39C. Duplicate samples (0.1 ml) taken every 15 minutes for the next
2 hours were pipetted onto 0.45 pm Whatman filter paper discs and immediately immersed in cold 10% trichloroacetic acid (TCA). All of the
discs were washed twice more in cold 10% TCA and then taken through
three washes of cold 95% ethanol. The filter discs were dried and
placed into vials with cocktail for the determination of radioactivity.
The procedure for measuring DNA synthesis in filaments of strain 615A .is the same except all the labeling prior to the shift to 30C was done at the permissive temperature (39C).
DNA synthesis was also measured by pulse-labeling with 3H-thymidine.
Cultures of strains X462 and 615A were grown overnight at 39C in L-broth and diluted 1/400 into fresh medium. Incubation continued at 39C until
OD 550= 0.1. The culture was then split, one portion shifted to 30C,and
1 ml samples were pulsed 4 minutes at 30C or 39C with 2.5 PCi of 3Hthymidine per ml. Unlabeled thymidine was added to the samples at a final. concentration of 10 pg/ml. At the end of the pulse, the tubes were immersed in an ice bath and 1 ml of cold 10% TCA containing 400
vg of thymidine per ml was immediately added. After 1 hour in the ice
bath, the samples were filtered through Millipore type HA filters (Millipore
Corp., Bedford, MA) or Gelman Metricel GA-6 filters (Gelman Instrument
Company, Ann Arbor, MI) and washed 3X with cold 107 TCA and 3X with cold
70% ethanol. After drying, the filters were placed into vials for the
determination of radioactivity.
Measurement of RNA Synthesis
Cultures were grown as described for pulse-labeling of DNA synthesis.




9
3
One ml samples were pulsed for 4 minutes with 2.5 uCi of 3H-uracil per ml (0.5 vg/ml). The cold 10% TCA contained 200 pg of uracil per ml. Measurement of PhospholipidSynthesis
Cultures of strain X462 and strain 615A were grown as described for pulse-labeling DNA synthesis. Ten ml samples at 39C or 30C were pulse-labeled for 4 minutes with 4 pCi of 32P-orthophosphate per ml. At the end of this period of labeling, 1 ml of cold 50% TCA was added and dispersed throughout the sample. The samples were then centrifuged at 8000 rpm for 15 minutes, the supernatant removed,and the insoluble material resuspended in methanol-chloroform (2:1). Lipids were allowed to extract overnight at room temperature. The next day, 1 ml distilled water and 1 ml chloroform were added; the solution was mixed vigorously and centrifuged at 8000 rpm -or 15 minutes. The upper aqueous phase was discarded and the lower lipid phase was removed for determination of radioactivity.
Measurement of Phospholipid Turnover
Strains 615A and X462 were grown overnight at 39C in L-broth,
diluted 1/200 into fresh, prewarmed medium,and reincubated at 39C until an OD 550 0.4. This culture was then diluted 1/20 into broth and grown for 1-2 generations in the presence of 32P-orthophosphate (8 pCi/ml) at 39C, shifted to 30C,and grown for 3 more generations. After the incubation at 30C to induce filaments, cells were spun down by centrifugation, washed twice,and resuspended in L-broth. One-half of the culture was incubated at 30C and the other half at 39C. Two ml samples were taken every 15 minutes for phospholipid analysis. Cells were precipitated with 57 TCA, centrifuged,and pellets resuspended in 1 ml methanol and 2 ml chloroform. Lipids were extracted overnight at room temperature




10
as described by Kanfer and Kenned (49). The 2 ml lipid layer was removed and a 0.2 ml sample was counted to determine total radioactivity. The remainder of the sample was evaporated to near dryness with dry N2 and spotted on silica gel G thin-layer chromatography plates (Analtech, Inc., Newark, DE). These plates had been preactivated in acetone and dried 15-60 minutes in a desiccator. The chromatograms were run in a solvent system of chloroform-methanol-glacial acetic acid (65:25:8, v/v) as described by Ames (5). Major phospholipids (phosphatidylethanolamine, phosphate idylglycerol, lysophosphatidylethanclamine,and cardiolipin) were visualized with iodine vapors, scraped into vials ,and counted for radioactivity.
Bacteria! Matings
Matings between freshly reisolated Hfr strains and strain 615A, one of the low-temperature conditional cell division mutants, were done as described by Miller (62). Donors growing exponentially in L-broth (2 X 108 cells/ml) were mixed with 2-4 X 108 cells/ml of strain 615A (F-) at a ratio of 1/20. The mixture was incubated with slow shaking for 90 minutes at 37C after which appropriate dilutions were plated on selective media.
Transductions
P1 transducing lysates were prepared by the confluent lysis
technique as described by Marsh and Duggan (59). For transduction experiments, the recipient cells were grown in Z-broth to a density of 10' cells/ml. The transducing lysate was diluted to yield a multiplicity of exposure of 0.1-0.5. Phage were allowed to adsorb at 37C for 10 minutes. Unadsorbed phage were removed by centrifugation. Bacteria were resuspended in sterile buffer and plated on appropriate selective media (10-1




-21
and 102 final dilutions).
Light and Electron Microscopy
The morphology of the cells were examined using a Zeiss photographic microscope. Nuclear stains were done by the Giemsa technique as described by Fuhs (26). For electron microscopy, cells were fixed in 1' osmium, embedded, sectioned,and examined using a Hitachi HUIIE electron
microscope (42).
Chemicals
All amino acids and vitamins were obtained from Calbiochem (San Diego, CA) or the Sigma Chemical Co. (St. Louis, MO). Streptomycin sulfate and nalidixic acid were obtained from the Sigma Chemical Co. The following radioactive chemicals with their specific activities were obtained from Amersham/Searle Co. (Amersham, England): [6-3H] thym1i'ne (25.6 Ci/mmol), [6-3H] uracil (23 Ci/mmol),and [ 32P orthophosphate (75 Ci/mg P).
Results
Isolation
Initially, 1,624 colonies were screened for poor growth on mini-al
plates at 25C. Of these, 162 grew poorly, and they were further ex-_ined in broth for their ability to form filaments at 30C and grow normaly,at 37C-39C. All of the 162 mutants exhibited this general characteristic and 19 were selected for characterization of growth and cell division. Four of these produced minicells at both temperatures and will be characterized in subsequent studies.
General Characterization
In both complex and minimal broth, the remaining 15 low-tempers:ure




12
conditional cell division mutants formed filaments at 30C (Fig. 2A). Upon shifting to the permissive temperature, all the mutants exhibited a coordinated burst in cell division, thus forming short cells at 39C (Fig. 2B). Nuclear stains of the filaments at 30C (Fig. 2C) revealed segregated masses of nuclear material throughout the length of the filament in all mutants. By light microscopy 1-2 completed septa per filament were frequently observed. Ultrastructural examination confirmed the presence of occasional septa and the absence of multiple, incomplete cross-walls.
The mutants could be divided into two basic groups based on their colonial morphology on minimal agar plates at 370. The majority of the mutants produced colonies similar to that of the parent, strain X462: circular, entire, convex,and, smooth. However, 4 mutants (JS1, JS5, JS8, JSlI) produced colonies at 37C that were irregular, undulated, flat,and wrinkled in appearance.
In a variety of other mutant systems (23,36,41,46,58,60,67,85,88,
91,104), high concentrations of salt and/or sucrose prevented the phenotypic effects of the cell division mutation at the nonpermissive temperature. The 15 low-temperature conditional cell division mutants were plated onto complex and minimal media plus and minus NaCl (1.5%) or sucrose (107.) and incubated at both the permissive and restrictive temperatures. The concentrations of salt and sucrose tested on our mutants were the maximal concentrations found in the literature to be effective for other mutant systems (58,88). Sucrose was effective in restoring colony-forming ability at the nonpermissive temperature to strains JS5 and JS14. These mutants were able to form colonies at the nonpermissive temperature in the presence of 10% sucrose. None of the other additives




13
phenotypically rescued mutants.
Effects of Temperature Shifts anud Other Treatments Wh:.ch Have Previously
Been Shown to Stimulate Cell Division
The kinetic aspects of cell division in each of the 15 mutants were examined. The results of one representative, strain 615A, is shown in Figure 3. Upon shifting to the restrictive temperature, cell division does not cease immediately, but continues at the same rate as the nonshifted control for 30-45 minutes (Fig. 3A). The cells remain viable for at least 180 minutes after the shift to the nonpermissive temperature (Fig. 4), but do not significantly increase in cell number after the residual 30-45 minutes of division (Fig. 3A). A coordinated burst in cell division was observed after 30-45 minutes upon shifting filaments to the permissive temperature (Fig. 5A).
Membrane-active agents such as ethanol (43) and protein synthesis inhibitors such as chloramphenicol (30,104,119-121) have been shown to stimulate cell division in several mutant systems. The effects of these two agents as well as the effects of salt and sucrose on the cell divi-sion ability of all the mutants were examined in liquid culture. None of these agents stimulated cell division in broth at the restrictive temperature in any of the 15 mutants. Sensitivity of Strain 615A to Deoxycholate and Antibiotics
Sensitivity to deoxycholate has previously been interpreted as evidence for an alteration in the bacterial envelope (3,23,24,35,36,38,51, 58,70,87-89,103). The ability of the mutants to form colonies on L-agar plates containing deoxycholate (0.25%) was tested. Five out of the 15 mutants, strains JSl, JS9, JSII, JSl2, and 615A, did not form colonies on this medium at either temperature. Neither the sensitivity to deovcholate nor the phenotypic rescue by sucrose correlated with either type




of colonial morphology.
Strain 615A was further examined for defects in the cell envelope. This strain was also found to be more sensitive that the parent strain to deoxycholate when grown in broth (Fig. 6) and this sensitivity was exaggerated at the nonpermissive temperature (30C). Although increased sensitivity to actinomycin D (7,23,51,54,76,94,103) and lysozyme (51,94) has been interpreted as evidence for alteration in the outer envelope, neither the parent nor the mutant was found to be sensitive to actinomycin D (25 Pg/ml) or lysozyme (2.5 pg/ml).
There have been several reports in the literature of envelope
mutants whose growth is not only sensitive to detergents, but also to a variety of unrelated antibiotics (54,79,94). Using antibiotic disc assays, zones of inhibition were compared at the permissive temperature, 39C, and the restrictive temperature, 30C (Table 2). At the restrictive temperature, strain 615A is more sensitive than the parent to all of the antibiotics. At 39C strains 615A and X 62 have nearly the same degree of sensitivity to bacitracin, rifampin,and nalidixic acid. However, strain 615A was also found to be more sensitive to neomycin (5 pg and 30 pg), chloromycetin (30 Pg), penicillin (10 Units), polymyxin B (50 Units and 300 Units), novobiocin (30 Pg), tetracycline (5 jg and 30 jg), erythromycin (15 jg), cephalothin (30 jg), coly-mycin (10 Vg), and aureomycin (30 jg) at 30C. No differences in the sensitivities of strains 615A and X462 to penicillin (2 Units), novobiocin (5 vg), and erythromycin (2 jg) were observed at either temperature. Leakage of Periplasmic Enzymes in Strain 615A
Agar diffusion assays were performed for ribonuclease I, a periplasmic enzyme. These indicated that strain 615A leaked this enzyme,




15
especially when colonies were incubated at the nonpermissive temperature. Alkaline phosphatase activity, on the other band, was not released into the growth medium as assayed by both p-nitrophenol overlay or spectrophotometrically (116). The selective release of ribonuclease I as well as the increased sensitivity to deoxycholate and a variety of antibiotics is consistent with other reports of differential permeability changes in envelope mutants (56). The results with strain 615A indicate that there is an impairment in the integrity of the cell envelope. Characteriation of DNA Synthesis and RNA Synthesis in Filaments of Strain
615A
Many mutants of E. coli have been reported which form filaments as a result of a mutation that interferes with normal DNA synthesis (36,37, 102) or normal DNA segregation (36,37). However, filaments of strain 615A are not impaired in either of these processes. Filaments of strain 615A contain masses of nuclear material scattered throughout their length (Fig. 2C). Total DNA synthesis in strain 615A continues normally at the nonpermissive temperature, paralleling the increase in absorbance (Fig. 7). Pulse-labeling studies (Fig. 8) confirm that DNA synthesis continues at 30C without any apparent lag. In addition, the DNA per mass ratios of the parent and mutant strains (Table 3) are quite similar at both the permissive and nonpermissive temperatures.
Figure 9 is a comparison of RNA synthesis in cultures of strain
X462 and strain 615A. These cultures were grown at 39C, shifted to 30C at time zero,and pulse-labeled with 3H-uracil. RNA synthesis is normal and parallels growth in strain 615A at both the nonpermissive and permissive temperatures.
Effects of Inhibitors of Macromolecular Synthesis Upon Division of




16
Filaments at 30C
Many cell division mutants in E. coli have been examined for sensitivity to inhibitors of macromolecuLar synthesis during induced division. These experiments involve splitting a culture of filaments into a number of subcultures with the addition of antibiotics at various times relative to the shift to the permissive temperature. An example from this study (Fig. 10) shows the effect of chloraTphenicol on induced division. For comparative purposes, the increase in cell number 75 minutes after the shift to the permissive temperature has been chosen as a measure of temperature-induced cell division. Increase in cell number after this time represents subsequent divisions of short cells as well as division of residual filaments. However, the conclusions of the relative effects of an inhibitor are not different if 60 or 90 minutes is used as the reference point. An increase in cell number above 50% of the control (39C culture with no additions) indicates that the majority of the shift-induced divisions has become insensitive at the time of the addition of the antibiotic. The division of filaments of strain 615A at the permissive temperature is sensitive to the inhibition of.protein synthesis by chloramphenicol for as long as 30 minutes. Thus, filaments of strain 615A require 30 minutes of new protein synthesis to divide at 39C.
Figure 11 summarizes several similar experiments in which inhibitors of DNA synthesis (nalidixic acid), RNA synthesis (rifamycin SV),or protein synthesis (puromycin, chloramphenicol) have been added at various times after filaments were shifted to the permissive temperature. Instead of expressing the data as in Figure 10, the results are expressed as the percentage of increase in cell number relative to the untreated 39C con-




17
trol culture versus the time at which the antibiotic was added following the shift of filaments to the permissive temperature. From these results, we can conclude that filaments of strain 615A require approximately 12 minutes of new DNA synthesis and 36-44 minutes of new protein synthesis, in the absence of new RNA synthesis,for division at the permissive
*temperature. The requirement for DNA synthesis was also confirmed using mitomycin C (5 vg/ml) and phenethyl alcohol (0.3%). Effects of Penicillin on Induced Division of Filaments
Low levels of penicillin inhibit cell division in E. coli with little effect on elongation (98). Maximum sensitivity to killing by high levels of penicillin and maximum sensitivity of division by low levels of lenicillin occur at or shortly after the completion of rounds of chromosome replication (40). The timing of this is coincident with the maximal rate of murein synthesis during the cell cycle (40). When 25 Units of penicillin G per ml are added up to 20 minutes after the shift of filar-nts to the permissive temperature, temperature-induced division as measured by increase in cell number was significantly inhibited (Fig. 11). '-us, it appears that filaments of strain 615A are blocked at a point in the cell cycle well prior to the penicillin-sensitive step. DNA Synthesis, Phospholipid Synthesis and Phospholipid Turnover During
Induced Division
Strain 615A appears to have a full complement of nuclear material at both permissive and nonpermissive temperatures (Table 3). However, division of filaments of strain 615A is sensitive to inhibition by nalidixic acid for as long as 12 minutes after the shift to the permissive temperature. We examined DNA synthesis in strain 615A during induced division and found the change in the rate of DNA synthesis to be si-ilar to that of the parent following the same shift in growth temperature (Fig.




13
12).
Ohki (80) found that phosphatidylglycerol turned over in a ste:-dse manner 20-30 minutes before each cell division in a 1", Casaino acids medium. The turnover of individual phospholipid comLonents was raeasured in filaments of strain 615A and in temperature-induced division of filaments in rich medium (Fig. 13). However, no abrupt changes the turnover of phosphatidylglycerol were observed during iniuced division. No turnover of phosphatidylethanolamine was observed.
Phospholipid synthesis during induced division of fila:ents of strain 615A was also examined(Fig. 14). The increase in the rates cf phospholipid synthesis was similar to the increase in rates Cobserved with the parent. For both strains, phospholipid synthesis zrallelei the increase in growth.
Mapp ng of the Division Lesion of Strain 615A
Results from the 90-minute matings (Table 4) revealed that the Lowtemperature conditional division locus in strain 615A was lccated be=:ween minutes 68-79. Interrupted mating experiments performed using strains CSH47a, CSH64,and 615A narrowed the locus to the region between mall (min 74) and xyl (min 79). Table 5 shows the results of tramsductiznal analyses involving a P1vir lysate made on strain 615A malA lpD + ar:3+ asd and several E. coli strains that are either malt-, por ar-, and wild-type for division. The division lesion in strain 615A appears to cotransduce with malA at frequencies of 20-335, with glp2L at frequencies of 9-52%,and with aroB at a frequency of 21.55. IL a further attempt to locate the cell division lesion in strain 615A w-ith respect to these markers, a transduction with the same Plvir lysate and an &Sd strain (U482; Table 1) was done. However, no asd transduc:ants could




19
be recovered using this lysate despite many attempts. Strain 615A does not require diaminopimelic acid for growth in rich mediuri (phenotype of asd- strains) at either the permissive or nonpermissive temperatures. The addition of" dianinopimelic acid does not prevent growth of filaments at 30C. Thus, it is probable that the cell division lesion in strain 615A is not the asd lesion. One possible explanation for these results is that the asd lesion and the division lesion in strain 615A are not compatible in the same strain.
Analysis of Transductants and Revertants
Spontaneous revertants were obtained by growing strain 615A in Lbroth at 30C. The culture was diluted (1/200) into fresh broth at
regular intervals. After three days of growth, dilutions of the culture were plated on L-agar and incubated at 25C. Isolated colonies were examined for growth in broth at both the permissive and nonpermissive temperatures. Ten spontaneous revertants that divided normally at both temperatures were selected and examined for antibiotic sensitivity (plates) and sensitivity to deoxycholate in broth. All of the revertants were as insensitive to the antibiotics as the parent (representatives, Table 2). However, the revertants were found to be as sensitive to deoxycholate (0.255) at 30C and slightly more sensitive at 39C than strain 615A.
Discussion
Burdett and Murray (lh) have recently characterized the events of
septation in E. coli B and B/r strains. In accordance with the cell cycle of Jones and Donachie (47), they found that the classical D period is composed of events leading to cell compartmentalization, septationand separation. In rich medium septation is completed in approximately 13




minutes while 7 minutes in needed for daughter cells to separate. Thus, approximately 20 minutes must elapse from the visual initiation of a cross-wall to the separation of daughter cells.
Strain 615A can be characterized as a septum-initiation type mutant and positioned accordingly in the scheme of Jones and Donachie. Residual division at the nonpermissive temperature and lack of multiple, incomplete septa at 30C support this classification (121). Mutants have been isolated which can divide in the presence of chloramphenicol (30, 88, 10, 121). This indicates that the assembly of septum precursors can take place even in the absence of protein synthesis. Cell division in strain 615A is sensitive to the addition of chloramrphenicol for as long as 36 minutes and to the addition of puromycin for as long as 44 minutes after shifting filaments to the permissive temperature. Also, temperatureinduced division of filaments is prevented by the addition of concentrations of penicillin G which block cross-wall initiation for as long as 20 minutes after the shift to the permissive temperature. Thus, strain 615A appears to be blocked in cell division at a point well prior to the assembly of septum precursors. This bloc could be structural and alter the nature or production of only one precursor or it could be regulatory and alter the envelope such that septum precursors cannot be assembled(36).
The primary lesion in strain 615A does not appear to be in the events involving the synthesis of bulk DNA or nuclear segregation. Crumplin and Smith have recently provided evidence for a new step during the synthesis of the E. coli genome (17). They have shown that nucleotides are incorporated into Okasaki fragments which are ligased into 38S singlestranded fragments. These 38S fragments are subsequently converted into full-sized daughter molecules. It is this latter step, the conversion




21
into the full-sized daughter molecules, which is inhibited by nalidixic acid. Filaments of strain 615A incorporate 3H-thyridine into DNA with no detectable lag and contain the same amount of DNA per unit mass as normal cells. However, the division of filaments of strain 615A is sensitive to nalidixic acid for as long as .2 minutes after the shift to the permissive temperature. Thus, it appears that filaments of strain 615A may be blocked at a point between the incorporation of nucleotide precursors into DNA and the nalidixic acid-sensitive step. Alternatively, the required termination protein (47) generated by the completion of a round of replication may be unstable in strain 615A a30C and the completion of new rounds may be required to permit the
initiation of new divisions. However, this product is presumably stable in other morphologically similar mutants which have been reported to initiate and divide in the absence of new DNA synthesis (69,85,120).
Strain 615A requires a significant period of new protein synthesis in the apparent absence of new RIA synthesis for division of filaments at the permissive temperature. This implies that the mRNA translated during this period to produce the required protein(s) is more resistant to turnover than most other E. coli mRNAs (27,101). Messenger RNA for most membrane proteins, most notably Braun lipoprotein (32-34), and excreted proteins often appear to be less susceptable to turnover (50, 66,99). Alternatively, transcription of some mRNA(s) may be resistant: to rifamycin SV. The mRNA for lipoprotein is one example (34).
Strain 615A contains an envelope defect as evidenced by increased sensitivity to deoxycholate and many antibiotics as well as by leakage of ribonuclease I, a periplasmic enzyme. The possibility of changes in cell wall components will be explored in subsequent studies. Analysis




22
of wild-type transductants and revertants of strain 615A support the involvement of this envelope alteration in the division lesion. These strains not only divide normally at 30C and 39C, but also are as resistant as the parent organism to antibiotics at both temperatures (Table 2) and the wild-type transductants are resistant to deoxycho7ate. Kcwever, the revertants remain sensitive to deoxycholate at both the ncnpermissive and permissive temperatures. One explanation of this is
-that the division-competent revertants result from a second gene mutation. This second mutation could permit division without restoring normal envelope integrity. Suppression of a division defect in lon strains of E. coli by a second mutation, recA, is well documented (2;). Another possibility is that a second gene mutation in strain 615A may be responsible for deoxycholate sensitivity. If this is true, however, the lesion for deoxycholate sensitivity and the cell division lesion must be in the same region of the chromosome (within 1.8 min) (62). The third possibility is that the revertants contain an altered gene product which permits cell division without completely restoring envelope integrity.
Cross-wall formation in E. coli involves a number of genetic lcci (3,20,36,37,41,6o,88,95,108,109,120). Most of these loci have been identified using high-temperature conditional cell division mutants. In an effort to discover new and different loci as well as learn more about the roles of existing loci in the division process, we have isolated lowtemperature conditional. cell division mutants. Preliminary results indicate that 2 out of the remaining 14 mutants isolated have division lesions that are cotransducible with malA. Thus, it appears that this method of isolation and enrichment for low-temperature condi'zional di-.-i-




23
sion mutants does not result in mutants whose lesions are clustered in
a single region of the chromosome. Strain 615A appears phenotypically similar to many of the high-temperatui-e conditions mutants previously described: strain BUG6 (85), strains fts fts 7- fts (108), strains AX621, AX629, AX655 (3), strain MX74T2ts52 (120), strain ts-20 (69), ftsA-F strains (88), strain Ya6 (95), strain MAC! (20), strain fil ts (104), strain ts612 (60), and strain ASE124 (41). Strain SN29 is a lowtemperature conditional cell division mutant of Agqnenellum quadru)licatu
(43) that can be included in this group of morphologically similar mutants. None of these mutants is blocked in bulk DNA synthesis or nuclear segregation as evidenced by the formation of long, multinucleoid filaments lacking cross-walls when grown at the nonpermissive condition. Although these mutants appear to be defective in cross-wall initiation rather than cross-wall formation, the latter possibility cannot be completely excluded. Incomplete cross-walls undiscerned by light microscopy potentially could be resorbed by hydrolases or destroyed in the fixation procedure for electron microscopy as suggested by Slater and Schaechter (102). Thus, this group of morphologically similar strains can be described as multinucleoid, septation-initiation type mutants. These mutants can, however, be divided into several groups based on other characteristics. Strains BUG6, ASH124 and fil ts divide normally when dense cultures are shifted to the nonpermissive temperature. Strains ASH124, BUG6, ts612, and some of the fts strains described by Ricard and Hirota (88) are phenotypically rescued by salt and/or sucrose at the restrictive temperature. Strains MX7hT2ts52 and fil ts are stimulated to divide at the restrictive temperature by the addition of chloramphenicol. The addition




of adenine at the nonpermissive temperature prevents filament-for:ation in strain YI.6. Strain SN29 is induced to divide at the nonnermissive temperature by ethanol. Strain ts-20 and MACi divide synchronous]y when filaments are shifted to the permissive temperature. Strains ftsB and ftsC form minicells in addition to multinucleoid filnents.
These mutants can be further grouped according to the location of
their genetic lesions. The majority of these loci are cluszered cn either side of the replication origin (min 74) (62). As pointed :ut by 1achmann et al. (8), it would certainly be to the organism's advantaZe to haTe the potential to amplify important genes during periods of rapid gr wth. The part of the genome near the replication origin would be presen: in several copies per nucleoid due to multiple initiations of the DNA replication cycle. The lesion in strain ts612 maps at min 74.7-75.5. Strains ftsA, AX series, and fts2 fts and fts all -luster between minutes 1-2 on the Taylor and Trotter linkage map (106). Trains MACI and ftsC map in the region between min 3 and min 8. Two division =itants (strains ftsB and MX74T2ts52) have lesions between in 29 and min The division lesion in strain Y16 was designated ftsH by Santos ani Almeida (95) in accordance with the scheme of Ricard and Eirota (83). The map position of. the lesion in strain Y16 is min 61. Eclland aol Darby also designated the division lesion in their mutant, strain ASHI24, as ftsH which maps at min 80. This ftsH lesion is distinct from ftsP that was previously positioned at min 77.5. Presently, we are uncertain if the cell division lesion in strain 615A (min 64-67 on the :aylor and Trotter linkage map or min 72-74 on the revised map of Bacfmann et al.) is distinct from ftsE. Ricard and Hirota (88) have mapped three s:rains (MT99, i181, MT123) with the ftsE lesion between min 66 and min 6.




25
Complementation analysis beLwcen strain 615A and one or more of the ftsE strains would determine if the mutation in strain 625A is distinct from ftsE or within ftsE. These studies are complicated by a requirement for an intermediate temperature that is permissive for both the lowand high-temperature conditional strains and/or by the use of salt to phenotypically rescue the ftsE strains (MT99,MT123) at the permissive temperature for strain 615A.




SECTION II
BIOCHEMICAL CHARACTERIZATION OF TiE ENVELOPE IMPAIRMENT OF STRAIN 615A Introduction
The cell envelope of gram-negative bacteria such as Escherichia
coli is composed of an inner cytoplasmic membrane, a thin layer of peptidoglycan (murein),and an outer membrane (28,74,93,111). The outer membrane can be distinguished from the cytoplasmic membrane not only by its location but also by its functions and molecular composition. The outer membrane contains lipopolysaccharide and large amounts of four major proteins, including Braun lipoprotein. Although the outer membrane is not the major permeability barrier of the cell (71-75), it is primarily responsible for the intrinsic resistance of gram-negative organisms to antibiotics such as penicillin. However, the outer membrane is permeable to small hydrophilic molecules such as saccharides of molecular weights less than 550 daltons (19,71-73). Nakae has shown that the reconstitution of membranes permeable to small hydrophilic molecules requires the presence of an aggregate of phospholipids, lipopolysaccharide, and three outer membrane proteins in Salmonella typhimurium (71,73) or a single outer membrane protein in E. coli (72). Other outer membrane components serve as specific receptors for a variety of bacteriophages and colicins (18,25,82,83). In addition, the outer membrane contains little enzyme activity except phospholipase A1 (10) and lacks the electron transport system characteristic of the cytoplasmic membrane (64, 81).
26




27
Strain 615A is a low-temperature conditional cell division mutant that has been isolated and characterized by this author. During previous investigations, strain 615A was found to be more sensitive than its parent to deoxycholate and a variety of antibiotics. In addition, strain 615A leaked ribonuclease I, a periplasmic enzyme. These results suggest that strain 615A has an altered envelope. This section summarizes our results comparing the envelope components of strain 615A to its parent, strain X462.
Materials and Methods
Bacterial Strains
Strain 615A is a low-temperature conditional cell division mutant isolated from strain X462 following ultraviolet light mutagenesis. It grows as long, multinucleate filaments with 1-2 septa per filament at 30C and as short rods at 39C. Strain 71 is a transductant with normal cell division derived from strain 615A using a transducing lysate made from the parent, strain X462.
Media and Growth Conditions
Luria (L) broth (52) was used in most experiments. Minimal medium
(62) was used for experiments in which bacterial membranes were isolated. Both media were supplemented with 2 g of glucose per liter. When required, amino acids were added at a final concentration of 20 pg/ml.
Liquid cultures were grown overnight with forced aeration at 39C (the permissive temperature for strain 615A) in 22 X 175 mm culture tubes. Overnight cultures were diluted into fresh, prewarmed media and incubated at 39C until they were growing exponentially. These cultures were then used to inoculate fresh, prewarmed media for use in subsequent




28
experiments. Optical density of cultures was measured at 550 nm using a Spectronic 70 spectrophotometer.
Isolation and Comparison of Lipopolysaccharide
Exponentially grown cultures (200 ml) at 39C and 30C were harvested by centrifugation and lyophilized. Lipopolysaccharide (LPS) was extracted and purified as described by Westphal and Jann (1-14). After dialysis, nucleic acids were removed by enzyme digestion (55) and repeated ultracentrifugation (105,000 X g for 4 hours) until absorbance at 260 nm demonstrated less than 3% nucleic acid contamination.
Purified LPS was compared by thin-layer chromatography using silicic acid-impregnated glass-fiber paper (Gelmann Instrument Co., Ann Arbor, MI) as described by Buttke and Ingram (15). Comparison of Phospholipids.
Exponentially growing cultures at 39C were split, incubated at 39C and 30C with 4 pCi of 32P-orthophosphate per mland allowed to grow for 4-5 generations (final 0D550 = 0.5). Cultures were inactivated by the addition of trichloroacetic acid (TCA) at a final concentration of 5% and extracted overnight with chloroform-methanol as described by Kanfer and Kennedy (49). The lower chloroform layer (containing lipids) was removed and evaporated with dry N2. The dried lipid sample was redissolved in a small amount of chloroform-methanol (2:1) and applied to a silica Gel G plate (Analtech, Inc., Newark, DE). The chromatograms were run in a solvent system containing chloroform-methanol-glacial acetic acid (65:25:8) as described by G. Ames (5). Major phospholipid species (phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylethanolamine,and cardiolipin) were visualized with iodine vapors, scraped into vials,and the radioactivity determined.




29
Fatty Acid Analysis
Cells growing exponentially at 30C and 39C irere inactivated at
0D550 = 0.6 by the addition of' 5% TCA, harvested by centrifugation,and extracted into chloroform-methanol as described by Kanfer and Kennedy
(49). The washed lipid extract was transesterified in 27 H2S04 (v/') in methanol as described by Silbert,et al. (100). The fatty acid methyl esters were extracted into pentane and analyzed on a Tracor G;s Chromatograph (Tracor Instrument Co., Auztin,TX). The fatty acids were identified by comparison of retention times to those obtained with authentic fatty acid methyl ester standards. Isolation of Bacterial Membranes
Overnight cultures of strains X462 and 615A grown in minimal meiium were diluted 1/200 into fresh, prewarmed minimal media at 39C and gr:w-n until OD550 = 0.4. Arginine (2 pg/ml) was added to each culture and allowed to mix. Ten ml portions of each culture were incubated at 2C and 30C for 60 minutes. At this time, each culture was pulsed with either 14C-arginine (1 pCi/ml) or 3H-arginine (10 PCi/ml) for 10 minutes. To stop the incorporation of label,cultures were immersed in an ice lath and 20 ml of unlabeled carrier cells were added. Cells were centrif'ged at 3000 rpm for 10 minutes, washed once with cold saline,and then resuspended in 10 ml of cold 0.01 M Tris-HCl buffer, pH 6.8. The cells were broken using a Bronwill Biosonik III (Rochester, NY). Whole cells and debris were removed by a low-speed centrifugation and the supernatant dialyzed overnight at 4C against 100 volumes of 0.01 M Tris-HCl buffer, pH 6.8. The membrane fraction was collected in an ultracentrifuge at 90,000 X g for 60 minutes.
Isolation of Inner and Outer Membranes




30
The procedure used to isolate bacterial membranes and separate the
inner and outer fractions has been described in detail by Duckworth and Dunn (22). This procedure differs from the above procedure (isolation of total membranes) in several ways: (i) strains are labeled in minimal medium with tyrosine and leucine [strain 615A or 71 with 3H-amino acids; strain X462 with 14C-amino acids], (2) strains are harvested in late log phase rather than exponential phase,and (3) cells are broken by several passages through the French pressure cell rather than by a Bronwill Biosonik III tissue disrupter. In addition, the total membrane preparation was separated into outer and inner fractions by the selective solubilizatio' of the inner membrane in Triton X-100. Analysis of Membrane Proteins
The membrane preparations described above were prepared for electrophoresis by the method of Schnaitman (96) as described by Duckworth and Dunn (22). Gels were prepared according to the procedure of Maizel (57). These were sliced, solubilized and counted for radioactivity according to Duckworth and Dunn (22). Analysis of Bound Lipoprotein
The total membrane preparations described were also used to assay
for the amount of bound lipoprotein as described by Torti and Park (107). Analysis of Peptidoglycan
Exponentially growing cultures of strain X462 and strain 615A were harvested by centrifugation at an OD550 = 0.6. The pellets were resuspended in 25 ml of 0.1 M sodium phosphate buffer, pH 7.2,and heated at 60C for 30 minutes to inactivate autolysins. Cells were broken with 30 second pulses from a Bronwill Biosonik III tissue disrupter (Rochester, NY). Unbroken cells were removed by centrifugation (3000 rpm for 10




31
minutes) and the supernatant was centrifuged at 10,000 rpm for 20 minutes. The pellet (containing peptidoglycan) was resuspended in 5 ml of phosphate buffer and added dropwise to 30 ml of boiling 10% sodium dodec:! sulphate (SDS). This solution was mixed for 20 minutes more and then centrifuged at 18,000 rpm for 60 minutes. The SDS extraction was repeated and this pellet was resuspended in 10 ml of phosphate buffer :or)taining pronase at a final concentration of 100 pg/ml. After heatinthe sample overnight at 60C, the SDS was removed by dialyzing agains: 100 volumes of water for 2 days (2 water changes per day). A final pellet was obtained by centrifugation at 18,000 rpm for 60 minutes. These pellets were lyophilized and weighed; samples (10 mg) were hy'rosyzed in 5 ml of 4 N HCl at 105C for .11 hours. Each hydrolysate was flash-evaporated to dryness, washed with water to remove residual E~h, and resuspended in 25 ml of 0.01 N HCi. Amino acid analysis (65) was performed in a JEOL Model JLC-6AH (Japan Electron Optic LaboratorieS, Cranford, NJ).
Assay for Cross-linking In Vivo
Overnight cultures grown at 39C in L-broth were diluted 1/50 into fresh, prewarmed L-broth. Growth was allowed to continue at 39C until
3
cultures were growing exponentially. H-diaminopimelic acid (25 PCi/ml) was added to 2 ml cultures at 39C and 30C. After 2.5 hours, incorporation was stopped by the addition of 5% TCA. The samples were treated with trypsin and lysozyme and analyzed for cross-linking by descending chromatography as described by Kaniryo and Strominger (48).. Chemicals
All ainino acids were obtained from Calbiochem (San Diego, CA) 7r the Sigma Chemical Co. (St. Louis, MO). The following radioactive com--




32
pounds were obtained from Amersham/Searle Co. (Amersham, England): [G-3H] 2,6-diaminopimelic acid dihydrochloride (300 mCi/mmol), L-[U- IIIC arginine monohydrochloride (324 YrCi/rmol),and 32P-orthophosphate (81 Ci/mg P). L-[3H] arginine (7Ci/nmol), [ 14CItyrosine (460 mCi/mmol), S14C] leucine (309 mCi/mmol), [311] tyrosine (40 Ci/imol), and [3H] leucine (59 Ci/mmol) were obtained from Schwarz/Mann Co. (Orangeburg, NY). The scintillation fluid used was toluene-based Omnifluor (New England Nuclear, Boston, IA) in all cases except when processing samples of labeled membranes proteins.
RESULTS
Analysis of Lipopolysaccharide
Lipopolysaccharide (LPS) was purified from strains X462 and 615A and analyzed by thin-layer chromiatography (Fig. 15). This method can distinguish differences in LPS structure such as those seen in "rough" and "smooth" strains (15) as well as interspecific differences. Using this technique, no differences were observed in the mobility of the LPS extracted from strain X462 and strain 615A at the permissive or nonpermrissive temperature.
Analysis of Lipids
Major phospholipid components were extracted from strain 615A, strain x462 and strain 71, a transductant with normal cell division, and analyzed by thin-layer chromatography (Table 6). No significant differences among any of the strains were observed at either the permissive or nonpermissive temperatures. In addition, no significant differences in fatty acid composition were observed between strains X462 and 615A (Table 7).




33
Analysis of Peptidogly an d Cross-linking
Strains x462 and 615A were examined for qualitative and/or quantitative differences in peptidoglycan composition (Table 8). Cells of either strain grown at 30C or 39C contained approximately the same amounts of li-acetylglucosamine, N-acetylmuramic acid, glutamic acid, alanine,and diaminopimelic acid. Thus, there are no differences in peptidoglycan. composition between the parent and mutant strains.
Morphologically aberrant mutants of E. coli that synthesize either hypo- or hyper-cross-linked peptidoglycan at a nonperrissive temperature have been isolated(48). We examined the percentage of cross-linkage in strains 615A and X462 at both 30C and 39C (Table 9). However, no significant differences between the two strains were oberved at either temperature.
Analysis of Membrane Proteins
The division lesion in strain 615A does not appear to result from changes in LPS structure, phospholipid composition, fatty acid composition, peptidoglycan compositionor in the cross-linking of the murein. The proteins of both the inner and outer membrane fractions were isolated and analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 16). The data are plotted as differences profiles. A difference profile is obtained by subtracting the percentage of ll C-counts (strain X462) from the percentage of 3H-counts (strain 615A or 71) (after correcting for background and spillover in each gel slice) and plotting this difference versus the slice number (22).' No differences in the inner membrane protein composition between strains X462 and 615A were detected at either the permissive or nonpermissive temperature (Fig. 16A). However, the outer membrane protein profiles of the mutant (strain 615A) and the




314
parent, strain X462, at 30C differ in two regions (Fig. 16B): region a (molecular weight = 40,000) and region b (molecular weight = 7,000 10,000). The difference in region a did not appear to be involved in the division lesion of strain 615A because the difference was still apparent at 30C in strain 71, a transductant with normal cell division (Fig. 16C). However, the deficit in the region b protein(s) was absent in this comparison. Thus, the region b protien(s) was thought to be involved in the division defect in strain 615A. Lipoprotein is an envelope protein in the outer membrane with a molecular weight of 7200 daltons in its free form (11-13). This protein is the major component normally found in region b (11-13). Thus, it appeared that strain 615A might be deficient in the free form of lipoprotein. However, the technique used to prepare inner and outer envelope proteins utilized cells approaching stationary phase. To further explore the possible differences in free lipoprotein, membrane proteins labeled in exponentially growing cultures at 30C with 3H-arginine (strain 615A) and 14C-arginine (strain X462) were isolated and examined (Fig. 17). The two profiles are almost superimposable with only a slight difference in the area of free lipoprotein (region b). An average of three determinations revealed that strain 615A has approximately 17 more of its total membrane protein as free lipoprotein than does the parent. Thus, the small deficit in free lipoprotein originally observed in strain 615A grown at the nonpermissive temperature may not be indicative of the division defect. The differences in results between the two methods can be attributed to differences in growth conditions and sample preparation. Analysis of Bound Lipoprotein
Torti and Park (107) have isolated an E. coli mutant that has a




35
temperature-sensitive deficiency in bound lipoprotein. This mutant maps in the same region of the chromosome as strain 615A (min 72-75) and forms filaments at the nonpermissive temperature (107; personal communication). These studies prompted us to examine the amount of bound lipoprotein in strains 615A and X4162. Using their procedure (107), no significant differences in the amount of bound lipoprotein were seen at either the permissive or nonpermissive temperature (Table i0).
Discussion
Several mutants of Escherichia coli and Salmonella typhimurium that have increased permeability to dyes, detergents and antibiotics have been shown to have an altered LPS structure and/or a change in one or more of their buter membrane proteins (6,24,75,90,105,118). In addition, loss of components of the outer membrane by chemical removal (53,54,68) or by physical treatments such as freezing and drying or osmotic shock (7,31,33,84) result in the breakdown of the "natural resistance" of gramnegative bacteria to many inhibitory agents. However, another group of mutants believed to have mutations affecting their cytoplasmic membranes (38,89) have been shown to have increased permeability and sensitivity to deoxycholate.
Weigand and Rothfield (112) have recently isolated l mutants of Salmonella typhimurium that leak ribonuclease I. One of these mutants, lkyD, has a significant decrease in the bound form of lipoprotein with a corresponding increase in the free form of lipoprotein (113). In addition, this mutant is defective in the morphogenesis of the division septum, forming blebs of outer membrane at septal sites and failing to separate normally. E. coli mutants (envA or envC) morphologically




36
similar to the 1kyL Salmonella tpI hirmrium mutant have been described previously (61,78,91,92). However, the envA mulation does not result in a difference in the amount. of free and bound lipoprotein (113). In addition, the envA mutation differs from the Ilya in map position (77, 112) and by not leaking ribonuclease I. Recently, E. coli strains bearing the envA mutation were found to have a 6-fold reduction in the specific activity of the enzyme N-acetylmuraryl-L-alanine amidase (117). This enzyme defect leads to the formation of chains of unseparated daughter cells. Another cell wall enzyme, carboxypeptidase II, has
been implicated as the lesion in BUG6, a high-temperature conditional cell division mutant of E. coli (9). Upon shifting to the permissive temperature, there is a rapid resumption of division, accompained by a 10-fold increase in the specific acitivity of carboxypeptidase II.
E. coli strains that carry the envC lesion also form chains of
daughter rods. The envC mutation not only confers sensitivity to deoxycholate and antibiotics such as penicillin, D-cycloserine and rifampicin, but also results in an alteration of the phosphatidylglycerol/cardiolipin ratios in the inner and outer membranes (61,91,92).
Strain 615A appears to have an envelope defect as evidenced by its increased sensitivity to deoxycholate and antibiotics. The leakage of ribonuclease I at the nonperinissive temperature is consistent with the defect being outside the cytoplasmic membrane and probably in the outer membrane. Our investigation of the different envelope components in strains X462 and 615A failed to reveal differences in LPS structure, phospholipid composition, fatty acid composition, peptidoglycan composition, percentage of cross-linking ,or in the amounts of bound lipoprotein at either the permissive or nonpermissive temperature. In addition, no




37
significant differences were detected by SDS-polyacrylaide gel electrophoresis of inner and outer membrane proteins. However, the envelope defect could be a result of a chang( in the outer or inner membrane proteins which is obscured within a particular molecular weight class. Many of the bands observed in SDS gels are known to be composed of more than one protein (97). Alternatively, a particular membrane protein could be altered by a single amino acid substitution rendering its activity cold-sensitive. Such an altered protein would remain undetected by SDS gels unless produced in abnormal waounts.
Nikaido has recently proposed that there are at least two pathways for the diffusion of small molecules across the outer membrane, one for hydrophilic compounds, and one for hydrophobic compounds (75). The hydrophylic pathway is unaffected by the structure of LPS present in the outer membrane. The hydrophobic pathway is almost inactive in wild type strains producing complete 12S and becomes fully active only in deep rough mutasts or EDTA-treated cells where phospholipid bilayer regions are exposed. The hydrophylic pathway involves protein aggregates in the outer membrane which are presumed to form pores (19,71-73). Strain 615A has increased permeability to both hydrophilic and hydrophobic compounds as evidenced by its increased sensitivity to a variety of antibiotics and deoxycholate. Thus, it seems likely that strain 615A has an alteration in its outer membrane organization affecting both pathways.
Other possible differences in envelope structure which were not
eliminated by this investigation include: (1) quantitative differences in the amounts of LPS or minor changes in the LPS structure of the mutant,
(2) differences in lipid composition between the inner and outer membrane, and (3) differences in activities of autolytic enzymes thought to be involved in the division process (9,63,1i7).




38
Table 1. Bacterial strains.
STRAIN SEX GENOTYPE SOURCE X462 F- ara- leu- azi tonA- proA- lacZ tsxr A. C. Frazer and R.
purE- ysA strr 2 i mtl- metE Curtiss III
thi
x462mg F- ara- leu- azi r tonA proA lacZ- tsxr This paper
purE_ A- lys r tl eE
pE str mtl-metE
thi- malA615A F- ara- leu- azi r tonA =(IA- lacZ- tsxr Derived from x462;
___ str _VJ _tl- otE low-temperature conthi- ditional for division 615Am F- ara- len- azi r tonA- proA lacZ- tsxr This paper; low ter,purE ljysA- strr xvl- mtl- metE- perature conditional
thi- malA- for division AB2847 F- aroB- tsx ma.- supE(?)X r X- J. T. Park
U482 Hfr asd- thi- rel- A- J. T. Park LW3 F- L7 thi- J. E. Cronan LW3m F- f .- thi- malA- This paper CSH147a, Hfr sup J. Miller strain kit CSH60 H1fr s J. Miller strain kit CSH6h Hfr thi- J. Miller strain kit CSH67 Hfr lac- gl: .gi mtl- malA- thi' X- J. Miller strain kit CSH74 Hfr thi- J. Miller strain kit




39
Table 2. Antibiotic disc assays.
Antibiotic (Conc.)
Kanamycin Bacitracin Rifampin Nalidixic Acid Strain Tep.- (30 _) (10 Units) (5 Pg) (30 yg)
X462 39C 10.5, 15a 6 10 10, 18 x462 30C 9, 15 7 10.5 17, 23.5 615A 39C 14, 18 6 10 10, 20 615A 30C 20 14 15 25 Rev4 39C 11, 17 6 9.5 16, 18.5 Rev4 30C 10, 16.5 7 10 17 .ev8 39C 11, 17 6 9 16 Rev8 30C 10.5, 16 7 10 18 Trans4x 39C 9.5, 14 6 9 9, 19 Trans4x 30C 9.5, 16 6.5 10 14, 18.5 Trans~l 39C 10, 14 6 8.5 8.5, 17 Trans7l 30C 10, 15.5 7 10.5 10, 19 aTwo zones of inhibition were apparent. An outer zone with slight turbidity was present surrounding the clear inner zone. Strains Xh62, 615A, revertants (Rev4,8) and transductants (Trans4x, 71) were grown overnight in L-broth at 39C. Approximately 4 X 106 bacteria/ml of each culture was diluted into 100 ml sterile, precooled L-agar. 15 ml of the seeded agar was pipetted into petri dishes. After the agar had hardened and dried for 2-1/2 hours, anitibiotic discs (i per plate) were placed in the center and the plates were incubated at either 39C or 30C for 18 hours. Diameter of zones of inhibition was measured in mn (diameter of each disc is 6 i).




40
Table 3. DNA per mass ratios of strain X462 and strain 615A.
ST AIN 1IPERATUREa DNA/1JMSSb
X'462 39C 2577 615A 39C 2699 X462 30C 1882 615A 30C 2075
a nis is the temperature at which the culture was uniforrily labeled (Figures 7 and 12). bDNA/mass ratio is the average cpm/OD between the OD 550 range of
0.1 to 1.0 (Figures 7 and 12). 550/550




Table It. Results of Hfr and 615A uninterrupted matings.
% Coinheritance
Hfr Selected Number Unselected Characters Strain Marker Analyzed leu purE jetE lysA Ll f ilanentation
CSH6o leu 90 100 --- 0 ... ... 0 CSH74 lyssA 90 --- 0 --- 100 --- 0 CSH67 metE 90 --- 0 100 0 --- 8.9 CSH47a l 90 --- 0 0 11 100 49 CSH64 xyl 90 10 --- 42 0 100 69




42
Table 5. Transductional analyses.
Recipient Selected Number of % Cotransduction of Unselected Marker
Strain Marker Transductants malA _D fila-ment formation Xh62r n malA 179 ---- 98.9 19.6 X462mg 177 92.7 ---- 9.0 LW3rn imalA 180 ---- 89.0 33.0 LW3m glpD 179 87.7 52.0 AB2847 aroB 158 .... 21.5




43
Table 6. Phospholipid composition in strains X462, 615A and 71.
STRAIN GROWTH PERCENTAGE OF TOTAL TEMPERATURE CL PG PE LE X462 39C 0.90 17.2 76.9 5.00 615A 39C 0.80 17.1 77.5 4.70
71 39C 0.77 14.9 77.2 7.10
X462 30C 2.00 15.9 76.2 6.oo 615A 30C 1.90 15.4 79.2 3.60
71 30C o.9o 16.2 8o.6 2.60
CL = Cardiolipin
PG = Phosphatidylglycerol
PE = Phosphatidylethanolamine
LE = Lysophosphatidylethaolamine




44
Table 7. Fatty acid composition in strains X462 and 615A.
PERCENTAGE OF TOTAL
GROWT! _ __ _ _ __ _ _
STRAIN TE KPERATURE 12:0 14:0 16:o i6:1 A17 18:1 x462 39C 1.42 2.37 47.9 24.0 4.07 20.4 615A 39C 2.46 2.46 48.7 24.9 4.12 18.8 x462 30C 2.13 2.25 36.2 30.9 28.5 615A 30C 1.14 3.49 37.1 33.0 27.4




Table 8. Analysis of peptidoglycan in strains X462 and 615A.
Amino Acid or Amino Sugar in rnmol/mg (Ratia ) Growth DiaminopiStrain Temperature Glucosamine Muramic Acid Alanine melic Acid Glutamic Acid
615A 39C 525.3(.81) 606.3(.93) 1172.4(1.8) 646.5(.99) 625.5(1.0) 615A 30C 558.7(.82) 677.3(.99) 1167.6(1.7) 649.7(.95) 684.3(1.0) x462 39C 6o8.9(.86) 690.7(.97) 1166.8(1.6) 702.4(.99) 711.6(1.o) X462 30C 532.5(.80) 611.o(.92) 1096.0(1.6) 596.7(.90) 666.5(1.0) aRatio = nol/m amino acid or amino sugar
nmol/mg glutamic acid




46
Table 9. In vivo cross-linking analysis of peptidoglycan in strains
x462 and 615A.
Growth
Strain Temperature dimer/monomer
615A 39C .88
615A 30C .80
X462 39C .81
X462 30C .72
aThe dimer or cross-linked species of lysozyme-digested peptidoglycan migrates with
an Rf = 0.2 when applied to Whatman no.31M
filter paper and subjected to descending chromatography in isobutyric acid-iN ammonium hydroxide (5:3). The monomer or uncross-linked species migrates with an
Rf =0.4 in this system.




Table 10. Analysis of bound lipoprotein in strains X462 and 615A.
Growth
Strain Temperature Percentage of Boid Lipoproteina
615A 39C 5.0 615A 30C 4.5 x462 39C 4.4
X462 30C 4.3
aEach sample was normalised by comparing each bound lipoprotein value to total envelope counts applied to the chromatogram. This assay is based on the observation 'that bound (murein-linked) lipoprotein can migrate on
chromatograms in an isobutyric acid-i N ammonium hydroxide
solvent after it is released from murein by lysozyme
digestion.




Figure 1. Linkage map of Escherichia coli K-12 showing the origins and directions of transfer of
Hfr strains (arrows).




/-. ,. H47a ,rCSH" gll t" CSH74,




Figure 2. Micrographs of strain 615A, a representative low-temperature
conditional cell division mutant and its parent, strain X462.
A, filaments of strain 615A at 30C after 2 hours; B, strain
615A 2 hours after shifting filaments (2A) to 39C; C, nuclear
stain of strain 615A after 2 hours at 30C; D, strain x462
at 39C, for comparison. Bar represents 10 p-meters.




51




Figure 3. Cell division following a shift from 39C to 30C. Cultures of strain 615A (A) and
strain X462 (B) growing exponentially at 39C were split and one-half of each culture
was incubated at 30C. Increase in cell number was monitored for 3-1/2 hours following
the shift to 30C. Symbols: ( @ ) 39C control culture, ( 0 ) 39C-30C shift culture.




300 A 100 B
100/9/ 4( /"
0 0
n / Ln
0 00 0 "
T- 4
X X (0 201 8/ 7
/ A ,
1 30 90 150 210 0 0 150 210 TIME (min) TIME(min)




Figure 4. Viability of strain 6l5A following a temperature shift from 39C
to 30C. An exponentially growing culture of strain 615A at
39C was split and one-half of the culture was incubated at
30C. Viability was monitored for 3 hours following the shift
to 30C by plating samples on L-agar and incubating the plates
for 18 hours at 37C. Symbols: ( 0 ) 39C, ( 0 ) 30C.




VIABLE CELLS X 105
cm 0 9
0 0
0
00
0
OD
Or-




Figure 5. Cell division following a shift from 30C to 39C. Filaxents of strain 615A were induced by growth for 2 hours at 30C. Cultures of strain 615A (A) and strain X462 (B)
at 30C were split and one-half of each culture incubated at 39C. Increase in cell number was monitored for 2 hours following a shift to 39C. Symbols: ( ) 30C-39C
shift culture, ( 0 ) 30C control culture.




CELL NUMBER X 106
C)
CELL NUMBER X 106
-In
0
FlN




Figure 6. Growth of strains 615A and X462 in the prresence and absence of deoxycholate. Deoxycholate (0.25%) was added to exponentially growing cultures at 30C (A) and 39C (B)
and absorbance at 550 nm was monitored for 2-1/2 hours. Syribols: ( 0 ) strain
X462, ( 0 ) strain X462 + deoxycholate, ( M ) strain 615A, ( C ) strain 615A + deoxycholate.




A 2.5 B
E /* C1.0 E 1.OLU 10
C) U) ,I < 0. 9 0) /M.0 E)- 0.0 15
030 90 150 090 190
TIME(MIN) TIME(miN) V




Figure 7. Effects of a shift from 39C to 30C on growth and DNA synthesis
in strains 615A (A) and x1462 (B). Cultures were uniformly prelabeled at 39C with 3H-thymidine, split and one-half of
each culture was incubated at 30C. Absorbance of each culture was recorded and. duplicate samples were processed for measurement of DNA synthesis. Symbols: ( 0 ) increase in absorbance
at 39C, ( 0 ) increase in absorbance at 30C, ( a ) 3H-thymidine
incorporated into DNA at 39C, ( 0 ) 3H-thymidine incorporated
into DNA at 30C.




2.0- A
If)
()
04
0 0
1 0.4- o
010
50
cyU0)0
ElI
U-.-
:z 4.0-7 s)o
000
1.C V 0 30 60 90 120
TIME(min)




62
2.0
E
0
LO1.0
Lo
0 0
8 00
6 0
0..7
0
X wool 0----ooo Y-5.0'
0 30 60 90 120
I IME(min)




Figure 8. Pulse-label measurements of DNA synthesis in strains 615A (A) and X462 (B) f'oloving
a shift from 39C to 30C. Exponential cultures at 39C were split and one-half of each culture was shifted to 30C. Samples were removed and oulsed for h minutes. Symbols:
( ) incorporation of 3H-thymidine at 39C, ( ) incorporation of 3H-thymidine at 30C.




70 A 150 B
20- 400
* 00
z Iq
()X
0
20. =- = 4(
0120 0 60 120
TI ME(min) TI ME(min)




Figure 9. Pulse-label measurements of RNA synthesis in strains 615A (A) and X462 (B) following
a shift from 39C to 30C. Exponential cultures at 39C were split and one-half of each culture was shifted to 30C. Samples were removed and pulsed for 4 minutes. Symbols:
( ) incorporation of 3H-uracil at 39C, (0) incorporation of 3H-uracil at 30C.




400 A 300 B
[
/
100 / 100
CN 0
/ /1
//*
101p a
0 60 120 0 60 120
TIME~min) TIME(min)




Figure 10. Effect of chloramphenicol on induced division of filaments
of strain 615A. Filaments of strain 615A were induced in Lbroth at 30C for 2 hours and subdivided. At time zero, all
but one subculture, 30C control with no additions, were shifted
to 39C. Chloramphenicol (CM) (100 pg/ml) was added at various
times after the shift to 39C. Increase in cell number was
monitored using an electronic particle counter. Symbols:
(0) 30C control with no additions, (*) CM at 15 minutes,
(*) CM at 30 minutes, ( U) CM at 45 minutes, ( D ) CM
at 60 minutes, 0 ) 39C control with no additions.




CELL NUMBER X 106
--O
0 I
A&
mU ell" II *I OR ~
01 0 ON




Figure 11. Summary of the effects of antibiotic additions on induced cell
division of strain 615A. The data are expressed as the percentage of the control (39C control with no additions) increase in cell number 75 minutes after the shift of filaments from 30C to 39C versus the time of addition of the antibiotic. Symbols:
( ) puromycin (330 Vg/ml), ( 0 ) chloramphenicol (100 pg/m-),
( U ) nalidixic acid (10 pg/ml), ( 0 ) penicillin (25 Units/ml),
(* ) rifamycin SV (200 pg/ml).




PERCENTAGE INCREASE IN CELL NUMBER
mo c 000
UnI




Figure 12. Measurements of growth and DNA synthesis in strains 615A (A)
and X462 (B) following a shift from 30C to 39C. Cultures were uniformly prelabeled with 3H-thymidine at 30C during
growth and the induction of filaments (strain 615A). Cultures
were split at time zero and one-half of each culture was
shifted to 39C. Absorbance of each culture was recorded and duplicate samples were processed for measurement of DNA synthesis. Symbols: ( 0 ) increase in absorbance at 30C, ( )
increase in absorbance at 39C, ( 0 ) 3H-thymidine incorporated
into DNA at 30C, ( 0) 3H-thymidine incorporated into DNA at
39C.




T2
2.0 A
o o
E 7
Zo04 0.
<0
C)
m
< Aoo
0.1 ,
50f
15 a
X /m0m o 0,0,U0 a-4.01.0 30 60 90 120
TIME(min)




73
2.0 B 0"0
c 0p
0
>< ox .000,10 C.) 0. 0/0
Z 0.4-1l
x 0000
I m/
00
,mm
0 30 60 90 120 T IME(m in)




Figure 13. Phospholipid turnover during induced division in strain
615A. Filaments were prelabeled with 32p-orthophosphate, washed and incubated in fresh growth medium at 39C (solid
lines) and 30C (dashed lines) at time zero. Symbols:
( ) lysophosphatidylethanolamine, (0 ) phosphatidylethanolamine, ( U ) phosphatidylglycerol, ( D ) cardiolipin.




PERCENTAGE OF TOTAL PHOSPHOLIPID
- V
I'I|
-iti II
I I0
L- I60
L1tl




Figure 14. Pulse-label measurements of phospholipid synthesis in strains 615A (A) and X462 (B)
following a shift from 30C to 39C. Cultures were grown at 30C with the induction
of filaments in strain 615A. At time zero, the cultures were split and one-half
of each culture was shifted to 39C. Samples were removed and pulsed for h minutes.
Symbols: ( 0 ) incorporation of 32p-orthophosphate into phospholipids at 30C,
0 ) incorporation of 32p-orthophosphate into phospholipids at 39C.




150 A 200
AB 0
0
CN
CN 0
040 0 X 50
X E 0
a.. o 0
00 0
0 60 120 0 60 120 TIME(min) TIME(min)




Figure 15. Thin-layer chromatography of LES isolated from strain x462
and strain 615A, Chromatography of whole LPS from cultures
of strain x462 and 615A grown at 30C or 39C was identical.




LO
CD
OL




Figure 16. Difference plots of membrane protein profiles from strains X462, 615A and 71. A,
Comparison of inner membrane proteins from cultures of stain x462 and strain 615A
grown at 30C; B, comparison of outer membrane proteins from cultures of strain x462
and strain 6l5A grown at 300; C, comparison of outer membrane proteins from cultures of strain X462 and strain 71 grown at 30C. The positions of Regions a and b are assigned by comparison of membrane protein profiles before the difference plots are generated. The bottom gel (C) has fewer slices because this gel did not electrophoresize as far as the other 2 gels.




A
".2
. B a b
0 A
.4'.4a b
0
0 V m" A-- v A y 4: "1-,-I v """
0 20 40 60 80 SLICE NUMBER
Co
H




Figure 17. Comparison of membrane proteins from cultures of strain X462 (dashed line) and
strain 615A (solid line) grown at 30C. Membrane proteins of strain 615A labeled
with 3H-arginine and membrane proteins of strain X462 labeled with 14C-arginine
were electrophoresized on SDS-polyacrylamide gels. The gels were sliced, the
radioactivity in each gel slice determined and plotted.




100
80
S60 uj
>40 < 'i
rr-20\
'IVI
N .
O0 10 20 30 40 50 60 70
.SLICE NUMBER
co




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Full Text

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LOW-TEKPZfl.,\TFR E CONDITIONAL CELL DIVISION MUTANTS OF ESCHERICHIA COLI By JOYCE ANNE S'.I'm:GEClN A DISSEli'l'ATIO:N PP.ESE:t.TJ'ED TO THE GRADUATE COUNCIL OF TEE u~HVERSITY OJ' FLORIDA JJ-l" PAP.TIA L FULFILLMENT OF THE REQUIREME.l1.T'!1 S FOR THE .:;)EGFE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF r LORIDA 1977

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ACKNOWLEDGMENTS The time and effort that I have invested in my work and the -wri ti:ng of this dissertation are really the products of several people that are close to me. :My parents helped to shape my attitudes and study r.e.bits from an e arly age by never appearing bored whenever I asked questions. My husband helped to develop my confidence and provided. ad.vice and er_ couragement when problems seemed unsurmountable. I dedicate my love :::.nd this dissertation to these three people. I want to acknowledge and thank the entire faculty, staff,and gr":.duate students of the Department of Mkrobiology and Cell Sdence for their friendship. I will sorely miss the conversations and compa n i o~ship inherit in this department. I have worked in the laboratories of Drs. Achey, Duckworth, Pre~~c, Preston, Duggan, Smith,and Bleiweis. I would like to specifically tt:::.nk all of these people for the use of their facilities and space. In a5.-di tion, special tharJ~s goes to Glenda Dunn for her excellent instruc~ fons on several techniques, most notably disc gel electrophoresis. Steve Hwst was most helpful in his explanations of the amino acid analyze~. Ed Hendrickson enlightened me with his experience of E. coli genetics. Both Dr.Aldrich and Bill Doherty have provided electron micrographs cf my cell division mutant. Tom Buttke was particularly kind to do the lipopolysaccharide analysis of strains x462 and 615A. Pam Spoutz an;i Dixie Donovan always provid~d me with the essentials of bacterial lEemedia and culture tubes. Most recently, Tom Hallam and John Silber ::'.l.ve ii

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introduced me to the technique of sucrose gradient centrifugation.and some special equipment in Dr. Acbey s laboratory. My committee members,Drs. Aldrich, Bleiweis, Duckworth, Duggan, and Hoffmann, have provided many help:1-'ul comments on my work and the writing of my dissertation. My warmest thanks, however, goes to Dr. Ingram, the chairman of my crn.:Jill..i.ttee, for his enthusiasm, patience ,and continual support. Finally, I want to thank tl1.ree special friends for their advice and encouragement throughout my graduate career--Nola Masterson, Carolyn Napoli ,and Ru.th Rmya.ni toff. iii

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TABLE O F CONTENTS Page ACKJ-IOWLEDGMENTS .. ii LIS'l' OF rrABLES .............. V LIST OF' FIGUP.ES vi ABSTR.AC'l' viii GEJ\i'ERAL INTRODUCTION 1 PHYSIOLOGIC JiJID GENETIC CHARACTERIZATION OF LOW-TEMPERATURE CONDITIONAL DIVI SION MUTANTS OF ESCHERICHIA COLI........... 3 Introduction. . . . . . . . . . . . 3 Materials and Methods. ... . . . . . . . . 4 Re s uJ.ts.................................................. 11 Discussion............................................... 19 BIOCHEMICAL CHARACTERIZATION OF THE ENVELOPE IMPAIRMENT OF STRAIN 615A. 26 Introduction. . . . . . . . . . . . 26 Materials and Methods .......................... : . . 27 Results.................................................. 32 Discussion. . . . . . . . . . . . 35 LITERA'l'URE CITED. 84 BIOGRAPHICAL SKETCH. 94 iv

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LIS'l' OF T.AJ3r.,ES 'l'able Page 1 Bacterial strains....................................... 38 2 Antibiotic disc assays .................................. 39 3 DNA per mass ratios o f s train x462 and strain 615.-'-.. . 40 Results of Hfr and 615A uninterrupted mati!"',gs........... 41 5 Transductional analyses. . . . . . . . . 42 6 Phospholipid comoosi tion in strains x462, 615A ar.:i 71... 43 7 Fatty acid com.position in strains x462 and 615A......... 44 8 Analysis cf peptidoglycan in st:rains x462 and fl;.-\....... 45 9 In vivo cross--linking analysis of peptidoglycan :.:1 strains x462 and 615A.. . . . . . . . . 46 10 Analysis of bound lipoprotein in strains x462 anc. 615A.... . . . . . . . . . . 4 7 V

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Figure 1 2 3 4 5 6 7 8 9 10 11 LIST OF FIGURES Linkage map of Escheri_chia coli K-12 showing the origins and d.irections of t1ansfer of Hf'r strains ( ar:cows) ............................................. Mj_crographs of straoi_n 615A, a rcpresentati ve lo'..rtemperature conditional mutant and its parent, strain x462 ................................................. Cell division following a shift from 39C to 30C ...... Viability of strain 615A following a temperature shift from 39C to 30C ................................ Cell division following a shift from 30C to 39C ...... Growth of strains 615A and x462 in the :9resence and absence of deoxychola~e ............................. Effects of a shif't from 39C to 30C on growth and DNA synthesis in strains 615A (A) and x462 (B) ......... Pulse-label measurements of DNA synthesis in s-:rains 615A (A) and x462 (B) following a shift from 39C to 30C .................................................. Pulse-label measurements of RNA synthesis in strains 615A (A) and x462 (B) following a shift from 39C to 30C .................................................. Effect of chloramphenicol on induced division of filaments of strain 615A ............................. Summary of the effects of antibiotic additions on ind~ced cell division of strain 615A ................ 12 Measurements of growth and DNA synthesis in strains 615A (A) and x462 (B) following a shift from 30C to 39C .................................................. 13 Phospholipid turnover during induced division in Page 51 53 55 57 59 61,62 64 66 68 70 72,73 strain 615A........................................... 75 vi

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Figure 14 15 J.6 17 LIST OF FIGURES--CONTINUED Pulse-label measurements of phospholipid synthesis i n strains 615A (A) and x462 (B) following a shift from 30C to 39C .................................... Thin-layer chromatography of LPS isola.te d frbm strain x462 and strain 615A ................................ Difference plots of rriembrane protein profiles from strains x462, 615A ar. d 71 ........................... Comparison of membrane proteins from cultures of strain x462 (dashed line) and strain 615A (solid line) grown at 30C ......................... ; ........ vii Page 77 79 81 83

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Abstract of Dissertation Pr~sented to the Graduate Council of the University of Florida in Pe, r tia. l Fu_lfillm:nt of the R eq_uirements for the Degree of Doctor of P hilosophy LOW-TEMPERA'I'URE CONDITIOIJAL C ELL DIVISIOX MUTANTS OF ESCHERICHIA COLI By Joyce Anne Sturgeon June 1977 Chairman: Lonnie O. Ingram Major Department: Microbiology and Cell Science Fifteen low-temperature conditional division mutants of Escherichia coli K-12 have been isolated. These grow normally at 39C and form filaments at 30C. AJ.l exhibit a coorc'limi ,ted burst of cell division upon exposing filrunents to the permissive temperature. None of the various agents which stimulated cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability or stimulating cell division in broth. One of these mutants, strain 615A, appears to have an altered cell envelope as evidenced by its increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonuclease I, a periplasmic enzyme. However, no significant differences between the parent and mutant strains are observed in lipopolysaccharide structure, phospholipid composition, fatty acid composition, peptidoglycan composition, or in the percentage of cross-liIL~ing. In addition, no differences are detected between the two strains in the inner and outer membrane protein compositions. This mutant has normal rates of deoxyribonucleic acid synthesis, ribonucleic viii

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acid synthesis, and phospholipid synthesis for division of filaments at the permissive temperature. However, strain 615A requires new protein synthesis in the apparent absence of new ribonucleic acid synthesis for division of filaments at the permissive temperature. The division lesion in strain 615A is cotransducibJ.e with malA, a.ro~, and ~]2Q_, and maps within minutes 72-75 on the E. coli chromosome. ix

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GENERAL INTRODUCTION Cell division in bacteria is a complex process involving the coordination and regulation of deoxyribonucleic acid synthesis, nuclear segregation, septation,and the physical separation of newly formed daughter cells. One method of studying bacterial cell division is the isolation cf mutants blocked in different recognizable aspects of the cell cycle. The majority of division mutants isolated and characterized in Escherichia coli have been high-temperature conditional cell division mutants (2,4,20,35-37 ,41r,67 ,69,85,108,115,121). A number of genetic loci involved in cross-wall f ormation has been identified using these mutants (3,30,36,37,41,60,88,95,l08,109,120). In an effort to discover new and different loci as well as learn more about the roles of existing loci in the division process, we have isolated low-temperature conditional division mutants in E. coli K-12. The main thrust of our efforts has been the physiologic and genetic characterization of one of these mutants, strain 615A. Section I contains these results as well as a comparison of the phenot;r~ic and genotypic properties of our mutants with previously isolated division mutants. The isolation and characterization of strain 615A has possibly revealed another locus involved in cell division in! coli (Section I). This division lesion appears to involve an impairment in the integrity of the envelope as evidenced by the increased sensitivity of strain 615A to deoxycholate and antibiotics as well as leakage of ribonuclease I, a periplasruic enzyme. It seemed logical to investigate the nature of l

PAGE 11

2 this envelope lesion by systematically analyzing the major co:iponenc:s of the envelope. Section II is the results and discussion of our effo:cts. The regulation of cell division is a fundamental precess of all living organisms. Currently very little is understood about t he re~.11.ation of any step in the cell division process of any organism Thus, the elucidation of the division process remains one of the most imp-:~tant areas of investigation in molecular biology today.

PAGE 12

SECTION I PHYSIOLOGIC AND GENETIC CHARACTERIZATION OF LOW-'l1EMPERATURE CONDITIONAL DIVISION MUTAl\JTS OF ESCHERICHIA COLI Introduction Ce11 division in bacteria is a complex process involving the coordination and regulation of DNJ1. replication, nuclear segregation, septation ,and the physical separation of newly formed daughter cells. Jones and Donachie (47) have e~visioned the cell cycle of Escherichia coli as involving two parallel series of events: chromosome replication and the synthesis of division proteins. Both series are seen as capable of proceeding independently to produce products which must act in a coordinate fashion leading to cell division. DNA synthesis, nuclear segregation, RNA synthesis,and prote~n synthesis are capable of proceeding normally in the absence of cell division (4,16,20,35,36,41,60,67,85,88, 109,121). However, a strict requirement for an event related to DNA syntnesis has been observed for division except under very unusual conditions where DNA-less cells are produced (l,35-37,39,li4,45). One method of studying bacterial cell division is the isolation of mutants blocked in different recognizable aspects of the cell cycle. Nmethyl-N'-nitro-N-nitrosoguanidine has been frequently employed by many investigators (2,4,20,67,69,85,92,103,108,115,121). It suffers from the disadvantage of inducing multiple, closely linked mutations (62). Using this mutagen, a variety of high-temperature conditional cell division mutants (2,4,20.,35-37,44,67,69,85,108,115,121) have been isolated. Such 3

PAGE 13

4 mutants are generally accepted as resulting from rnissense mutations which lead to the production of less stable proteins unable to function properly at the elevated temperature ( 32 ,8). However, this interpretation is not as readily applicable to "cold--sensitive mutants"(llO). Thus, it seemed possible th3.t the isolation and characterization of low-temperature conditiona l cell division mutants might reveal new and different loci involved in the cell division process. This paper describes the physiology and genetic lesion in a UV-induced "cold-sensitive" mutant of Escherichia coli K-12. Materials and Methods Bacterial Strains The bacterial strains and their genotypes are listed in Table l. In addition, the approximate origins and direction of transfer of the Hfr strains are shown in relation to the genetic markers of the parent strain (Fig. 1). Genetic designations are according to Taylor and Trotter (106). ~.lap positions of amino acids, sugars,and antibiotics are in accordance vith the revised linkage map of Bachmann, Low, and Taylor (8). Whenever possible, the revised map positions will be used. However, in discussing the genetic lesions in division previously described, we have used the former linkage map. Bacteriophage Pl. was part of the strain vir kit obtained from Cold Spring Harbor Laboratory (Cold Spring Harbor, NY). Strains x462mg, 615Am,and LW3m are rnalAderivatives of strains x462, 615A,and LW3, respectivel;Y. The malAlesion was introduced into these three strains by 2-aminopurine mutagenesis (62) and subsequent selection of malcolonies on MacConkey agar supplemented with 0.2% maltose. The~lesion was introduced into strain x462malAin an

PAGE 14

analagous f ashion. M edia 5 Dix and H e lmst ette r minimal medium (HM) (21) and Lur i a ( L ) brot::. (52) w e r e u sed f o r isolation, enrichment,a nd subsequ e n t expeTiments i n volvi n g the low-temper ature conditiona l c ell d i vision rmt ants Bott -..rere supplemented ,Ii th glu cose (2 g/1) a nd thiamine (10 mg/ 1). L--arnino c:.dds w ere a dd e d t o min imal medi a a t a final c o nc e n t r a tion of 40 g/ml. -Strept o myci n sulfate (100 g/ml) w a s used as a do no r c ountersel ectiT = agent in mating experiments. Solid mi n imal m e dium was prepared by mixing equal volur:es of 2::,: Dix and Helmstette r minimal medium and 2X agar (1.5% f i nal c cncentrc:.-:ion) after autoclaving L--agar was p repare d by adding agar to L--jroth a ~ a final concentratio n of l. 5 % b efore autoclaving. L--NaCl or !,'.::,!-NaCl r::::i i a were pre p a r e d b y adding 15 g o f NaCl p e r liter to the above ::--ecipes ::;:rior to autoclaving L-sucrose and MM-sucrose media w e r e prepared by ad~ng sucrose at a final concentration of 10% to the aga r s olution s after autoclaving s eparately. L-deoxycholate and MM-deoxycholate jedia c ~2-taine d 0.25% sodium deoxycholate. Selective plates for all genetic experiments were prepared as described by Miller (62). Growth Conditions Liquid cultures were grown in 22 X 175 mm tubes w ith c c~stant,:orced aeration. Overnight cultures gro~'Il at 39C were diluted into fresh, prewarrr.ed medium. Incubation was continued at 39C until cultures .. e.re growing exponentially. Studies involving temperature shifts plus c~ minus various a gents were generally conducted as follows: filaments vere induced by growth at 30C for 1-1/2 to 2 hours in L-broth or 3 to 4 '::curs in MM. Thes e cultures along with appropriate controls were then sr::.it,

PAGE 15

treated with various agents,and incubated at 30C and 39C. Bacterial cell numbers w ere determined with a Celloscope electronic particle counte r (orifice, 24 1.11n; amplifi.cation, 1; electro zone, 24; lower threshold, 18). Optical density of bacterial cultures was mea-sure d at 550 nm using either a. Spectronic 70 spectrophotometer or a Beckman Model 25 spectrophotometer. Isolation and Enrichment 6 'l'he low-temperature conditional cell division mutants were originally derived from E scherichia coli K-12, strain x462 (Table 1), a generous gift from A. C. Frazer and R. Curtiss III. A log-phase culture was mutagenized with ultraviolet light (1.24% survival) and allowed to grow overnight in the dark at 37C, Thermosensitive cell divisio n mutants were enriched using a procedure modified fro m van de Putte et al. (108). The mutagenized culture was :initially diluted 1/20 and allowed to reach an OD550 between 0.4 and 0.6 (log phase) at 37C. This culture was then filtered through a 3 pore size nuclepore filter (Nuclepore, General Electric). The filtrate, containing small cells, was used as the inoculum into fresh, prewarmed broth at 30C (J./20). After this culture had reached an OD550 between 0.4 and 0.6, 10 ml was filtered and washed with an equal volume of fresh medium through a 5 pore size nuclepore filter. 'I'he filter, containing filamentous cells, was used to inoculate a fresh culture tube at 37C. This description of the filtration procedure constituted one cycle; five such cycles of filament enrichment followed before dilutions of the i.st c~lture at 37C were plated onto Ir-agar for isolated colonies. At no time throughout the enrichment procedure were the cultures allowed to reach late log to stationary phase. 'l'his was done to ensure that a physiologically uniform period was employed during

PAGE 16

both filament formation and stimulated cell divi'sion. Enzyme and Antibiotic Disc Assays Release of ribonuclease I was determined by the method of Lopes et al. ( 56). L-agar plates were overlaid with 2. 5 ml of top agar conta:.:1ing 1. 5 % RNA (pH adjusted to 7. 0 J. The parent or mutant strain was spot-::ed onto the h ardened overlay and. after incubation at 37C or 25C for 2 h::urs, the plates were flooded with 0.1 N hydrochloric acid. Areas of RNA dgestion were clearly visible in the opaq_ue background. Alkaline phosphatase activity was assayed by the p-nitrc:;ihenol overlay method and spectrophotometrically as described by Willsky e t al. (116). Sensitivity to antibiotics was determined using disc assays. la.gar was seeded (4 X 106 cells/ml) with an overnight culture (parent, mutant, revertant, or transductant strain). Portions of 15 rJ. each -.. -ere poured into sterile petri plates and allowed to harden. Antibiotic impregnated discs (Difeo Laboratories, Inc., Detroit, MI) were placet in the center of individual plates. After 18 hours of incubation at either 39C or 30C, the diameter of the zone of clearing was neasure~. Measurement of DNA Synthesis DNA synthesis was measured in two ways in the parent and mutant strains. The first method was to sample cultures that had been uni~ormly labeled with 3H-thymidine. To measure DNA synthesis in filanents i~iuced to divide by a temperature shift to 39C, an exponential culture at : 9 C was inoculated into prewarmed medium to which thymidine (1 Ci/ml, 5 g/ml) and deoxyadenosine (250 g/ml) had been added. The cultures were allowed to grow in the presence of the label for 4-5 generatic~s by incubating the culture at 39C for 2 generations and then shiftir:~ to

PAGE 17

30C for 3 generations. After this period, cuJ.tures of st!'ain 615 A h a d formed filaments. Cultures wer e spl:i.t and one portion was incuba t e d at 39C. Duplica t e samples ( 0.1 ml) taken every 15 minutes for the next 2 hours were pipetted onto 0.45 m Whatman filte r paper discs and immediately irmnersec. i n c old 10% trichloroacetic acid (TCA). All of the discs "Were washed twice more in cold 10% TCA and then taken through three washes of cold 95% ethanol. The filter discs were dried and placed into vials with cocktail for the determination of radioactivity. The procedure for measuring DNA synthesis in filaments of strain 615A is the same except aJ.l the labeling prior to the shift to 30C was done at the permissive temperature (39C). DNA synthesis was also measured by pulse-labeling with 3H-thymidine. CuJ.tures of strains x462 and 615A w ere grown overnight at 39C in L-broth and diluted 1/400 into fresh medium Incubation continued at 39C until OD550= 0.1. The cult ure was the n split, one portion shifted to 30C,and 1 ml samples were pulsed 4 minutes at 30C or 39C with 2.5 Ci of 3 Hthyrnidine per m1. Unlabeled thyraidine was added to the samples at a final concentration of 10 g/ml. At the end of the pulse, the tubes were immers e d in an ice bath and 1 ml of cold 10% TCA containing 400 8 g of thymidine per ml was immediately added. After 1 hour in the ice bath, the samples were filtered through Millipore type HA filters (Millipore Corp., Bedford, MA) or Gelman Metricel GA-6 filters (Gelman Instrument Company, Ann Arbor, MI) and washed 3X with cold 10% TCA and 3X with cold 70% ethanol. After drying, the filters were placed into vials for the determination of radioactivity. Measurement of RNA Synthesis Cultures were grown as described for pulse-labeling of DNA synthesis.

PAGE 18

One ml samples w ere pulsed for 4 rt1Lnutes with 2 5 uCi of 3H-uracil per ml (0.5 g / ml). The cold 10% TCA contained 200 g of uracil per ml. M easure ment of Ph ospholipid Synthe~2;~ Cultures of str ain x 46 2 and strain 615A were g rown as described for pulse-labeling DNA synthesis. 'l'en ml sample s at 39C or 30C were 32 :pulse-labeled for 4 minutes with 4 Ci of P-orthophosphate per ml At the end of this period of labeling 1 ml of cold 50 % TCA was added and dispersed throu ghout the sample. The samples w e r e then c entrifuge d at 8000 rpm for 15 minutes, the supernatant removed,and the insoluble material resuspended in methanol-chloroform (2:1). Lipids were allowed to extract overnight at room temperature. The next day, 1 ml distilled water and 1 ml chloroform were added; the solution was mixed vigorously and centrifuged at 8000 rpm f'or 15 minutes. The upper aqueous phase was discarde d and the lowe r lipid phase was removed for determination of radioactivity. M easurement of Phospholipid 'l'urnover 9 Strains 615A and x462 were grown overnight at 39C in L-broth, diluted 1/200 into fresh, prewarmed medium ,and reincubated at 39C until an OD550 = 0. 4. This culture was then diluted 1/20 into broth and growr1 32 for 1-2 generations in the presence of P-orthophosphate (8 Ci/ml) at 39C, shifted to 30C,and grown for 3 more generations. After the in cubation at 30C to induce filaments, cells were spun down by centrifugation, washed twice,and resuspended in L-broth. One-half of the culture was incubated at 30C and the other half at 39C. Two ml samples were taken every 15 minutes for phospholipid analysis. Cells were precipitated with 5 % TCA, centrifuged,and pellets resuspended in 1 ml methanol and 2 nu chloroform. Lipids were extracted overnight at room temperature

PAGE 19

a.s described by Kanfer and Kennedy ( 119). 'l'he 2 ml lipid layer was re-moved and a 0.2 ml sample was counted to determin e tota l radioactivity. The remainder of the sample was e vaporated to near dryness w:lth dry N 2 and spot ted on silica gel G thin--laye r chromatography plates ( Ana l t ech, Inc. Newark, DE). These plates h a d been preacti vated in acetone and dri.e d 15-6 0 rn:i.nutes in a desiccator. The chromatograms were run in a 10 solvent system of chloroform-methanol-glaci a l acetic acid (65:25:8, v/v) as describe d b~,r Ames ( 5). Major phospholipids (phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidyletha.nolamine,andcard iolipin) were visualized with iodine vapors, scraped into .. vials a nd counte d for radioactivity. Bacterial Matin~ Matings between freshly reisolated Hfr strains and strain 615A, one of the low-temperature conditio n a l cell division mutants, were done as described by Miller (62). D o n o r s growing exponentially in L-broth (2 X 108 cells/ml) were mixed with 2-4 X 108 cells/ml of strain 615A (F-) at a ratio of 1/20. The mixture was incubated with slow shaking for 90 minutes at 37C after which appropriate dilutions were plated on selective media. Transdu c t i.ons Pl transducing lysates w ere prepared by the confluent lysis Vlr technique as described by Marsl1 and Du.~gan ( 59). For transduction exo periments, the recipient cells were grown in Z-broth to a density of 10~ cells/ml. The transducing lysate was diluted to yield a multiplicity of exposure of 0.1-0.5. Phage were allowed to adsorb at 37C for 10 minutes. Unadsorbed phage were removed by centrifugation. Bacteria were resuspended in sterile buffer and plated on appropriate selective media (10-l

PAGE 20

-2 and 10 final dilutions). Light and Electro n Microscopy '11he morphology of the cells were examined using a Zeiss photogrc.::;:,hic microscope. Nuclear stains were d one by the Giemsa technique as described by Fuhs (26). Fo r electron rr~croscopy, cells were fixed in 1 % osmium, embedded, sectioned,and examined using a Hitachi HUll E electr: n microscope ( lt2). Chemicals All amino acids and vitamins were obtained from Calbioche m (San Diego, CA) or the Sigma Chemical Co. (St. Louis, MO). Streptomycin sulfate and nalidixic acid were obtained from the Sigma Chemical Co. The following radioactive chemicals with their specific activities ";:::-e o~taine d from Amersham/Searle Co. (Amersham, England): [6-3H ] thymi:~ne 3 32 (25.6 Ci/mrnol), [6-H] uracil (23 Ci/mrnol),and [ P] orthophosphate (75 Ci/mgP). Results Isolation Initially, 1,624 colonies were screened for poor growth o n mini-cl plates at 25C. Of these, 162 grew poorly, and they were further exc.:::ined in broth for their ability to form filaments at 30C and grow normal},:, at 37C-39C. All of the 162 mutants exhibited this general character:stic and 19 were selected for characterization of growth and cell divisic::. Four of these produced ruinicells at both temperatures and wi l l be ct.2.r acterized in subsequent studies. General Char acterization In both complex and minimal broth, the remaining 15 lo-...-temper::.:ure 11

PAGE 21

conditional cel. l division mutant~~ formE:d filaments at 30C (F'i g 2A). Upon shifting to the permissive temperature, all the mutants exhibited a coordinated. burst in cell division, thus forming short cells at 39C (Fig 2B). Nuclear stains of the f:ilaments at 30C (Fig. 2C) revealed segregated masse,.; of nuclear material throughout the length of the filament in all mutants. By ligh t microscopy 1-2 completed septa per filament were frequently observed. Ultrastructura.l examination confirmed the presence of occasional septa and the absence of multiple, incomplete cross-walls. The mutants could be divided into two basic groups based on their colonial morphology on minimal agar plates at 37C. 'I'he majority of the mutants produced colonies similar to that of the parent, strain x462 : circular~ entire, convex,and smooth. How!2ver, 4 mutants (JSl, JS5, JS8, JSll) produced colonies at 3'(C that were irregular, undulated, flat ,and wrinkled in appearance. In a variety of other mutant systems (23,36,41,46,58,60,67,85~88 91,104), high concentrations of salt and/or sucrose prevented the pheno typic ef:fects of the cell division mutation at the nonpermissive temperatU1:e. The 15 low-temperature conditional cell division mutants were plated onto complex and minimal media plus and minus NaCl (1.5%) or sucrose (10%) and incubated. at both the permissive and restrictive tem perature3. The concentrations of salt and sucrose tested on our mutants were the maximal concentrations found in the literature to be effective for other mutant systems (58,88). Sucrose was effective in restoring colony-forming ability at the nonpermissive temperature to strains JS5 and JS14. These mutants were able to form colonies at the nonpermissive temperature in the presence of 10% sucrose. None of the other additives 12

PAGE 22

phenotypicaJly rescu e d mutants. Ef'fects of T empe r ature Shif'i~~i':!}~ Other Treatment~, W.h5 ch Ha.ve Previously Been Shown t o Stimulate Cel l Divj_sion The kinetic aspects of' cell division in each of the 15 mutants w ere examined. The results of one r epresentative, strain 615A, is shown in Figure 3. Upon shifting to the restrictive temperature cell division does not cease iIILmediately, but continues at the same rate as the nons hifted control for 30-45 minutes (Fig. 3A). The celJs rema .in viable for at least 180 minutes afte r the shift to the n onpermissive temperat ure ( Fig. 4), but do not significantly increase in cell n\lffiber after the residual 30--45 minutes of division (Fig. 3A). A coordinated burst in cell d ivision was observe d after 30-45 minutes upon shifting filaments to the permissive temperature (Fig 5A). M embrane-active a gents such as ethanol (43) and protein synthesis inhibitors such as chloramphenicol (30,104,119-121) have been shown to stimulate cell division in several mutant systems. The effects of these two agents as well as the effects of salt and sucrose on the cell divi-sion ability of all the mutants were examined in liquid culture. None of these agents stimulated cell division in broth at the restrictive temperature in any of the 15 mutants. Sensitivity of Strain 615A to Deoxycholate and Antibiotics Sensitivity to deoxycholate has previously been interpreted as evi dence for an alteration in the bacterial envelope (3,23,24,35,36,38,51, 58,70,87-89,103). The ability of the mutants to form colonies on L-agar plates containing deoxycholate (0.25%) was tested. Five out of the 15 mutants, strains JSl, JS9, JSll, JSl.2, and 615A, did not form colonies on this medium at either temperature. Neither the sensitivity to deo:xycholate nor the phenotypic rescue by sucrose correlated with either type 13

PAGE 23

14 of colonial m o r phology. Strain 615A was further examined for defects in the cell envelope. This strain w a s also found to be more sensitive that t h e parent strain to cleoxycholate when grown in brot h (Fig. 6) a nd this sensitivity was exaggerate d a t the nonpermissive temperature (30C). Althou g h increased. sensitivity to actinomycin D (7,23,51,54,76,9l1,l03) and lysozyrne (51,91;.) has been interpreted as evidence for alteration in the outer envelope, neither the p arent nor the mutant w a s found to be sensiti.ve to actinomycin D (25 g/ml) or lysozyme (2.5 g/ml). There haye been several reports in the literature of envelope mut ants whose growth is not only sensitive to detergents, but also to a variety of unrelated antibiotics (54,79,94). Using a ntibiotic disc assays, zones of inhibition were compared at the permissiv e temperature, 39C, and the restrictive temperature, 30C (Table 2). At the restrictive temperature, strain 615A is more sensitive than the parent to all of the antibiotics. At 39C strains 615A and x462 have nearly the same degree of sensitivity to bacitracin, rifampin,and nalidixic acid. However, strain 615A was also found to be more sensitive to neomycin (5 g and 30 g), chloromycetin (30 g), penicillin (10 Units), polymyxin B (50 Units and 300 Units), novobiocin (30 g), tetracycline (5 g and 30 g), erythromycin (15 g), cephalothin (30 g), coly-mycin (10 g), and aureomycin (30 g) at 30C. No differences in the sensitivities of strains 615A and xL162 to penicill:i.n (2 Units), novobiocin (5 g), and erythromycin (2 g) were observed at either temperature. Leakage of Periplasmic Enzymes in Strain 615A Agar diffusion assays were performed for ribonuclease I, a periplasmic enzyme. These indicated that strain 615A leaked this enzyme,

PAGE 24

especially when colonies were incubated at the nonpermissive temperature. Alkaline phosphatase a.ctiyity, on the othe r hand, was not released into the growth medium as assayed by both p-nitrophenol overlay or spectrophotometricalJ.y (116). 'rh e selective release of ribonuclease I as well as the increased sensitivity to deoxycholate and a variety of antibiotics is ccnsistent with other reports of differential p ermeability changes in enve!ope mutants (56). The results with strain 615A indicate that there is a.n impairment in the integr-ity of the cell envelope. Characteriation of DNA Synthesis and RNA S;ynthesis :i.n Filaments of Strain 615A_ Many mutants of E. coli have been reported which form filaments as a result of a mutation that interferes with normal DNA synthesis ( 36, 3'i', 102) or norm a l DNA segregation (36,37). However, filaments of strain 615A are not impaired in either of these processes Filaments of strain 615A contain masses of nuclear material scattere d throughout their length (Fig. 2C). Total DNA synthesis in strain 615A continues normally at the nonpermissive temperature, paralleling the increase in absorbance (Fig. 7), Pulse-labeling studies (Fig. 8) confirm that DNA synthesis continues at 30C without any apparent lag. In addition, the DNA per mass ratios of the parent and mutant strains (Table 3) are quite similar at both the permissive and nonperrnissive temperatures. Figure 9 is a comparison of RNA synthesis in cultures of strain x462 and strain 615A. These cultures were grown at 39C, shifted to 30C at time zero,and pulse-labeled with 3H-uracil. RNA synthesis is normal and parallels growth in strairr 615A at both the nonpermissive and permissive temperatures. Effects of Inhibitors of Macromolecular Synthesis Upon Division of 15

PAGE 25

Filaments at 30C M any cell division mutants in E coJ.i have been examined for sensitivity to inhibitors of m acrorrolecu1ar synthesis during induced division. These experiments involve splitting a culture of filaments into a number of subctLltur2s with the additio n of antibiotics at various times relative to the shift to the perm issive temperature. An example from this study (Fig. 10) shows the effect of chlormnphenicol on induced division. For comparative purposes, the increase in cell number 75 minute s after the shift to the pennissive temperature has been chosen as a measure of temperature-induced cell division. Increase in cell numbf r after this time represents subsequent division s of sho!t cells J.6 as well as division of residual filaments. However, the conclusions of the relative effects of an inhibitor are not different if 60 or 90 minutes is used a s the reference point. An increase in eel: number above 50% of the control (39C culture with no additions) indicates that the majority of the shift-induced divisions h a s become insensitive_ at the time of the addition of the antibiotic. The division of filaments of strain 615A at tl1e permissive temperature is sensitive to the inhibition of.protein synthesis by chloramphenicol for as long as 30 minutes. Thus, filaments of strain 615A require 30 minutes of new protein synthesis to divide at 39C. Figure 11 summarizes severa l similar experiments in which inhibitors of DNA synthesis (nalidixic acid), RNA synthesis (rifamycin SV)~or protein synthesis (purornycin, chlorampqenicol) have been added at various times after filaments were shifted to the permissive temperature. Instead of expressing the data as in Figure 10, the results are expressed as the percentage of increase in cell number relative to the untreated 39C con-

PAGE 26

17 trol culture versus the time at which the antibiotic was added follc.. -,d.ng the shift of' filaments to the pennissi ye temperature. From these rc3lLl ts, we can conclude that filaments of strain 615A require approxinately 12 minutes of new DNA synthesis and 36-44 minutes of new protein synU,c:sis, in the absence of new RNA synthesis,for division at the permissive temperature. The requirement for DNA synthesis was also confirmed using mitomycin C (5 g/ml) and phenethyl alcohol (0.3%). Effects of Penicillin on Induced Division of Fila:nents Low levels of penicillin inhibit cell division in E. coE with ::..i.ttle effect on elongation (98) Maximum sensitivity to killing by high :=.-::vel s of penicillin and maximum sensitivity of division by low levels of ::-::rricillin occur at or shortly after the completion of rounds of chrom o~:m e replication (40). The timing of this is coincident with the naxirus~ rate of murein synthesis during the cell cycle (40). When 25 Units of p-::::ii cillin G per m.l are added up to 20 minutes after the shift of filan:~::ts to the permissive temperature, temperature-induced division as meas:ed by increase in cell number was significantly inhibited (Fig. 11). ~~us it appears that filaments of strain 615A are blocked at a poin t in ~ie cell cycle well prior to the penicillin-sensitive step. DNA Synthesis, Phospholipid Synthesis and Phospholipid Turnover Dur:~g Induced Division Strain 615A appears to have a full complement of nuclear mater:al at both permissive and nonpermissive temperatures (Table 3). Howeve>!', division of filaments of strain 615A is sensitive to inhibition by ~alidixic acid for as long as 12 minutes after the shift to the permiss:ve temperature. We examined DNA synthesis in strain 615A during induc2'i division and found the change in the rate of DNA synthesis to be si=.ilar to that of the parent following the same shift in gro,rth temperatur::> (Fig.

PAGE 27

1 2). Ohki (80) f ound t hat phosphl1tidylglycer o l turneri over b a s t er-..;ise man n e r 20-30 minutes b e fore each cel l d i v ision i n a 1 % Casar:.:.no aci c 3 medium. The turnover o f ind ividual phos phol ipid compnents -,.-as meas ured in filaments of strnin 615A and in temperature-::. n duced d i v i s ic:-~ o f f i laments in r i c h mediwn (Fie;. 13). However, n o 2:orupt c:: ,2.nges i~ the turnover of phosphatidylglycerol were o bserved durin g ir::.uced d:. ;-i sion. No turnover of phosphatidylethan o l2.:.'1ine was oc,.=;erved. Phospholipid synthesi s during induced di v ision c-:: fila::= ~ts of strain 6l5A was also examined(Fig 14). The increase in the rates c : pho spholipid s ynthesis was similar t o the increase i n rates :;oserve'.:. with the parent. For both s t r a ins, phospholipicl synt:1esis :;:::.ra llel e:. the increase i n g rowth. Mapping of t h e Division Lesion of Strain 615A 1 8 R esult s from the 90-minute mating s ( Table 4) reYeal e d t::at the :owt em p e rature conditiona l division locu s in str a i n 615A was lc2ated b~:een minutes 6 8-79 Interrupted mating experinents perforJed usi~g stra::.~ s CSH47 a C S H64 ~ and 615A narrowe d the locus t o the region bet;,een ma1..:_ (min 74) and~ (mi n 79). T able 5 shows t h e r esults of trs~sducti:~al + + + analyses invo J _ving a Pl lysat e m a d e on s t r ain 615;,_ malA ~lpD a::-:B Vlr + asd and seve r a l ~coli strains that are either mal..',_-, g l pl: ,or ar:3-, and: wild--type for division. The division l esion in ~ train =-.5A a p J:c'=.r s to cotransduce with malA at frequencies of 20-33% with glp~ a t frequencies of 9-52%,and with aroB a t a frequency of 21.5% I~ a furt~er attempt to locate the cell division lesion in strain 615A w:.-:h r espect to these markers, a transduction with the same P l lysate s.nd an :..::d -vir + strain ( U482; T able 1) was done. However, no a s d t~ansduc: ~nts c o.:=-.J.

PAGE 28

be recovered using this lysate despite many attempt~;. Strain 615A does not require d.iarninopimelic acid fer grcwth in ric!i mediwn (phenotype of asdstrains) at either tbe permissive or nonpermissive temperatures. The addition of diaminopimelic acid does not prevent growth of filaments at 30C. Thus, it i s probable that the celJ. division lesion in strain 615A is not the asd lesion. One possible explanation for these results j_s that the asd lesion and the di vision lesion in strain 615A are not compatible in the same strain. Analvsis of Transductants and Revert.ants Spontaneous revert.ants were obtained by growing strain 615A in Lbroth at 30C. The cult~e was diluted (1/200) into fresh broth at regular intervals. After three days of growth, dilutions of the culture were plated on 1-agar and incubated at 25C. Isolated colonies 19 were examined for growth in broth at both the permissive and nonpermis sive temperatures. Ten spontaneous revert.ants that divided norr.ially at both temperatures were selected and examined for antibiotic sensitivity (plates) and sensitivity to deoxycholate in broth. All of the revert.ants were as insensitive to the antibiotics as the parent (representative~, Table 2). However, the revert.ants were found to be as sensitive to deoxycholate (0.25% ) at 30C and slightly more sensitive at 39C than strajn 615A. Discussion Burdett and Murray (14) ha..ve recently characterized the events of septation in ~coli B a...n.d B/r strains. In accordance with the cell cycle of Jones and Donachie (47), they found that the classical D period is composed of events leading to cell compartmentalization, septation,and separation. In rich medium septation is completed in approximately 13

PAGE 29

minutes wbile 7 minutes in needed for daughter cells to separate. Thus, approximately 20 minutes mm;t elapse from the visual initiation of a cross-wall to the separation of daughter cells. 20 Strain 615A can b e characterized as a septum-initiation type mutant and positione d accordingly in the scbeme of Jones and Donachie. Residual division at the nonpermissive temperature and lack of multiple, incomplete septa at 30C suJJport this classification (121). Mutar.ts have been isolated which can divide in the presence of chlorampheni co l (30~ 88, 104 121). This indicates that the assembly of septmn precursors can take place even in the absence of protein synthesis. Cell division in strain 615A is sensitive to the addition of chlorampheni.col for as long as 36 minutes and to the addition of puromycin for as long as 44 minutes after shifting filaments to the permissive temperature. Also, temperature induced division of filaments is prevented by the addition of concentratj_ons of penicillin G which block cross-wall initiation for as long as 20 minutes after the shift to the permissive temperature. Thus, strain 615A appears to be blocked in cell di.vision at a point well prior to the assembly of s eptu..rn precursors. This bloc}: could .be structural and alter the nature or production of only one precursor or it could be regulatory and alter the envelope such that septum precursors cannot be assembled(36). The prin~ry lesion in strain 615A does not appear to be in the events involving the synthesis of bulk DNA or nuclear segregation. Crumplin and Smith have recently provided evidence for a new ste p during the synthesis of the! coli genome (17). They have shown that nucleotides are incorporated into Okasaki fragments which are ligased into 38S singlestranded fragments. These 38S fragments are subsequently converted into full-sized daughter molecules. It is this latter step, the conversion

PAGE 30

into the full-sized daughter molecules which is inhibited by nalidix::.s acid. Filaments of strain 615A incorporate 3H-t hyT:idine into D~IA wi tr. no detectable lag and cc, ntain the same amount of DUA per unit mas s as normal cells. Howe v er, the division of filaments o f strain 615A is sensitive to nalidixic acid for as long as 12 minutes after the shift to the permissive temperature. Thus, it appears that filaments or' strain 615A rna:;,r be blocked at a point between the incorporation of .nucleotide precursors into D N A a nd the nalidix:Lc acid-sensitive step. Alternatively, the required termination protein (47) generated by thE: completion of a round of replication may be unstable in strain 6 15 A a,_ 30C and the completion of new Younds m a y be required to permit the initiation of n e w di visions. However, this product is presmnably sta: ::.e in other m orphologically similar mutants which have been reported to ini.tiate and divide in the absence of 1,ew' Dr!A synthesis (69,85,120). Strain 615A requires a significant period of new protein synthes~s in the apparent absence of new RNA synthesis for division of filament;; at the permissive temperature. This implies that the mRNA translated during this period to produce the required protein(s) is more resista::.t to turnover than most other ~coli mRNAs (27,101). Messenger RNA f o:tuost m e mbrane proteins, most notably Braun lipoprotein (32-34), and excreted proteins often appear to be less susceptable to turnover (50, 66,99). Alternatively, transcription of some mRNA(s) may be resiste.n: to rifamycin SV. The mRNA for lipoprotein is one example (34). Strain 615A contains an envelope defect as evidenced by increase5.. sensitivity to deoxycholate and many antibiotics as well as by leakage of ribo nuclease I, a periplasmic enzyme. The possibility of changes : n cell wall components will be explored in subsequent studies. Analysi~ 21

PAGE 31

of wild-type transductants and revertan t s o f strain 61 5 A sup:r,".lrt tt.e involvement of this envelope altera1~ion in the division lesion. These strains not only divide normally at 30C and 39C, but aJ.so are a s res:'..::;tar.t as the parent organism to antibiotics at both temp eratures (Tabl'= 2 ) Dnd the wi.1d-type transductants are resistant to de oxy cho2.e.te. i-:-:.-.rever, the revertants rem ain sensitive to deoxycholate at both the n o::-_p e rmissive and permissive temperatures. One explanation of tiis is -that the division-cor:ipetent revertants result from a second gene mut:;, tion. This second mut ation could permit division without res-:.oring normal envelope integrity. Suppression of a division defect in lon strains of! coli by a second mutation, recA, is well docUITie~ted (2?). Another possibility is that a second gene 1rntation in strain 615A m2.:,22 b e responsible for deoxycholate sensitivity. If this is true, howeY'=~ the l2sion for deoxycholate sensitivity and the cell divisio~ lesio~ =ust be in the same region of the chromosome (within 1. 8 min) ( 62). The third possibility is that the revertants contain an altered 6ene pro~uct which permits cell division without completely restoring envelope ir: tegrity. Cross-wall formation in coli involves a number of ge:c:.etic lo~i (3,20,36,37,41,60,88,95,108,109,120). Most of these loci ha\-e been identified using high-temperature conditional cell division ~utants. In an effort to discover new and different loci as well as lear~ more c:out the roles of existing loci in the division process, we have isolatec lowtemperature conditiona l cell division mutants. Prelit~nary !'esults =-~ dicate that 2 out of the remaining 14 mutants isolated have civision lesions tha t are cotransducible with nalA. Thus, it appears that tt:s m ethod of isolation and enrichment for low-temperature condi-:::onal c:. .-i-

PAGE 32

s i on mutants does not result ir! mutants 1.-1hose lesions are clustered in a single reg i o n of the chrom osome. St:i:ain 615A apvears phenotypical:ly similar to many of the high-tempf,ratufe conditiona l mutants previcusly describe d : s t rain BUG6 ( 85) stra,ins ft~?.-' fts7-, fts8 -(108), strains AX621 A X 629 AX.655 ( 3), strain MX74T;!ts52 (120), strain ts-20 ( 69), ft s1l-I strains ( 88) strain Yl6 ( 95), s train MA.CI ( 20), strain fil ts (104), strain ts612 ( 60), and strain ASH12l ~ ( 41). Strain SN29 i s a lowtemperature conditiona l cell division mutant o f Agrnene lJ.um _922:.adr2:Plicatu:m 2 3 ( 43) that can be included in this group of morphologically similar mutant s N o n e of these mutants is blocked in bulk DNA synthesis or nuclear segre-gation as evidenced by the formation of long, multinucleoid filaments : lacking cross-walls when grown at the nonpermissive condition. Although these mutants appear to be defective in cross-wall initiation rather than cross-wall formation, the latter possibility cannot be completely excluded. Incomplete cross-walls undiscerne d by lig h t microscopy potentially coul d be resorb e d by hydrolases or d estroyed in the fixation procedure for electron microscopy as suggested by Slater and Schaechter (102). Thu s this group of morphologically simila r strains can be describe d as multinucleoid, septation-initiation type mutants. These mutants c an, howe ver, be divided into several groups based on other characteristics. Strains BUG6, ASH124 and fil ts divide normally when dense cultures are shifted to the nonp ermissive temperature. Strains ASH124, BUG6, ts612, and some of thefts strains described by Ricard and Hirota (88) are phenotypically rescued by salt and/or sucrose at the restrictive temperature Strains MX74T2ts52 and fil are stimulated to divide at the restrictive temp e r ature by the addition of chloramphenicol. The a.ddi tion

PAGE 33

of adenine at the n onpermissi v e temperature P"' ~ v ents fila.JL":clt-forr:.::.:ion in strain Yl6. Strain SN29 is induced to divide at the no~~ermiss:7e temperature by ethanol. Strain ts-20 a.rid MACI di vi-::e sync:..:'.' o n o usl:, whe n filaments are shifted to the permissive tempen.ture. Strains ::tsB and ftsC form minicell s in addition to multinucleoid. filar:=:::its These mutants can b e further groupe d according t o the locatiu::. of 24 their genetic lesions. The majority of t h ese loci 2.re clu.::::ered c~ either side of the replication origin (min 74) ( 62). As p:iinte d -::;.1t by :E.::.2h.man:: et _?-1. ( 8), it would certainly b e to the organi sm s a.dvant'::.5e to ts-,-e the potential to amplify important genes duri::ig per:ods of ~apid ~~vth. The part of the genome near the replication origin -,;-ould c::: presen-:: in several copies per nucl eoid due to multiple i nitiat::.ons of c.:he DNA replication cycle. The lesion in strain ts61 2 maps at min 74.7-75.5. Strains ftsA, A.'\.. seri:=s and _fts2 .f_ts 7-, and fts8 -; all ::luster ~,::twee:1 minutes 1-2 on the Taylor and Trotte r linkage map (l06). :::rains :'...':.CI and ftsC rnap in the region between min 3 and I!lin 8. Two c..:-.rision ::.-.it ants ( strains ftsB and MX74T2ts52) h ave lesions between 1::in 29 s.."ld min The division lesion in strain Yl6 was designated ftsH by Ss:1tos a c ~ Almeida (95) in accordance with the scheme of RicarJ and Lrota (c:) The map position of. the lesion in strain Yl6 is min 61. E:::lland a:::i Darby also designated the division lesion in their mutant, strai:.::. ASH124, as ft.sH which maps at min 80. This ftsH lesion is dis-'.;inct f::1m ftsr ":hat was previously positioned at min 77. 5. Preser.tly, we are ..:..1certai::. if the cell division lesion in strain 615A (min 64-67 o n the :-aylor a::..i Trotter linkage map or min 72-74 on the revised map of Bac:-..::iann et al.) is distinct from ftsE. Ricard and Hirota (e8) have mappec -'.;hrce s:~ains (MT99, t-'IT1181, M'l.'123) with the ftsE lesion betwee n :::in 66 s:, d min -:9.

PAGE 34

25 Complcm e ntati,::m analysis between-strain_ 615A and one or more of the ftsE strains would determine if the mutation in strain 615A is distinct from ftsE or within ftsE. These stt;_dies are compUcated by a requirement for an intermediate temperature that is permissive for both the low and high-temperature conditional strains and/or by the use of salt to phenotypically rescue the ftsE strains (MT99,MT123) at the permissive temperature for strain 615A

PAGE 35

SECTION II BIOCHEMICAL CHARACTERIZATION OF THE ENVELOPE I.MPAIR.ViEIVi.' OF STRAIN 615A Introduction The cell envelope of gram-negative bacteria such as Escherichia coJi. i s composed of an inne r cytoplasmic membrane, a thin layer of pep-tidoglycan ( mure i n),.and an outer membrane ( 2 8,74,93,111 ) The outer membran e can b2 distinguished from the cytop l asmic membrane not only b y its location but also b y its functions and molecula r c o nposition. 'I'he oute r membrane conta:i.ns lipopolysaccharide and large a.mounts of foill major proteins, includ::ng B:::-aun lipoprotein. Although the outer membrane is not the major permeability barrier of the cell (71-7 5), it is primarily responsible for the intrinsic resistance of gram -negative organisms to antibiotics such as penicillin. However, the outer mem-brane i s permeable to small hydrophilic molecules suc h as saccharides of molecular weights less than 550 daltons ( 19,71-73). Na.1<-ae has shown that the reconstitution of m f:Illbranes permeable to s:r.iall hydrophilic mole cules require s the presence of an agg1egate of phospholipids, lipopolysacchari de ,and three outer membrane proteins in Salmonella typhimurium (71,73) or a sing l e oute r membrane protein in~coli (72 ). Other oute r membr a n e components serv e as specific receptors for a variety of bacteriophages and colicins (18,25,82 ,83). In addition, the outer membrane c on ta:i.ns little e n zyme activity except phospholipase A1 (10) and l acks the electron transport system characteristic o f the cytoplasmic membrane ( 6!1, 81). 26

PAGE 36

Strain 615A is a low-temperature conditional cell division mutant that has been isolated and characterized by this author. During previous investigations, straj_ n 615A wan found to be more sensitive than its parent to deoxycholate and a variety of antibiotics. In addition, strain 615.A le&.ked ribonucleas e I, a periplasm.ic enzyme. 'I'hese results suggest that strain 615A bas an altered envelope. This section summarizes our reEults comparir:g the envelope ccmpon2nts of strain 615A to its parent, strain x462 Material.2 and MethoJ.s Bacterial Strajns Strain 615 A is 2, low-temperature conditional cell division mutant isolated from strain x462 following ultraviolet light mutagenesis. It grows as long multinucleate filc1ments with 1-2 septa per filament at 30C and as short rods at 39C. Strain 71 is a transductant with norm a l cell division derived from strain 615A using a transducing lysate made from the parent, strain x462. Media and Growth Conditions Luria (LJ broth (52) was used in most experiments. Minimal medium 27 ( 62) was used for experiments in which bacterial mE:,mbranes were isolated. Both media were supplemented with 2 g of glucose per liter. When required, amino acids were added at a final concentration of 20 g/ml. Liquid cultures were grown overnight with forced aeration at 39C (the permissive temperature for strain 615A) in 22 X 175 mm culture tubes. Overnight cultures were diluted into fresh, prewarmed media and incubated at 39C until they were growing exponentially. 'I'hese cultures were then used to inoculate fresh, prewarmed media for use in subsequent

PAGE 37

28 experiments. Optical density of' n:J tu.res was measured at 550 nm using a Spectronic 70 spectrophotometer. Exponentially grown cu..lture: 3 ( 200 ml ) at 39C a nd 30C w e r e harvested by centrifugation and lyophilized. Lipopolysaccharide (LPS) was extracted and purified as described by Westphal and Jann (114). After dialysis, nucleic acids w e r e r emoved b y enzym e digestion (55) and repeated ultracentrifugation (105,000 X g for 4 hours) until absorbance at 260 nm demonstrated less than 3% nucleic acid contamination. Purified LPS was compared by thin-layer chromatograpby using silicic acid-impregnated glass fiber paper (Gelmann Instrument Co., Ann Arbor, MI) as described by Buttke and Ingram (15). Co.!!U2arison of Phospholipids Exponentially growing cultures a t 39C were split, incubated at 39C 32 and 30C with 4 Ci of P -orthophosphate per ml ,and allowed to grow for 4-5 generations (fina l OD550 = 0.5). Cultures w ere inactivated by the addition of trichloroacetic acid (TCA) at a fina l concentration of 5 % and extracted overnigh t wi t:t chlorofo rm-methanol as described by Kanfer and Kennedy (49). The lower chloroform layer (containing lipids ) was removed and e vaporated with dry N 2 The dried lipid sample was r edissolved in a small amount of chloroform-methanol ( 2:1) and applied t o a silica Gel G plate (Analtech, Inc. N ewark, DE). The chrom atograL1s were run in a solvent system containing chloroform-methanol-glacial acetic acid (65:25:8) as described by G. Ames (5). M ajor phospholipid species (phosphatidylethanolamine phosphatidylglycerol, lysophosphatidylethanolamine ,and cardiolipin) w ere visualized with iodine vapors, scraped into vials,and the radioactivity determined.

PAGE 38

Fatty Acid Analysis Cells growing exponent iaJ -:i.;/ at 30C and 39C were inactivated at on550 = 0.6 b y the addition of 5% TCA, harvested by centrifugation,a~d extracted into ch.loroform-methanol a.s described by Ka.nfer and Kennec.:, ( 49). The washed lipid extract was transesterified in 2% H 2so4 (v/v) in methanol as described by Silbert,i al. (100). The fatty acid methyl esters were extracted into pentane and analyzed on a Tracor G::.s Chromatograph (Tracor Instrume::1t Co., Austh1,'rX). The fatty acids -,.--=T e identified by comparison of retention t imes to those obtained with authentic f a tty acid methyl ester standards. Isolation of Bacterial Membranes 29 Overnigh t cultures of strains x462 and 615A grovm i n minima l me::hnn w e r e diluted 1/200 into fresh, prevarmed minimal media at 39C and gr:wn until O D 550 = 0.4 Arginine ( 2 g/ml) w a s added to each culture anc. allowed to mix. 'l'en ml portions of each culture were incubated at 3?C and 30C for 60 minutes. At this time, each culture was pulsed with eithe r 14c-arginine (1 Ci/ml) or 3 H-arginine (10 Ci/ml) f o r 10 mi:r:.1tes To stop the incorpo ration of l abel,cultures w ere i mmersed in an ice ~~th and 20 ml of unlabeled carrier cells were added. Cells were centrir.1ged at 3000 rpm for 10 minutes, w ashed onc e with cold saline ,and then resuspe nded in 10 ml of cold 0.01 M Tris-HCl buffer, pH 6.8. The cells were broken using a Bronwill Biosonik III (Rochester, NY). Whole cells and debris w ere removed by a low-speed centrifugation and the superr.s.tant dialyze d overnight at 4C against 100 volumes of 0. 01 M Tris -HCl buf:'-2r, pH 6.8 The membrane fraction was collected in an ultracentrifuge a: 90,000 X g for 60 minutes. Isolation of Inner and Outer Membranes

PAGE 39

The procedure used to isolate bacterial membr anes and. separate the inner and outer fractions b,:i.s bee n de::,cribed in detail by Duckworth and Dunn ( 22). This procedure differs from the above procedure (isolation of total membranes) in several ways: (1) strains are labeled in minimal medium with tyrosine and l eucine [strain 615A or 71 with 3 tt-amino acids; strain x462 with 14c--amino acids], (2) strains are harvested in late log phase rather than exponential phase,and (3) cells are broken by several passages through the French pressure cell rather than by a Bromrill Biosonik III tissue disrupter. In addition, the tota l membrane preparation. w a s separated into outer and inner fractions by the selective solubilization of the inner membrane in Triton X-100. Analysis of Membrane Proteins The membrane preparations d escribed.above were prepared for electrophoresis by the method of Schnaitman (96) as described by Duckworth and Dunn (22). Gels were prepared according to the procedure of Maizel (57). These were sliced, solubilized,and counted for radioactivity according to Duckworth and Dunn (22). Analysis of Bound Lipoprotein 30 The total membrane preparations described were also used to assay for the amount of botmd lipoprotein as described by Torti and Park (107). Analysis of Peptidoglycan Expon~ntially growing cultures of strain x462 and strain 615A were harvested by centrifugation at an OD550 = 0.6. Th e pellets w ere resuspended in 25 ml of 0.1 M sodium phosphate buffer, pH 7.2,and heated at 60C for 30 minutes to inactivate autolysins. Cells were broken with 30 second pulses from a Bronw:ill Biosonik III tissue disrupter (Rochester, NY). Unbroken cells were removed by centrifugation (3000 rpm for 10

PAGE 40

31 minutes ) and the supernatant was ce.ntrifuged at 10,000 r p m for 20 m.:.~_utes. The pell e t ( containing peptidoglyco.n ) was resu spe,1d f : d in 5 ml of ph-::.2 phate buffer and added dropwise t o 30 ml of b0Din1:, 10% sodiun dodee::rl sulphate ( SDS). This solution was mixed for 20 minutes more and th:::: centrifuged at 18,000 rpm for 60 minutes. The SDS extraction was r 2 peated and this pellet was resuspended in 10 ml of phosphat e rmffer ::on t aining pronase at a f:inal concentration of 100 g/ml. After heati:_~ the sample overnight at 60C, the SDS was removed by dialyzing again:o-:: 100 volumes of water for 2 days ( 2 w ater chang,es per day). J._ final p ellet was obtaine d by centrifugati on a t 18,000 rpm for 60 minutes. These pellets were lyophilized and weighed; sar. 1 ples ( 10 mg) were hy::.::--o syzed in 5 wi of 4 N HCl at 105C for 11 hours. Each hyclrolysate flash-evaporated to dryness, washed witr~ water to remov e residual c=-., and resuspende d in 25 m l of 0.01 N HCl. Amino acid analysis (65) V':.3 performed in a JEOL Model JLC-6.A.H (Japan Electron Optic L a boratorie3, Cranford, NJ). Assay for Cross-linking In Vivo Overnight cultures grown at 39C in L-broth were diluted 1/50 i::::.o fresh, prewarmed L-broth. Growth was allowe d to continue at 39C un-'::il cultures were growing exponentially. 3H-diaminopimelic acid (25 Ci/ml) was added to 2 ml cultures at 39C and 30C. After 2.5 hours, incorp~~ation was stopped by the addition of 5% TC.A.. The samples were treated ,n-::2 trypsin and lysozyme and analyzed for cross-linking by descending c:::.ro matogrn.phy as described by Kruuiryo and Strominger ( 48). Chemicals All amino acids were obtained from Calbiochem ( San Diego, CA) ~! the Sigma Chemical Co. (St. Louis, MO). 'l'he following radioactive ~0m-

PAGE 41

32 pound s w ere obtained from Amershe.rn/f.,E:a:tle Co. (Arner sham Eng l and): [G3 H ) 2,6-diaminopimelic aci d dihydro~h1orid.e ( 3 00 rnCi/mm oJ.), L-[U-11 1c) arginine monohyd r ochloride ( 3211 mCi/mmoJ) .and 32P-orthophosphate ( 81 Ci/mg P). ~[3 H ) arginine ( 7Ci/mmol), [ 1 4c)t.yrosine (460 mCi/nn.1J.ol), [14c) leucine ( 309 mCi/mmol), [3H) tyrosine ( 40 Ci/rrJnol), and [3H] leucine (59 C i /mrnol) w ere obtained from Schwarz/Mann Co. (Orangeburg, NY). The scintillation f luid used was to.luene-based Omnifluor (New Eng land Nuclear, Boston, MA) in all cases except w hen processing samples of labeled membranes proteins RESULTS Analysis of Lif'opolysaccharide Lipopolysaccharide (LPS) was purif{ed from strains x46 2 and 615A and analyzed by thin-layer chro:umtog:rnphy ( F i g 15). This method can dis-tinguish differences in LPS structure such as those seen in "rough11 and "smooth" strains (15) as w ell as interspecific differences. Using this technique, no differences w ere observed in the mobility of the LPS extracted from strain x 462 and strain 615A at the permissive or nonp er niissive temperature. Analysis of Lipids Major phospholipid components were extracted from strain 615A, strain x462 and strain 71, a transductant with normal cell division, and analyzed by thin-layer chromatography (Table 6). No significant differences among any of the strains were observe d at either the perrnis-sive or nonpermissive temperatures. In addition, no significant differences in fatty acid composition were observed b etween strains x462 and 615A (Table 7).

PAGE 42

Analysis of P~ptidoglyca n a n d Cr oss-linking Strains x462 and 615A were examined. for qualitative and/or quantitative diffe r ences in peptidoglyca n composition (Table 8) Cells of either strain grmm at 30C or 39C contained approximately the same amounts of H a c etylglucosarnine N-acetylmuramic acid, glut&11ic acid, alanine ,and diam inopimeJj_c acid. 'I'hus tbere are no differences in peptidoglyca n composition between the parent and mutant strains. 33 Morphologically aberrant mutants of! coli that synthesize either hypo-or hyper-cross-linked peptidoglycan at a nonpernJissi ve temperature have been isolated(48). WP. examined the percentage of cross-linkage in strains 615A and x462 at both 30C and 39C (Table 9). However no significant differences between the t,m strains were oberved at either temperature. Analysi.s of Membrane P roteins The division lesion in strain 615A does not appear to result from changes in LPS structure, phospholipid composition, fatty acid composition, peptidoglycan composition or in the cross-linking of the murein. The proteins of both the inner and outer membrane fractions were isolated and analyzed by SDS-polyacrylamide ge l electrophoresis (Fig. 16). The data are plotted as differences profiles. A difference profile is ob 1!1 tained by subtracting the percentage of C-counts (strain x462 ) from the percentage of 3H-counts (strain 615A or 71) (after correcting for background and spillover in each gel slice) and plotting this diffe r ence versus the slice number ( 22). No differences in the inner membrane protein composition between strains x462 and 615A were detecte d at either the permissive or nonpermissive temperature (Fig. 16A). However, the outer membrane protein profiles of the mutant (str a i n 615A) and the

PAGE 43

34 parent, strain x462, at 30C differ in two regions (Fig. 16B): region a (molecular weight= 40,000 ) nnd region b (moh:c:ular weight= 7,000 10,000). The difference in region a did not appe:ar to b e involved in the division lesion of strain 615A because the difference was still apparent at 30C in strain 71 a transductant with norma l cell division (Fig 16c). However, the deficit in the region h protein( s ) was absent in this comparison. Thus t h e region b protien(s) was thought to be involved in the division defect in strain 6 15A. Lipoprotein is an envelope protein in the outer membrane with a molecular weight of 7200 daltons in its free form (11-13). This protei ~ is the major component normally found in region b (11-13). Thus, it appeared that strain 615A might be deficient in the free form of lipoprotei n However the t e chnique used to prepare inner and outer envelope proteins utilized cells approaching stationary phase. T o further explore the possible differences i n free lipoprotein, membrane proteins labeled i n exponenti3 14 ally growing cultures at 30C with H-arginine (strain 6 15A) and C-ar-ginine (strain x462) were isolated and examined (Fig. 17). The two profiles are almost superimposable with only a slight diffe r ence in the area of free lipoprotein (region b). An average of three determinations revealed that s train 615A l 1as appr oxim ately 1% more of its total mem-brane protein as f ree lipoprotein than does the parent. Thu s the small deficit in free lipoprotein originally observed in strain 615A grown at the nonpermissive temperature may not be indicative of the division d e f ect. The differences in results between the two methods can be attriouted to differences in growth conditions and sample p reparation. Analysis of Bound Lipoprotein 'Torti and Park (107) h ave isolated an E coli mutant that hn.s a

PAGE 44

35 temperature-sensitive deficiency in bound lipoprotein. This mutant maps in the same region of the chromosome as strain 6J.5A (mj_n 72-75) and forms filaments at the nonpermissive temp-2rature (107; personal communication). These studj_es prompted us to examin e the amount of bound lipoprotein in stra.ins 615A and x!1 62 Using their procedure (107), no significant differences in tte amount of bound lipoprotein w ere seen at eithe r the permissive or nonpermissive temperature (Table 10). Discussi on Severa.J. mutants of Es cherichiEt coli and Salmo n ella t.y phim ur i um that have increased 1->e r meability to dy e s d e t ergent s ,and antibiotics have been sho,m to h a v e an altered LPS structure and/or a cha nge in one or more of their outer membrane proteins (6,21-r, 7 5 ,90,105, 118). In addition, loss of components of the outer membrane by c hemical remova l (53,51+, 68) or by physical treatments such as freezing a nd drying or osmotic shock (7,31,33, 84) result in the breakdown of the "natura l resistance" of gram negative bacteria to many inhibitory agents. However anothe r group of mutant s believed to have mut ations affecting their cytoplasmic membranes (38,89) have been sho,m to have increased p e rmeability and sensitivity to deoxychola.te. Weigand and Rothfield (Jl2 ) have recently isolated lky mutants of Salmon ella tYPhirnurium that leak ribonuclease I. One of these mutants, lkyD, h a s a significant decrease in the bound form of lipoprotein with a corresponding i nc rease in the free form of lipoprotein (113). In addition, this mutant is defective in the morphog enesis of the division septwn forming blebs of outer membrane at septal sites and failing to separate normally. E. coli mutants ( envA or envC ) morphologically

PAGE 45

similar to the lkyD SalmonellB:_ :~yphirnur~um muta .nt have been described previously (61,78,91,92). Hov,r:1ver, the _envA nutation does n o t result in a. difference in the amount::; of f;ee and b ound lipoprotein (113). In addHion, the ~nvf)._ mutation differs from the lkyD in map position (77, 112) and by not leaking ribonuclease L Recently, coli strains bearing the envA mutation were found to have a 6-fold reduction in the specific activity of the enzyme N-acetylm.urarrwl-L-alanine amidase (117). This enzyme defect l eac.s to the formation of chains of unseparated daughter cells. Another cell wall enzyme carboxypeptidase II, has been implicated as the lesion in BUG6, a high -"temperature conditional cell division mutar,t of coll_ ( 9 ). Upon shifting to the permissive temperature, there is a rapid resumption of division, accompained by a 10-fold increase in the specific acitivity of carboxypeptidase II. E. col!_ strains that carry the envC lesion also form cha .ins of daughter rods. The envC mutation not only confers sensitivity to d.eoxycholate and antibiotics. such as penicillin, D -cycloserine and rifrunpicin, but also results in a n alteration o f the phosphatidylglycerol/cardiolipin ratios in the inner and outer membranes ( 61,91,92). Strain 615A appears t o have an envelope defect as evidenced by its increased sensitivity to d eoxiJcholate and antibiotics. The leakage of ribo nuclease I at the nonpermissiv e temperature is consistent with the d efect being outside the cytoplasmic membrane and probably in the outer membrane. Our investigatio n of the diffe rent envelope comp onents in strains x 46 2 and 615A failed to reveal differences in LPS structure, phoE pholipid composition, fatty acid composition peptidoglycan c omposition, percentage of c1oss-linking ,or in the amounts of bound lipoprotein at either the permissive or nonpennissive temperature. In addition, no

PAGE 46

3'( significant differences were detected by SDS-polyacryla mide gel electrophoresis of inner and outer membr a ne proteins. However, the envelope defect could be a rcsillt of a chaneC in the outer or inner membrane proteins which is obscured within a particula r molecular w e i ght class. M any of the bands observed in SDS gels are known to l>e composed of mor e than one protein (97). Alternatively, a particular membrane protein cou .. ld b e altered by a sing l e amino acid substitution rendering its activity cold-sensitive. Such an altered protein would rer.1g,in ur1detectec. by SDS gels unless produced in abnormal awounts. Nikaido has recently proposed that the::re are at least two pathways for the diffusion of small molecules across the outer menbrane, one for hydrophilic compounds and one for hydrophobic compounds (75). 'I11e hydro phylic pathway is unaffected by the structure of LPS present in the outer membrane. The hydrophobic pathwa y is almost inactive in wild type strc:dns producing complete LPS and becomes fully active only in deep rough muta 11ts or EDTA-treated cells where phospholipid bilayer regions are exposed._ The hydrophylic pathway involves protein aggregates in the outer membran e which are presumed to form pores (19,7 1-73). Strain 615A has increased permeability to both hydrophilic and hydrophobic compounds as evidenced by its increased sensitivity to a variety of antibiotics and deoxycholate. Thus, it seems likely that strain 615A has an alteration in its oute r membran e organization affecting both pathways. Other possible differences in envelope structure which were not eliminated by this investigation include: (1) quantitative differences in the amounts of LPS or minor change s in the LPS structure of the mutan t ( 2 ) differences in lipid composition b e tw een the inne r and outer membrane and (3) differences in activities of autolytic enzymes thought to be involve d in the division process (9,63, 1i7 ).

PAGE 47

38 T able 1. Bacterial strains. STHAIN S E X GENO'l1YPE SOURCE ---x46 2 -. r -?I.::._') AlacZ r F ara l e u azi ton.A tsx ------A C. Frazer and R, ]'.' purE A lys~ str .2S'll mtl r netE Curtiss III thj_ x462m g -. r -lacZ -r F ara leu 8.ZJ. ton!\. pro.I\ tsx This paper -r -purE l_ysA str &.l mtl metE -thi -~ -malA --615A -. r J2!:.?A-r F ara 1eu azi tonA lacZ tsx -----Derived f rom x462; purE-" lysAr str &1_ mtl rnetE --1,!.., ___ low-temperatUl'e con--thi ditiona l for division 615Am F .r proA-r ara l e u azi tonA l acZ tsx This pauer low tern-" purE-" lysAr -str gl mtl m etE ----~ ~ perature conditiona l -thi m a lA fo1 ~ division -----AB2847 F -supE (?) "r /.. aroB tsx maJ. -----J. T Park ul-1-82 Hfr -thi >.. asd rel J T Park --LW3 glpDthi -F J. E. Cron a n LW3m F -elpD~ thi m alA -This paper CSH47a Hfr s~ J Miller strain kit CSH60 Hfr sup J. Miller strain kit CSH64 Hf'r thi J Miller strain k i t CSH67 Hfr EY!--lac gal mtl malA thi >.. J Miller strain kit CSH74 Hfr thi J. Miller strain kit

PAGE 48

Table 2. Antibiotic disc assays. Antibiotic (Cone.) Kanamycin Bacitracin Ri f ampin Nalidixic Acid Strain 1'em:e. (30 g ) (10 Uni~s ) ( 5 g ) ( 30 g ) x462 39C 10. 5, 15a 6 10 10, 18 x462 30C 9, 15 7 10.5 lr{, 23 5 615A 39C 1lr, 1 8 6 10 10, 20 615A 30C 20 14 15 25 Rev4 39C 11, 17 6 9 5 16, 18.5 Rev4 30C 10, 16. 5 7 10 17 ;. ev8 39C 11, 17 6 9 16 Rev8 30C 10. 5, 16 7 10 18 Trans4x 39C 9 5, 14 6 9 9, 19 Trans4x 30C 9 ,5, 16 6.5 10 14, 18. 5 Trans 71 39C 10, 14 6 8.5 8 .5, 17 Trans71 30C 10, 15.5 7 10. 5 10, 19 aTwo zones of inhibition were apparent. An outer zone with sligh t turbidity was present surrounding the clear inner zone. Strains xl162, 615A, revertants (Rev4 ,8) and transductants (Trans4x, 71) wer e grow n overnight in L-broth at 39C. Approximately 4 X 106 bacteria/ml of each culture was diluted into 100 ml sterile, precooled L-agar. 15 ml of tr, e seeded agar was pipetted into petri dishes. After the agar had hardened a nd dried for 2-1/2 hours, anitibiotic discs ( 1 per plate) were placed in the center and the plates w e r e incubated a t either 39C or 30C for 18 hours. Diameter of zone s of inhibition was measured in mm (diruuete r of each disc is 6 mm). 39

PAGE 49

Table 3 DNA per mass ratios of strain x462 and strain 615A. STRAIN 'l'EMPERATUREa DNA/MASSb xJ162 39C 2577 615A 3 9 C 2 699 x 4 62 30 C 1 882 615A 3 0 C 20 -r5 aT'nis is the t emperature at '\-:"bich the cul tare was uniforml y labeled (.F'j gvres 7 and 12). bDNA/ m ass ratio is the average cpm/OD550 between the OD550 rang e of 0.1 t o 1.0 (Figure s 7 and 12). 4 0

PAGE 50

~l Table !1. ResuJ.t s of Hfr and 615A uninte i r upted m ating s % Coinh erita nc e Hfr S eJ.e cted Number Unselected Cha r acters Strain M a r ker Analyzed l ~ u purE met~ .;!;xsA Nl fUamentation CSH60 leu 90 100 0 0 CSH74 lysA 90 0 100 0 CSH67 metE 90 0 100 0 8 9 CSH47a El! 90 0 0 11 100 49 CSH6l+ 90 10 42 0 100 69

PAGE 51

h2 Table 5 Transductiona l a0.alyses. R ecipient Selected Numbe r o f of Cotransduction of Unselected Marker /0 Strain Marke r 'l'ransductants rnaJ.A _gl]2Q fil8_1nent formation x~62mg malA 179 98. 9 19.6 xlr62mg f.:1:pii 177 9 2 .7 9.0 LW:?-,m malA 180 89. 0 33. 0 LW3m 179 87. 7 52. 0 AB2847 aroB 158 21.5

PAGE 52

43 Table 6 Phospho1ipid composition in strains x462 615 A and 71. GROWTH fEHCENTAGE OF TO'l'AL STRAIN 'I'EMPERATURE: C L PG PE LE xl-162 39C 0.90 17 2 7 6 9 5 .00 615A 39C 0.80 l'"!.l 77 5 4 70 7 1 39C 0. 7 7 14 9 77 2 1 .~o x462 30C 2 00 15 9 76 2 6 .00 615A 30C 1.90 15 4 7 9 2 3 .60 71 30C 0.90 16 2 80.6 2.60 CL= Cardiolipin PG= Phos phatidylglycerol P E = Phosphatidylethanolami ne LE= Lysophosphatidy1ethanolamine

PAGE 53

Table 7 Fatty acid composition in strains x462 and 615A. PEHCF.NTAGE OF TOTAL GHOW'r!:[ S'l'RAIN TEHf'ERATUR E 12:0 111 :0 16 : 0 16: 1 til7 18: 1 xii.62 39C 1.42 2 .37 4'(. 9 2h.o 4.07 20, 1r 61.;iA 39C 2 .4 6 2.1+6 48. 7 24.9 1 r 1 2 18.8 x462 30C 2 13 2 .25 36. 2 30,9 28.5 615A 30C 1.14 3 l19 37. 1 31.0 27.4

PAGE 54

Table 8 Analysis of peptidoglycan in strains x462 Amino Acid or Amino Growth Strain Temperature Glucosamine Muramic Acid 615A 39C 525 3( 81 ) 606,3(.93) 615A 30 C 558.7(.82 ) 677. 3( 99) x46 2 39C 608 9 (.86) 690 .7(.97) x462 30C 532. 5 (. 80) 611. 0( 92) aRatio = nmol /mg amino acid or amino sugar runol/mg glutamic acid and 615A. Su ga r in ru:iol/mg (Ratio a, ) Diaminopi -Alanine melic A cid Glutamic Acid 1172 !;( 1. 8) 646 5( 99) 625,5(1.0) 1167 .6(1. 7) 649 ,7(.95) 684 .3(1.0) 1 1 66 .8(1. 6 ) 702 4( 99) 711.6(1.0) 1096.0(1.6) 596 7 ( 90) 666.5(1.0)

PAGE 55

T able 9. In vivo cro s s-link ing an8.l ysis of p e p t i doglyca n in strains x462 and 615 A G ro1-rt h Strain 'l'empe ratm' e dim er/monomer 615A 39C .88 6J.5A 30C 8 0 xi1G 2 39C 8 1 x462 30C r(2 a'l'he dim e r or cross-linked species of lysozyme -digest e d peptidog 1ycan migrates with a n Rr:::; 0 2 when applied to Whatman n o 3MM filte r paper an d subjected. t o descending chr o mat o~raphy in isohutyri c acid-lN am moni 1-1m hydroxide ( 5 : 3 ) The m o nomer or uncross-linke d species migrat e s with a n Rr =:; 0.4 in this system a 46

PAGE 56

'I'able 10. Analysis of bound lipoprotein in strains xl162 and 615A. Growth Strain Temperature Percentage of Bound Lipoprotein 615A 39C 5.0 615A 30C 4.5 x462 39C 4.4 x462 30C 4.3 aEach sample was normalised by comparing each bound lipoprotein value to total envelope counts applied to the chromatogram This assay i s based on the observati on that bound ( murein-linked) Jipoprotein can migrate on chromatograms in an isobutyric acid-1 N ammonium hydroxide solvent after it is released from murein by l ysozyme digestion. a

PAGE 57

Figure 1. Linkage map of Escherichia coli K-12 showing the origins and directions of transfer of Hfr s~rains (arrows).

PAGE 58

.... )(YI ... I mal~ asd I I I 73 aroB glpD }s

PAGE 59

Figur_ e 2 .Micrographs of strain 615A, a representative low-temperature condit ional cell division mutant and its parent, strain x462. A, filaments of stn:d n 615A at 30C afte1 2 hours; B strain 615A 2 hours after shifting filaments ( 2A) to 39C; C nuc lear stain of strain 615A after 2 hours at 30C ; D, strain x462 at 39C, for comparison. Bar r epresents 10 --meters.

PAGE 60

51 I ,, ( I (

PAGE 61

Figure 3. Cell division followj_ng a shift from 39C to 30C. Cultures of strain 615A (A) and strain x462 (B) grcwing exponen+,j_ally n.t 39C we:?:e split and one-half of each cu.1-:.ure was incubated at 30C. Increase in cell number was monitored for 3-1/2 hours following the shift to 30C. Symbols: ( ) 39C control culture, ( 0) 39C-30C shift culture.

PAGE 62

300 A 100 B / / 100 / 4 ; / I.I') / $ It, /. 0 0 ./ / 0 ,. I ,>< >< ./ / / 0 w 20 ./ w 8 / 0 cc ./ m /"//' _,,o :::, / o -o_,,.,JJ :::, z / .,,,,-0 0 / z _,.0--,::; // ..., .,,,,,,.o ..., ..., 5 j,o_,,o ..., 2 w w // u u I ~:/ I at / 1 30 90 150 210 0.40 30 90 50 210 TIME {min) TIME(min) \J7 w

PAGE 63

Figure 4. Vi abilHy of str6-i n 6.15A fol.lowing a temperature shift from 39C to 30C. An exponentially g r owi n g culture of straj_n 615A at 39C was split and one-half of the culture was incubated at 30C. Vi ability was monitored for 3 hours following the shift t o 30C by p lating samp les on L-agar and incubating the plates for 18 hours at 3'7C. Symbols : ( 0 ) 39C, ( 0 ) 30C,

PAGE 64

400 It) 2 100 >< Cf) _J _J w u L1J _J 20 (D <( > 55 Q. 60 120 180 TIME (MIN)

PAGE 65

Figure 5. Cell di vision follo,dng a shift from 30C to 39C Fila.'11.ents of stntin 615A were induced by growth for 2 hours at 30C. Cultures of st. :::-ain 615A (A) and strain x462 (3) at 30C were split .qnd one-half of each cul tw:e incubated at 39C. Increase in cell rmrr:ber was monitored for 2 hours following a shift to 39C Symbols: ( ) 30C-39C shift culture, ( 0 ) 30C control culture.

PAGE 66

90 A 5 8 / I (0 I (0 0 0 I X I X I ffi 25 Ck: 15 I LLJ 0 cc LU I / :E 0 :::> :::> i /I z z I /0 _J _J _J _J 5 / LJ.J 7 o-o.,,O LJ.J /; ttd!J/o-o/ u u 41) / _,,,,. J>o II 60 120 60 120 TIME(M1N) TIME(M1N)

PAGE 67

Figure 6. Growth of strains 615A and x462 in the r,tesence and absence of deoxycholate. Deoxv cholate (0.25% ) was added to exponentially growing cultures a.t 30C (A) and 39C (B) and absorbance at 550 run was monitored for 2-1/2 hours. Symbols : ( ) strain x462, ( 0 ) strain x462 + deoxycholate, ( ia ) strain 615A, ( 0 ) strain 615A + deoxy cholate.

PAGE 68

2.5 2.5 A B ,.....i....-~ / i 11" E 1.0 (9 ....... c1. ;~ ~ / / 0 / ? lt') .-,@ Lt) .1./ LJJ (' / Lt) / w u / .,... u ;/ z z 40.4 (It "' .r CD /11/ <( 0. / o-D""o 0::: .. / CD a-:: o' 0 Ill> /-0-D__.. II) C/) CD / / D <{ II) CD 0/ .15 / <:( :15 I/ _,,,,o,.....o 0 --0-0-0 'o 'o-o-o 30 90 1 .crto 0 90 150 TIME(M1N) TIME(M1N) \.Jl \0

PAGE 69

Figure 7. Effects of a shift from 39C to 30C on growth and DNA synthesis in st:cair:.s 615A (A) and xL162 ( B) Cultures were unifo::-m.ly prelabeled at 39C with 3H-thymidine, split and one-half of each culture was incubated at 30C. Absorbance of each culture was recorded and dur,:licate samples were processed for measurement of DNA synthesis. S:yn1bols : ( 0 ) increase in absorbance at 39C, ( 0 ) increase in absorbance at 30C, ( ti ) 3H-thymidine incorporated into DNA at 39C, ( D ) 3Hthym idine incorporate d into DHA at 30C.

PAGE 70

2.0 E C O 1.0 Lt) II) w u z <( 0.4 CD g CD <( N 0 >< 15 6 1 A 60 90 120 TIMECmin)

PAGE 71

2. E C 0 LO 1.0 Lt) w u z <( 0 CD 0 Cl) en < L a_ 5.0 u / / /. /. /0 / /0 /0 / o--o /. / ..,.......-o ___...--,o D 1 5o ______ 3 __ 0 __ 6__,j0..__ ____.9i.-0--1_.2_0 TIMECmin) 62

PAGE 72

Figure 8. Pulse-label measurements of DNA synthesis in strains 615A (A) and xi+62 (B) :'ollowi.ng a shift from 39C to 30C. Exponential cultures at 39C w ere split and one-half of each culture was shifted to 30C. Sam:iles were remoyed and pulsed f"or 4 ~im;.tes. Symbols: ( ) incorporation of 3H-thymidine at 39C, ( 0 ) inco;poration of 3H--:hy ,:iidine at 30C.

PAGE 73

70 150 A B 40 N N 0 0 X X L a.. a.. u u 1 60 120 60 120 TIMECmin) TIMECmin)

PAGE 74

Figure 9,Pulse-label measurements of RNA synthesis in strains 615A ( A ) and x462 (B) following a shif~ from 39C to 30C. Exponential cultUYes at 39C were split and one-half of each cuJ_ture was shifted to 30C. Samples were r emoved and pulsed for 4 minutes. Symbols: ( @ ) incorporation of 3H-uracil at 39C, ( 0 ) incorporation of 3H-uracil at 30C.

PAGE 75

400 A 300 B I / 100 I 100 N N 0 0 r--1 t--4 >< >< l 40 C.. 30 (.) u 0 1/ 0/ / / 10 9 0 60 120 0 60 120 TIMECmin) TIMECmin) (J'\ 0\

PAGE 76

Figure 10. Effect of chloramphenicol on induced division of filaments of strain 615A. Filamen t s of strain 615A were induce d in Lbroth at 30C for 2 hours and subdivided. At time z ero, all but one subculture, 30 C control with no additions were shifted to 39C. Chlora.mphenicol (CM) (100 g/ml) was added at v arious times afte r the shift to 39C. Increase in cell numbe r was monitored using an electronic particle counter. Symbols: ( 0 ) 30C control with no additions, ( ) CM at 15 minutes, ( "Ct) CM at 30 minutes (II) CM at 45 minutes, ( 0) CM at 60 minutes, ( 0 ) 39C control with no additions.

PAGE 77

68 60 0 (0 0 X Q: w (D 15 ::, z .....J --' w u 3._,____....___.....__~_ 0 30 60 90 TIMECmin)

PAGE 78

Figure 11. Slli!L11.ary of the effects of antibiotic additions on induced cell division of strain 615A. The data are expressed as the percentage of the control (39c control with no additions ) increase in cell number 75 minutes after the shift of filaments from 30C to 39C versus the time of addition of the antibiotic. Symbols: ( ) puromycin (330 g/ml), ( 0) chloramphenicol (100 g/rru), l II) nalidixic acid (10 g/ml), ( 0) penicillin (25 Units/ml), : ( lt ) rifarnycin SV ( 200 g/ml).

PAGE 79

7 0 w 100 *,---------* CD L ::) z 80 _J /2(9 _J w u 0 0 z w en <( w e::: u z w 20 z w 00 w 15 30 45 60 (L TIMECmin) -

PAGE 80

Figure 12. Measurements of growth and DNA synthesis in strains 615A (A) and xLr62 (B) following a shift from 30C t o 39C. Cultures were uniformly prelabeled with 3H-thymidine at 30C during growth and the induction of filaments (strain 615A). Cultures were split at time zero and one-half of each culture was shifted to 39C. Absorbance of each culture was recorded and duplicate samples were processed for measurement of DNA synthesis. Symbols: ( @)increase in absorbance at 30C, (0) increase in absorbance at 39C, ( II ) 3H-thymidine incorporated into DNA at 30C, ( 0 ) 3H-thymidine incorporated into DNA at 39C.

PAGE 81

E C 0 Lt) It) 2.0 A w u z <( 0.4 CD cc <( N 0 >( 0.1 r---------'----'----'-so 15 1.ooo-laiool-~60n-,9~0:----.......__ 120 TIMECmin) 7 2

PAGE 82

2.0 E C f5 Lt) w u Z 0.4 <( CD Ck: 0 Cf) al <( N 0 >< :l: 20 CL U 5.0 B 1.50:;--~:0-f~-----=:1-=--..L-J 30 60 90 120 T IMECmin) 73

PAGE 83

Figure 13. Phospholipid turnover during induced division in strain 615A. Filaments were prelabeledwith 3 2P-orthophosphate, washed and incubated in fresh growth medium at 39C (solid lines) and 30C (dashed lines) at time zero. Symbols: ( G ) lysophosphatidylethanolamine, ( 0 ) phosphatidylethanola.mine, ( U ) phosphatidylglycerol, ( 0 ) cardiolipin.

PAGE 84

80 -g--o o =--.. 0 ......... ---0 -=--------------0 0 a.. :::i 60 0 :r: a.. en 0 :r: a.. _J ;:! 40 LL 0 w l!) ~20 U -=--------------.--11 .... -----------0:: ---w a.. 75

PAGE 85

Figure 14. Pulse-label measurements of phospholipid synthesis in strains 615A (A) and x462 (B) following a shift from 30C to 39C. Cultures were grown at 30C with the induction of filaments in strain 615A. At time zero, the cultures were split and one-half of each 2ulture was shifted to 39C. Samples were removed and pulsed for 4 minutes. Symbols: ( 8) incorporation of 32P-orthophosphate into phospholipids at 30C, ( 0) incorporation of 32P-orthophosphate into phospholipids at 39C.

PAGE 86

150 200 A B N N 0 0 p-f ,-f ><40 >(50 :E c.. Cl. t) (.) 60 12 0 60 120 TIMECmin) TIMECmin)

PAGE 87

Figure 15. Thin-layer chromatography of LPS isolated from strain x462 and strain 615A. Chromatography of whole LPS from cultures of strain x462 and 615A grown at 30C or 39C was identical.

PAGE 88

LPS FRONT I I x462 0 ... .. I 0 615A 0 '(9

PAGE 89

Figure 16. Difference plots of membrane protein profiles from strains x462, 615A and 71. A, Comparison of inner membrane proteins from cultures of stain x462 and strain 615A grown at 30C; B, comparison of outer membrane proteins from cultures of strain x462 and strain 615A grown at 30C; C, comparison of outer membrane proteins from cultures of strain x462 and strain 71 grown at 30C. The positions of Regions a and b are assigned by comparison of membrane protein profiles before the difference plots are generated. The bottom gel (C) has fewer slices because this gel did not electrophoresize as far as the other 2 gels.

PAGE 90

.2 A a b 0 -.2 .4 8 a b u 0 T-0 -0 I -.4 I .4 a b M C 0 -0 0 -.41---L--....1.-------'--__.__.....____,_ __,___....___ 0 20 40 60 80 SLICE NUMBER

PAGE 91

Figure 17. Comparison of membrane proteins from cultures of strain x462 (dashed line) and strain 615A (solid line) grovm at 30C. Membrane proteins of strain 615A labeled with 3H-arginine and membrane proteins of strain x462 labeled with 14c-arginine were electrophoresized on SDS-polyacrylamide gels. The gels were sliced, the radioactivity in each gel slice determined and plotted.

PAGE 92

100 80 60 u UJ 40 \ r' <{ 1 1 uJ \ a: 20 \ I 10 I I I l I 20 30 40 SLICE NUMBER I I \ I \ \\ I .\_ 50 60 70 0::, w

PAGE 93

LITERA'EURE CITED 1. Adler, H I., W. D. Fisher, A Cohen, and A A Hardigree. 1967 .Minature Escherichia coli cells deficient in DNA. Proc. Natl. Acad. Sci. U. S A 57: -3 21-326 2 Ahmed, N and R. J Rowbury 1971. A temperature-sensitive cell division component in a mutant of Salmonella tYPhimurium J. Gen. Microbial. 67: 107-115 3. Allen, J. S., C. C. Filip, R A. Gustafson, R G. Allen, and J. R. Walker. 1974. ReguJ.ation of bacterial cell division: Genetic and phenotypic analysis of temperature-sensitive, muJ.tinucleate filament-forming mutants of Escherichia coli. J. Bacterial. 117: 978-986. 4. Allen, R. G., J A. Smith, R C Knudsen, and J. R. Walker. 1972. Initial charac+~erization of temperature-sensitive cell d i vision mutants of Escherichia coli. Biochem Biophy. Res Com.~. 47: 1074-1079. --5. Ames, G. 1968. Lipids of Salmonella !l:Phimmium and Escher:i.chia coli: Structure and function. J. Bacterial. .22_: 833 -81+3. 6. Ames, G F., E N. Speidich, and H Uikaido. 1974. Prote:fo composition of the outer membrane of Salmonella typhimurium: Effect of lipopolysaccharide mutations. J. Bacterial. J.l 7 : !106-416 7. Anr a.ku Y. and L. A Heppel. 1961. 0:;-i the nature of the changes induced in Escherichia coli by osmotic shock. J. Biol. Chem. 2Lr2: 2561-2569 8. Bachmann B J., K B. Low, and A. L. Taylor. 1976. Recalibrated linkag e map of Escherichia coli K-12 Bacterial. Rev 40: 116-167. 9. Beck, B D. and J. T Park. 1976. Activity of three murein hydro lases during the cell division cycle of Escherichia coli as measured i n toluene-treated cells. J. Bacterial. 126: 1250-1260. 10. Bell, R. M., R D. Mavis M, J. Osborn, and P. R. Vagelos. 1911. Enzymes of phospholipid metab olism: Loc a lization in the cytoplasmic and outer membr ane of the cell envelope of Escherichia coli and Salmonella typhimuri u.rn. Biochirn Biophy. Acta 21.19: 628-635, 11. Braun, V. and V Bosch. 1972, Sequence of the murein-lipoprotein a.nd the atto.chmcnt site of the lipid. Eur. J. Biochern. 2 8: 51-69. 84

PAGE 94

1 2 Bra.un V. and U Sieglin. 19'(0 'J'he covalent mm-ein-lipoproteir. structure of the Ese:hc r iclJ J .F.I. coli c ell w all. Th e atta chr.::ent sit e -----85 of the lipoprotein on the ~u rein. Eur. J Bioche m 13: 336-346. 1 3 Br a u n V. and H. W olff. 19'( 0 'l h e mur ein--lipoprot ein linkage fo the cell w all of E s cherchia coli. Eur. J. Biochcm. 14: 3 87-391. 14. Burdett, I. D J and R G E Murr a y 1974. study of septlUn f o rmation i n Escherichia c o~i duxing synchronous growth. J. Bac:teriol. 119: Electron mic:roscc~ e strains Ban d B / r 1039-1056. 15. Buttke, T M ru1d L 0 Ing ram. 1975, Comparis o n of lipopolyse.c charicles fro m Agmen e1J.um q_ua d r uplicat~ to Esche richia c oli and S a lmon ella !_;[phi murium by using thin-la y e r chromatography. J Bacterial. 124: 15 66-1573, 16. Cl ark, D J. 1968. Regulation of deo:x--yribonucleic acid replica tion and cel l di visio n i n Escherichia c oli B / r J. Bacte~iol. J i : 1214-122)~. 17. C rurnplin, G. C. and J T. Sm:i.th. 1976. Nalidixic acid and bacterial chromosome replication. N ature 260 : 643-644. 18. D avies, J K a n d P Reeves. 1975, G enetics of resista n c e to colicins in Escherichia coli K 12 : Cr oss-resistance among colici'-s of group B J. Bacterial. 123: 96-101. 19. Deca d G. M. and H Nikaido. 1976. Out e r membrane of grclil.-nege._-:ive b acteria. XII. Molecular-seiving f u nction of cell wall. J. Bacteria] 1 28 : 325 336 20. D e Pedro, M. A., J.E. Lla.mas and J. L Ca.novas. 1975. A timi=g control of cell division in Escherichia coli. J. Gen. ~licrobiol. 91: 307-314. 21. D i x D. E and C E. Helmstetter. 1973. Cou pling between chror::.o some completion and cell di vision in Escherichia coli. J. Bacte~iol. 115 : 78 6-795. 22. Duckworth, D. H. and G. B. Dunn. 1976. Membrane protein biosynthe s i s in T5 bacteriophage-infected Escherichia coli. Arch. Bioche= Biophy. 172: 319 328 23. Egan, A. F. and R. B. Russell. 1973. Conditional mutations affecting the cell envelope of Es c h erichia coli K-12 Genet. Res 21 : 139152 24. Eriksson-Greenberg, K G., K Nordstrom, and P Englund. 1971. Resistn.nce of E scherichia coli to penicillins. IX. Genetics ani phy siolog y of class II ampi cillin-resistant mut ants that a.re gals.c tose negative or sensitive to bacteriophage C21, or both. J. B acterial. 108: 1210-1223

PAGE 95

86 25. Feige, U. and S. Stirm. 1976 On the structure of the E scherichia coli C cell wall lipopolysaccharid. e core and on its cpxrrh receptor region. Biochem Biophy. Per; Comm 71: 566--573. 26. Fuh s C. W. 1973, Cytochernical examination of blue-green algae, p. 117-143. In N. G. C arr a nd B A Whitton ( ed.), The biology of the blue -green algae, Vol. 9 Univerisity of California Press, Berkeley and Los Angelos, California. 27. Geiduschek, E P. and R Has elLorn. 1969, M essenger RNA. Annu. Rev. Bioche m 38: 647-676, 28. Ghuysen, J.M. and G. D. Shock.man. 1973 Biosynthesis of peptidoglycan, p. 37--130. In I.. Leive (ed.), Bacteria l m embranes and walls, Vol. 1. Marcel Dekker Inc., New York. 29. Gre en, M. H. L., J Greenl,erg, and J. Donch. 1970, Effect of a recA gene on cell divisio n and capsular polysaccha.ride production in a lon strain of E sch2richi,':!:_ coli. Genet. Res. Camb. H: 159 -169. 30. Grula, E A. and M. M. Grula. 1 962 Cell division in a species of Erwinia. V. Effect of metabolic inhibitors on terminal divi------sion and composition of a "division" medium. J. Bacteriol. 8 4 : 492-499. 31. Hambleton, P. 1971. Repair of wall damage in Escherichia coli recovered from an aerosol. J. Gen. Microbial. : 81-88~ ---32. Havthorne, D. C. and J. Friis. 1964. Osmotic remedial mutants. A new classification for nutritional mutants in yeasts. G enetics 50: 829-839. 33. Heppel, L.A. 1971. The concept of periplasmic enzymes, p. 223-247. In L. I. Rothfield (ed.), Structure and function of biological membranes, Vol. 1. Academic Press, New York. 34. Hirashima, A. and M. Inouye. 197 3 Specific biosynthesis o f an envelope protein of Escherichia coli. Nature 24 2 : 1105-407. 35. Hirota, Y., .J. Mordoh, and F. Jacob. 1970, On the process of celluar division in Escherichia coli. III. Thermosensitive mutants of Escherichia colj_ altered in the process of deoxyribonucleic acid initiation .J. Mol. Biol. .2]_: 369-387, 36. Hirota, Y., M. Ricard, and B. Shapiro. 19'(1. The use of thermosensitive mutants of Escherichia coli in the analysis of cell division, p. 13-31. In L. A. Manson~), Biomembranes, Vol. 2. Plenum Press, New York. 37, Hirota, Y., A. Ryter, and F. Jacob. 1968. Thermosensitive mutants of Eschc>ric h i a coli affected in the process of deoxyribonucleic acid synthesis and cellular division. Cold Spring Harbor Symp.

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Quant. Biol. 33_: 677-693. 38. Hirota Y S W&kil, T3. Shapiro, A. Hyter, J". H urwitz, and F Jacob. 1969. Sur un mut ar : t t:1e:r;nosensible (t I Es r.:h<::richia coli des anomalies d e la membrane C.R. Acad. Sc. Par:i.s ~69: 1346-1348 39. Hoffmcin, H and M. E Frank. 1963 Time-lapse photomicrography 8 7 of the formation of a free sph erica l granule in an E sche6chia coli cell end. J Bacterial. 86: 107 5 -1078 40. Hoffmann, B., W J'1csser and U Schw arz. 1972. Regulation of polar cap formation in the life cycle of E scheri~hia coli. J Suprarn Struc 1 : 29-37 41. Holland, I B. and V Darby. 1976 G enetica l and physiological studies on a thermosensitive mut ant o f Escherichia coli defective i n cell di vision J. Gen. Micro biol. _2~: 156-166. 42. Ingram L 0 and H C .. Aldric h 1971L Cell separation in blue green bacteria J Bacteriol. 118: 708-716. 43. I ngra.m L 0 and W. D. Fisher. 1973. Sti~ulation of cell division by membrane-active agents. Biochem Biophy. Res Comm .2.Q: 200210. 4 4 Inouye, M. 1969 Unlinking of cell division from deoxyribonucleic acid repl i c ation in a t e"1.perature-sensitive dem, yTibonucJ.eic acid synthesis mutant of Escherichia coli. J. Bacteriol. 9 9: 842-850 45. Inouye M 1971 Pleiotrophic effect of the recA gene of Escherichia coli: Uncoupling of cell division from deoxyribonucleic replication.J: Bacteriol. 106 : 5395 42. 46. Inouye, M 1972 Reversal by NaCl of envelope protein change s related to deoxyribonucleic acid replication and cell division of Escherichia co li. J Mol. Biol. 63: 597-600 4 7 Jones, N C and W. D Donachie. 1973 Chrom o some replicati on transcription and control of cell. di vision of Escherichia coli. Nature New Biology 241: 1 00-103 4 8 Kanuryo T and J L Strominger. 1974 Penicillin-resistant temperature mutants of Escherichia coli which synthesize hypo-or hyper-cross-linked peptidoglycan. J. Bacterial. 117: 568 -577 49. Kanfer, J and E P Kennedy 1963. M e tabolism and function of bacterial lipids. I Met abolism of phospholipids in Escherichia coli B J Biol. Chem. 238: 2919-2922 5 0. L avalle, R and G De H auwe r 1968 Messenger RNA synthesis during runino acid starvation in Escherichia coli. J Mol. Biol. 37: 269-2 8 8. 51. Lazdunski, C and B M Shapiro. 197 2 Relat:i onship between permeability, c ell di vision and mur ein m etabolisr:.i in a mutant of E scherichia coli. J Bacteriol. 111: 499-509.

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90 77 Normark, S 1971. Genetics of a cha.in-forming mutant of Escherichia coli. Genet. Res. 16: 63-78. 78. Nor mark, S., H G. Boma n and G D Bloom. 1971. Cell division in a chain-forming envA mutant of Escherichia coli K-12. Fine structure of division sites a nd effects of EDTA, lysozyme and ampici)lin. Acta Fathol. Micro biol. ScancL Sect. B ]9: 651 664 79, Norrnark S H. G Boman, and E Matsson. 1969. Mutant of J~scb erichia coli with anomalous cell division and ability to decrease episomally mediated resistance to ampicillin and several othe r antibiotics. J Bacterial. 97: 1334-13112 80. Ohki, .M. 1972. Correlation between metabolism of phosphatidylglycerol and membrane synthesis in Escherichia coli. J. Mol. Biol. 68 : 2119-264 81. Osborn, M. J., J.E. Gander, E. Parisi, and J Carson. 1972 M ec h anism of assembly of the outer membrane of Salmonella typhimuriurn J. Rial. Chem. 24 7: 3962-3972. 82. Pugsley, A. P and P. Reeves. 1976. Increased production of the outer membrane receptors for co.licins B, D and M by Escherichia coli under iron starvati0n. Biochem Biophy. Res Comm. 70: -8:.<). 83. Randall-Hazelhauer, L and M Schwarz. 1973. Isolation of the bacteriophage l ambda receptor from Escherichia coli. J. Bacterial. 116: 1!,36-1446. 84. Ray, B., J. J. Jezeski, and F. F. Busta. 1971. Repair of Injury in freeze-dried Salmon ella anaturn Appl. Microbial. 22 : 40l-lf07. 85 Reeve, J N., D J. Groves, and D. J. Clark. 1970, Regulation of ce11 division in Escherichia coli: Characterization of temperaturesensitive division mutants. J~acteriol. 104: 1052-1064. 86. Ricard, M. and Y. Hirota. 1969. Effet des sel s sur le processus de division cellularie d 'Escherichia coli. C.R. Acad Sc. Paris 268: 1335-1338. 87. Ricard, M. and Y Hirota. 1973. Effet des sels et autres composes sur le phenotype de mutants thermosensibles de Escherichia coli. Ann. Microbial. (Inst. Pasteur) 124A: 29-43 88. Ricard, M and Y Hirota. 1973. Process of cellular division in E sl!herichia_ coli: Physiological study of thermosensitive mutants defective in cell division. J. Bacterial. 116: 314-322 89. Ricard, M., Y Hirota, and F. J acob. 1970. Isolement de mutants d e membrane chez Escherichia coli. C. R. Acad. Sc Paris 27Q_: 2591-2593.

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103. Starke J.~ D. Di 1971 r Phenotypic E scherichia coli. Savino, G Michel, A Rodolakis, and P. Thomas. expressio n of an en vC-di vi,-;ion mutant of Ann. !-1icTo 1 :5.)l. ( J i1St. P a:~teur) )_;_25B: 227 232 lOL1 Stone A. B 1973. Regu1ation of c ell division in a temperature sensitive divisio n mutant of Esch erichia co1i. J. Bacterial. 116: ---------7111-75 0 105. T amald S T Sato, and M 11atsuhashi. 1971. Role of lipopolys accharide in antibiotic resis~anc e a nd b acteriophage adsorption of Escheri chi3. c oli K -12. J Bacterial. )-05: 968-975. 92 106 T aylor, A L and C. D Tr'.)tter. coli K-12 Bacteriol. Rev. 36 : 1972 Linkage map of Escherichia 504-524. 107. Torti, S. V. and J. T. Park. 1976. Lipoprotein of gram-negative b acteria is essential for e;r owth and d i vision. Na-1;,ure 263 : 3 23 3 26 108. v an de Putte, P., J; van Di llewijn, and A. Ror s c h 1964. The selection of mutants of Escherichia coli with impaired cell division a t e l e v a ted temperature s Mutat ~Res. 1: 1 21-128 109. Walker, J. R., A Kovar:i.k J. S. Allen, and R. A. Gustafson. 1975 Regulation of b acterial cell division: Temperature-sensitive mutants of Escherichia c oJi tha t are defective in septum-formation. J. B acterial. 12~: 693-703. 110. Wehr, T., L. Waskell, and D A Glaser. 1975, Characteristics of cold sensitive mutants of Escherichia coli K -J.2 defective in d eoxyribonucleic acid replication. J. Bacterial. 1 21: 99~107 111. Weid el, W. and H Pelzer. 1964 Bagshaped macromolecules--a nev outlook in bacterial cell walls, p. 193-232. In F. F Nord (ed.), Advances in enzymology, Vol. 26. Interscien c e Publishers Inc., New York. 112 W e igand, R. A. and L. I. Rothfield. 1976 Genetic and physiologic classification of periplasmic-leaky mutants of Salmonella typhimurium J. Bacterial. 1 25 : 3!10-345. 113. W e igand, R. A., K. D. Vinci, and L. I. Rothfield. 1976. genesis of the b acterial division septwn : A new class of d efective mutants. Proc. Natl. Acad. Sci. U. S. A. 73: 1886 Morpho sept ation-1882 -114. Westph al, 0. and K. Jann. Bacteria l lipopolysaccharides: Extraction with p h enol-water and fu:-the r applications of the procedure, p 83-91. In R. L. Whistler and M. L. Walfrom (ed.), Methods in carbohydrate chemistry, Vol. 5. Academic Press Inc., N e w York.

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115. Wij sma n H J. W 19'12. A genetic map of several mutations af fecting the m ucopeptide layer of Escherichia coli. G enet. Res. 20: 65-74. 93 116. Willsky, G. R R L. Bennett, and M H. M a la.my. 1973. Inorganic phosphat e transport in Ef;cherichia coli: Involvement of two genes which play a role in alkaline phosphatase reguJ_ation. J. Bacteriol. 113: 529-539. 117. Wolf-Watz, H and S. Normark 1976. Evidence for a role of Nacetylmw.arJ1yl-L-alanine amidase in septum separation in Escherichia coli. J. Bacteriol. 128: 580-586. 118. Wu, H. C 1972 Isolation and characterization of an Escherichia coli mutant with alteration in the outer membrane p r oteins of the c ell envelope. Biochim. Biophy. Acta 290: 27li-2 39. 119. Zusman, D R 1973. Membrane protein synthesis i n Esc h erichia coli: Sensitivity to chloramph enicol. Arch. Biochem Biophy. 159: 336-31.il. 1120. Zusman, D R., M Inouye, and A. B Pardee. 197 2 C ell division i n Escherichia coli: Evidence for regulation of septation by effector molecules. J. Mol. Biol. 69 : 119-136. 121. Zusrr.an, D R and D .M. Krotoski. 197 4 C ell division in ;Escherichia coli: Analysis of double mutants for filamentation and abnormal septation of DHA-less cells. J Bacteriol. 120: Jlt27-llI33.

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BIOG RAPHICAL SKF}"l'CH The a uthor was born Joyce Anne Stanfield on Novembe r 20 1951, in the Panama C a nal Zone. Sh e was rea r e d by her fat h e r and mothe r in Salina, Kansas, and Orlando, Florida She was graduat e d froru Edgewater High School, O r l ando, Florida, in 1969. She took a B S. degree cum l aude from the University of Florida in June, 1973. The author entered graduate schoo l at the University o f Florida late r that month and is presently a candidate for the Doctor of PhiJ.osophy degree in the Department of Microbiolog y and C ell Science The author is married to Roy Joseph Sturgeon who is currently a c andidate for the Doctor of Philosophy degree i n the Department of Pharmaceutical Chemistry at the University of Florida.

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I certify that I have read t : 1 i s study' a;. 1 d that in my opim .. on it co;1forms to a cceptable standards of scholarly presentation and i s fully adequate, in scope and quality, a s a dissertation for the degree of Docto r of Philosophy. J 'IC0"~' ~AL, V, \ ~-7'f---.,.~=----=<'---Lonnie O. IngraJU~~:n AssociatE:l Professor of Microbiology and iCell Science I c e~'.tify tha t I have read t his study atj.d that in my opini o n it c o ~ f orms to acceptable standards of scholarl~ prese ~ tation and is full y adequate, i n scope and quality, as a dissertation for the degree of Doctor of Ph~losophy. I I ----/ .. .Ji &1u:.4~'1,...::;_____._...c:..:-+,~,."'3~"""'.C..,,.~<---Henry C drich I Profess of Microbiology:and Cell I .. I 1 Sc1.ence I certify that I have read this study and that in my opinion it conf orms to accep table standards of scholarly presentation and is fully adequate, ::.n scope and y_uali ty, as a dissertation for the degree of Doctor of Philosophy. Arnold S. Bleiweis Professor of Microbiology and Cell Science I certify that I have read thi s stud y and that in my o pinion i t confor j : s t o acceptable standards of scholarly presentation and is fully adequate, i n scope and quality, as a dissertation for the degree of Doctor of Philosophy. ~ij/LC/~ D onn a H D uckworth Associ ate Professor of Medical Microbiology and Immunology

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I certif'r that I have read t his study and that in my opinion it conforms to acceptable standards of scho.:...arly :presentation and i s fully adequate, in scope and ~uality, as a dissertation or the degree of Doctor of Philosophy. Associate Mi~robiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, i n scope and quality, as a dissertJtion for the degree of I Doctor of Philosophy. Edward M. Hoffmann Associate Professor of Micro?iology and Cell Science This dissertation was submitted to the Graf.::.mte Faculty of the Department of tf icrobiology. a nd Cell Science in the C oi.lege of Arts and Sciences and to t~1e Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. June 1977