Abortive T5 bacteriophage infections of Escherichia coli containing the colicinogenic factor, ColIb

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Abortive T5 bacteriophage infections of Escherichia coli containing the colicinogenic factor, ColIb
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Glenn, Jerry, 1951-
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
    List of Figures
        Page vii
        Page viii
    Abbreviations used
        Page ix
    Abstract
        Page x
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
    Materials and methods
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Results
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
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        Page 33
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        Page 36
        Page 37
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        Page 39
        Page 40
        Page 41
        Page 42
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        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
    Discussion
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
    Bibliography
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
    Biographical sketch
        Page 101
        Page 102
        Page 103
        Page 104
Full Text













ABORTIVE T5 BACTERIOPHAGE INFECTIONS
OF Escherichia coli CONTAINING
THE COLICINOGENIC FACTOR, Col lb










By

JERRY GLENN













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



UNIVERSITY OF FLORIDA 1979











































To Steven E. Ross and the late James W. Dunlop Their accomplishments have given me inspiration, and

their advice and encouragement have given me direction.













ACKNOWLEDGEMENTS

I acknowledge the help and support of my parents and my wife. Without them, this dissertation would not be.

Also, I want to express my gratitude to the Department of Surgery of the University of Florida, College of Medicine. They have iven me the freedom to pursue an extremely unconventional

surgical training program.

Finally, I thank the people who have guided me in learning to answer questions in a scientific manner. My advisory committee --Drs. Donna Duckworth, Kenneth Berns, George Gifford, and Carl Feldherr -- was quite helpful. In particular, I thank Donna Duckworth for giving me an idea whose time had come and for guiding me in the right direction. Also, discussions with Kenneth Berns during Dr. Duckworth's sabbatical year were sustaining. Drs. William Holloman, Nicholas Muzcyzka, and Thomas Pinkerton contributed numerous suggestions. Glenda Dunn gave me invaluable technical instruction and assistance.













TABLE OF CONTENTS

ACKNOWLEDGEMENTS ......... ........................
LIST OF TABLES ........ ......................... ... vi
LIST OF FIGURES ........ ......................... ...vii
ABBREVIATIONS USED ....... ....................... ....ix
ABSTRACT ............ ............................ x
INTRODUCTION ............ .......................... 1
Abortive Infections Mediated by the
Collb Factor ........... ........................ 2
MATERIALS AND METHODS ....... ...................... 9
Organisms ............ ........................... 9
Media and Growth of Bacteria ....... .. ............... 9
Colicin Production ....... ...................... I.11
Phage Growth ........ ......................... I.11
Phage Infections ....... ....................... ....12
Macromolecular Synthesis ...... ................... ...12
Dye-Buoyant Density Equilibrium
Centrifugation of Bacterial DNA .... ............... ...13
Host-Cell DNA Breakdown ....... ..................... 15
Proline and Glutamine Uptake ........ ................. 15
o-Methylglucoside (oMG) Uptake ..... ................ ... 16
Fluroescence Experiments ...... ................... ...16
Potassium Efflux ........ ....................... ...17
Attempts to Prevent Abortive of T5
Infection in ColIb+ Hosts ..... .................. ...18
Gel Analysis ........ ......................... ... 18
Statistical Methods ......... ...................... 19
Materials ............ .......................... 20
RESULTS .......... ............................ ...21
Macromolecular Synthesis in T5 and T5hl2Infections of RM 42, RM 43, and RM 39 ............... ... 21
Confirmation that RM 39 Contains
Plasmid DNA ........ ......................... ... 28
Confirmation that T5hl2- Infections
Begin Promptly ........ ....................... ...31
Amino Acid Accumulation in T5 and T5hl2Infections of RM 42, RM 43, and RM 39 ............... ... 31
Confirmation that Inhibited Proline and Glutamine Accumulation is Not Due Solely
to Inhibited Protein Synthesis ..... ............... ...42
(Y-Methylglucoside Accumulation in T5 and T5hl2- Infection of RM 42, RM 43, and
RM 39 .......................................... ...42
Glucose Incorporation into Macromolecules in T5 and T5hl2 Infection of RM 42,
RM 43, and RM 39 ....... ...................... ...44



iv








Fluorescence Intensity of NPN During
T5 and T5hl2- Infection of RM 42,
RM43,andRM39 ................. ........ 50
Potassium Efflux During T5 Infection
of RM 42, RM 43, and RM 39 ........... ....... .. 53
Attempts to Prevent Abortion of T5
Infection of Collb-Containing Cells . . . . . 53
Macromolecular Synthesis in T5 Wild-Type
and T5A1- Infection of RM 42, RM 43,
and RM 39. ...... .. .......................... .. 57
Proline, Glutamine, and MG Accumulation
in T5 Wild-Type and T5aml6d-Infected
RM 42, RM 43, and RM 39......... .......... 59
Fluorescence of Membrane-Bound NPN
During T5A1- Infections . . . . . . . . 71
Potassium Efflux During T5aml6d Infections
of RM43 .....*-*........................... 71
Absence of Host DNA Breakdown During
T5aml6d or T5amH27 Infection . . . . . . . . 75
Gel Analysis of Phage Proteins . . . . . . .. . 75
DISCUSSION ................. ............... .. 81
BIBLIOGRAPHY ............... ................. ..93
BIOGRAPHICAL SKETCH ..................... 1. 01































v













LIST OF TABLES

1. Characterization of Bacteria and Phage . . . . 10

2. Proline Accumulation (in 60 seconds) by Infected RM 42, RM 43, and RM 39 . . . . . 37

3. Glutamine Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39 . . . . . 41

4. Proline and Glutamine Accumulation in Uninfected and Infected RM 43 After
1 Minute or 15 Minutes of Incubation
with Chloramphenicol ................... 43

5. a-Methylglucoside Accumulation (in 90 seconds) by Infected RM 42, RM 43,
and RM 39 . . . . . . . . . . . . 47

6. Potassium Efflux from T5-Infected RM 42, RM 43, and RM 39 ............ ... .56

7. Attempts to Prevent Abortion of T5 Infection of Collb+ Hosts . . ................ 58

8. Proline Accumulation (in 60 seconds)
by Infected RM 42, RM 43, and RM 39 . . . . . 66

9. Glutamine Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39 . . . . . 68

10. a-Methylglucoside Accumulation (in 90
seconds) by Infected RM 42, RM 43,
and RM 39 ..... ....................... .. 70

11. Potassium Efflux from TSAl--Infected
RM 42 and RM 43 ............ ............ 73












vi













LIST OF FIGURES

I. Uridine Incorporation into Acid-insoluble Macromolecules in Uninfected anc Infected
RM 42, RM 43, and RM 39 ....... ................. 22

2. Tyrosine Incorporation into Acid-insoluble Macromolecules in Uninfected and Infected
RM 42, RM 43, and RM 39 ..... ................. ...24

3. Proline Incorporation into Acid-insoluble Macromolecules in Uninfected and Infected
RM 42, RM 43, and R1 39 ....... ................. 27

4. Ethidium Bromide-Cesium Chloride Gradients of DNA Extracted from RM 42, RM 43, and RM 39 ... ...... 30

5. Host DNA Breakdown After T5hl2--Infection of RM 42, RM 43, and RM 39 .... ................ ...32

6. Proline Accumulation in 60 Seconds by Infected RM 42, RM 43, and RM 39 ..... ............. 36

7. Glutamine Accumulation in 90 Seconds by Infected RM 42, RM 43, and RM 39 ...... ............ 40

8. a-Methylglucoside Accumulation in 90 Seconds by Infected RM 42, RM 43, and RM 39 ... ........... ...46

9. Glucose Incorporation into Acid-insoluble Macromolecules in Uninfected and Infected
RM 42, RM 43, and RM 39 ....... ................. 48

10. Fluorescence Intensity of N-phenyl-lnaphthylamine During T5 and T5hl2
Infections of RM 42, RM 43, and RM 39 ... .......... ...52

11. Potassium Efflux from T5-Infected RM 42,
RM 43, and RM 39 ...... ..................... 55

12. Uridine Incorporation into Acid-insoluble
Macromolecules in Uninfected and TSAI-Infected RM 42, RM 43, and RM 39 ..... ............. 60

13. Tyrosine Incorporation into Acid-insoluble
Macromolecules in Uninfected and T5AlInfected RM 42, RM 43, and RM 39 ..... ............. 62



vii








14. Proline Accumulation in 60 Seconds
by T5Al--Infected RM 42, RM 43,
and RM 39 . . . . . . . . . ... .. 65

15. Glutamine Accumulation in 90 Seconds
by T5Al--Infected RM 42, RN 43,
and RM 39 .... . . . . . . . . . . 67

16. a,-Methylglucoside Accumulation in 90 Seconds
by T5A1 -Infected RM 42, RM 43, and RM 39 . . . 69

17. Fluorescence Intensity of N-phenyl-lnaphthylamine During T5Al--Infections
ofRM42, RM43, andRM39 ................ 72

18. Potassium Efflux from T5A1-Infected
RM 42 and RM 43 .......... .......... .74

19. Absence of Host DNA Breakdown During
T5Al--Infections of RM 42, RM 43,
and RM 39 .......... ..............76

20. Gel Analysis of Phage-Induced Proteins
During T5 and T5Al--Infections of RM 42
and RM 43 ............... ..... .... .80




























viii













ABBREVIATIONS USED

a-methylglucoside (a-inethyl-D-glucopyranoside) DCCD NN'.-dicyclohexylcarbodiimide

EDTA ethylenediamine tetraacetate

NPN N-phenyl-l-naphthylamine

ONPG o-nitrophenylgalactoside

SDS sodium dodecyl sulfate

SV40 simian virus 40

TCA trichloroacetic acid

TEMED NNN',N'-tetra-methyl-ethylenediamine

TES buffer Tris-EDTA-saline buffer

TMG thio-O-r.iethyl-D-galactoside


























ix








Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy




ABORTIVE T5 BACTERIOPHAGE INFECTIONS OF Escherichia coli CONTAINING
THE COLICINOGENIC FACTOR, ColIb

By

Jerry Glenn

June 1979

Chairperson: Donna H. Duckworth Major Department: Immunology and Medical Microbiology When bacteriophage T5 infect Escherichia coli containing the colicinogenic factor, ColIb, the infections are abortive. RNA and protein syntheses stop abruptly and simultaneously at about 12 minutes after adsorption, and no progeny phage are produced. In the current work, I have shown that numerous changes in membrane function appear at nearly the same time as cessation of macromolecular syntheses. These alterations include inhibition of glutamine and proline uptake, stimulation of o-methylglucoside uptake, increased fluorescence intensity emitted by a membrane-binding probe, and efflux of preloaded potassium ions. The combination of results suggests that the infectious process is halted because of membrane depolarization. The same pattern of pathophysiological changes occurs during infections of Collb+ hosts by T5 mutants deficient in second-step transfer of DNA, indicating that no early or late phage gene expression is necessary to elicit the abortive response.



x













INTRODUCTION

Extrachromosomal elements of bacteria carry from cell to cell a tremendous variety of genetic determinants. At least 30 types of lysogenized phage are found among coliforms (26), and over 250 kinds of plasmids are established in E. coli alone (83). Actually, such figures might grossly underestimate the true numbers, since modifications and rearrangements of nucleotide sequences within any single cell probably continuously yield new genetic combinations (22,89). These novel structures efficiently pass to other bactera -- even to unrelated bacteria. Then, the process is repeated, implementing rapid change in the genetic complement of bacterial populations.

Extrachromosomal elements are eminently suited, therefore, to serve as instruments for bacterial adaptation, and they do in fact often provide strong survival advantage to the cells in which they reside (89). In certain Enterbacterial strains, for example, some types of extrachromosomal elements enable their hosts to prevent replication of particular virulent bacteriophages. This can be accomplished in several ways. Some elements carry genetic determinants that alter specific viral receptors, making the cell resistant to the phage which normally adsorb to that site (1). Another mechanism is superinfection exclusion (2,3,31). Here, adsorption is normal. This term describes the ability of one phage -- the extrachromosomal element in this case -- to prevent entry of DNA from a second phage that is subsequently adsorbed. The genome of the




2


superinfecting virus is trapped within the cell envelope, preventing any phage gene expression. Yet another way that some genetic elements circumvent viral replication is by directing the synthesis of restriction endonucleases (72). These enzymes break down incoming DNA that is unmodified at specific restriction sites. Finally, there is abortive infection, during which adsorption and DNA entry are normal, and the viral genome remains intact. The initial stages of viral gene expression occur as usual, but the presence of a lysogenized phage or a plasmid in some way prevents the infecting virus from producing progeny.

Of these four mechanisms -- resistance, superinfection exclusion, restriction, and abortive infection -- abortive infection remains the least understood. This is the case despite extensive study of several abortive systems, among which are T7 infections of F (fertility factor)-containing E. coli (11,12,13,23,24,68,76), T-even rIl mutant infections of X lysogens (14,34,39,92), and T-even or T5 infections of P2 lysogens (8,9,10,35).

The long-term objective of my research is to elucidate the

mechanism underlying the abortive infection which occurs after T5, or its close relative BF23, infects E. coli containing the colicinogenic factor, Collb. Studies described herein answer several questions that are important steps toward realizing this goal.

Abortive Infections Mediated by the ColIb Factor

Strains of E. coli carrying the colicinogenic factor, Collb, are nonpermissive hosts for bacteriophage BF23 (80,97). However, bacteria containing the F-factor, the X prophage, or the P1 prophage are able




3


to support replication of this phage. Likewise, BF23 can replicate in the presence of ColB, ColV, ColEl-30, ColE2-P9, and Colla. Thus, the viral infection is not aborted because of the presence of extrachromosomal DNA in general; rather, specific genetic determinants on the plasmid are involved.

It is not surprising that T5 shares BF23's inability to grow in ColIb-containing cells (80). This characteristic is just one of many the two phages have in common (reviewed in 87). They also have colinear maps and undergo phenotypic mixing, gene product substitution, and genetic recombination.

Productive BF23 and T5 infections are very similar as well. Both viruses are unusual in that they inject their genomes in two steps (59,60,61,62). The first-step-transfer DNA, constituting only 8% of the whole, codes exclusively for class I (pre-early) proteins (70). These proteins are synthesized from about 1 minute until about 10 minutes after infection. One of them, the Al gene product, is required for host DNA degradation (62,71). The Al polypeptide and also the A2 gene product are required for entry of the remaining second-step-transfer DNA. Carrying all class II (early) and class III (late) genes, the other 92% of the genome enters the host at about 4 minutes after infection, several minutes before early protein synthesis begins (70). The early proteins are necessary for phage DNA replication, which begins about 9 minutes into the infectious process

(25). Late proteins, the structural proteins included, first appear at about 13 minutes and are synthesized until lysis, approximately

1 hour after infection (70).




4



Abortive infections begin in the same way, but become manifestly abnormal after a short time (79). Phage DNJA entry occurs normally, and the infecting genome remains intact (79,97). Pre-early gene expression proceeds as usual, resulting in degradation of the bacterial genome and death of -the host cell. Then, at about 8 to 12 minutes after adsorption, incorporation of RNA and protein precursors into macromolecules stops. Little early protein synthesis occurs, and no late proteins appear. Since early proteins are required for phage DNA synthesis (69), it is not surprising that phage DNA replication is prevented. Finally, some laboratories observe rapid cell lysis of infected nonpermissive cells 15 to 30 minutes after phage protein synthesis begins (80).

A few phage circumvent the abortive process, producing plaques

at an efficiency of plating about 106 relative to that on noncolicinogenic hosts (74,80). These host-range (h-) mutants, which are recessive to wild-type in mixed infections, have a mutation in a pre-early gene (5,74). In T5, the mutation lies in gene A3, and the mutants fail to produce a small protein with a molecular weight of 12,000 (98). The T5 A3 gene product is thought to be identical to PE5 seen on polyacrylamide gels after electrophoresis of infected-cell extracts (D. J. McCorquodale, personal communication). Similarly, BF23 host-range mutants have a mutation in gene P3 (corresponding to A3 of T5 on the colinear maps). Furthermore, the electrophoretic mobility of P115 derived from cells infected with BF23 h mutants is altered, consistent with the suggestion that PE5 is the P3 gene product. Hence, it appears that the A3 or P3 gene product must be present to elicit the abortive response.




5



It has been proposed that early protein synthesis is required in abortive TS infections (45). It had been shown that if 15 infects cells in the presence of very low calcium concentrations, only preearly proteins are expressed (77,78). When additional calcium is added later, however, early RNA and proteins quickly appear. If ColIb +hosts are infected with T5 in the presence of little calcium, allowed to synthesize only pre-early proteins for 12 minutes, then supplemented with additional calcium, RNA synthesis proceeds for only a short time thereafter; i.e., the infection aborts soon after calcium supplementation (45). If the same experiment is performed, except that chloramphenicol is added just prior to calcium addition, RNA synthesis continues much longer than when chloramphenicol is not added. The implication is that chloramphenicol, in the second case, prevents synthesis of an early protein(s) necessary for activating the abortive mechanism. At least one alternative explanation, however, is that a low calcium concentration present at the time of infection might also slow production of pre-early proteins (69). This could be expected to inhibit production of the A3 gene product and might delay cessation of macromolecular synthesis.

To determine if any early protein synthesis is indeed necessary to halt the infectious process, I have taken another approach using T5Ai mutants. Because a functional Al protein is lacking, these phage can synthesize only pro-early proteins; second-step DNA transfer does not occur (62,71). I will show here that the abortive response appears despite the absence of early proteins, suggesting that the only viral protein required in T5 abortive infection is the A3 gene product.





6



In addition to the phage's own contribution, host-determined factors are involved in abortive infection. One-step growth curves indicate that roughly 5% of the cells in a Collb' population will support phage replication, though the phage yield per infective center is decreased by 70-85% (75,80). Since the phage that do manage to grow do not grow well upon reinfection of Collb-containing cells, these phages are not host-range mutants; rather, their growth depends on a low level of permissiveness in a few cells among the host population. The degree of T5 inhibition is also dependent to some extent upon the particular host strain carrying the plasmid (79). For example, the yield of T5 infectious centers on E. coli W3110 (ColIb) is similar to that if T5 is grown on W3110 polAl (ColIb). The average phage yield per infective center, however, is 5-fold greater on the strain with normal ability to produce host DNA polymerase 1.

Furthermore, some Collb-containing strains are permissive because of mutations on chromosomal or plasmid DNA (50,69, Richard Moyer, personal communication). Genetic analysis of these strains reveals that mutations at two chromosomal loci are necessary to express a fully permissive phenotype. The first locus, designated cmrA (ColIb-mediated resistance) on the E. coli map, is 91% cotransducible with rspE and is proximal to aroE. The second, cmrB, is 75% cotransducible with rspL and is distal to aroE. Other strains are permissive because of plasmid-borne mutations, but these have not been mapped. Thus, taken together, the studies indicate that contributions by plasmid, host, and phage are all necessary to produce an abortive infection.





7



To determine how the various factors interact to arrest the infection is a complex problem. It had been hypothesized that the primary defect is in transcription (79). Fully active R14A polymerase is recoverable from infected ColIb-containing cells, however, indicating that this enzyme is not the target (99). Alterations of the DNA template have also been studied to see if there are any which could explain cessation of gene expression. Packaged DNA is nicked, but the genome is lighted soon after infection (45). Only in productive infections, however, is the DNA subsequently renicked, suggesting that the transcriptional program during abortive infection could be altered as a result. Since lighted DNA is transcribed normally in an in vitro system (54), though, the relevance of the finding is questionable. A direct approach would be to determine if DNA

extracted from abortively infected cells can direct in vitro transcription, but this has not been done.

It has also been proposed that the primary defect is at the translational level (50). The cmrA locus maps near the gene coding for a ribosomal protein. This suggests that the ribosomal apparatus might be implicated, but no study has determined if extracts from abortively infected cells can support -in vitro translation. Thus, while no sound basis for a primary transcriptional or translational defect has been found, the studies looking for such abnormalities have not been exhaustive and cannot, therefore, rule out these possibilities.

Since it has previously been hypothesized that membrane dysfunction causes the abortion of T7 infections of male E. coli (12,23)





8


and of T-even r1I mutant infections of A lysogens (34,39), another line of investigation has been to examine changes in the cell envelope during abortive 15 infection. Infected nonpermissive cells become sensitive to sodium dodecyl sulfate (SDS)-induced lysis, unlike their infected Collb- counterparts (19). Although indicative of structural alterations in the host's outer membrane which allows the detergent to reach the inner membrane, this change occurs later than the cessation of macromolecular synthesis. As mentioned previously, some laboratories also note early abortive lysis (80), but the meaning of this event is not clear either. No progeny phage are produced even when premature lysis is prevented by stabilizing the host cells in medium of high osmolarity. Furthermore, our laboratory does not observe the spontaneous lysis (19).

I have looked more closely at membrane function during T5

infections of ColIb hosts to determine if physiological alterations of the membrane could possibly account for the abortive response. The results suggest that host membrane depolarization is the primary event leading to cessation of the T5 infectious process.














MATERIALS AND METHODS

_Oranisms

The bacterial strains used for most experiments are characterized in Table 1. Richard Moyer supplied our laboratory with three strains which are isogenic. RM 42 contains no plasmid; RM 43 bears wild-type ColIb-P9; RM 39 has a mutant ColIb plasmid which renders the host permissive for T5 (Richard Moyer, personal communication). Other strains were occasionally used, as indicated. E. coli was originally obtained from M. J. Bessman and maintained for many years in Donna Duckworth's laboratory, whereas E. coli C(HF4704) was taken from a stock maintained by William Holloman. E. coli CR63 was the gift of M. L. Dirksen.

Rolf Benzinger provided wild-type bacteriophage T5; T5hl2,

T5aml6d, and T5amH27 were obtained from D. J. McCorquodale. Relative plating efficiencies of the phage stocks on the various bacterial strains are also shown in Table 1. T5hl2- has a mutation which allows it to replicate in Collb+ cells (74), whereas T5aml6d and T5amH27 each have an amber mutation in the Al gene (5).

Media and Growth of Bacteria

The growth medium used for most experiments was M9 phosphatebuffered, balanced salt solution (46), supplemented with glucose (0.5%), yeast extract (0.05%), CaCl2 (5 x 10-4 M), and thymine (501jg per ml). Other additives were present as indicated in the description of each experiment. Unless otherwise indicated, bacteria were grown from a



9







TABLE 1. Characterization of Bacteria and Phage Plating Efficiency Relative to CR63(su+) E. coli Colicin
Y train Genotype Production T5 VWild-type(T5wt) _T5hl2_ T5aml~d T.5arH27

RM 42 W3110 (thy-, collRI) -1 1 0- 10-5

RM 43 W3110 (thy-, ColiR, ColIb-P9) + 10-7 0.5 <10-9 <-10PM 39 W3110 (thy-, COIR 001Ib-Pyr2') + 1 3 10-5 105


iColIR indicated resistance to the external action of Colicin lb.

2ColIb-P9h- indicates a mutant ColIb factor which allows T5 replication.





11



5% inoculum of an overnight culture. Growth was followed by monitoring turbidity on a Klett-Summerson colorimeter (660 nm filter), which had been previously calibrated to numbers of bacterial colony formers.

Potassium efflux experiments were performed using dilute tryptone broth (4 g tryptone and 2.5 g IaCl per liter of medium) as growth medium. Hershey broth (8 g of nutrient broth, 5 g of peptone, 5 g of NaCl per liter) was employed for some purposes, where indicated. Growth in these media was followed in the manner outlined above.

Finally, Tris-buffered medium was used. Per liter, this medium

contains 2.0 g of NH4CI, 5.0 g of NaCl, 0.4 g of KCl, 0.01 g of MgCl2.6H20,

0.02 g of Na2SO4, 5.0 g of Casamino acids, 2.5 ml of 80% glycerol, 250 mg of thiamine, and 100 ml of Tris (1 M, pH 7.3).

Colicin Production
To determine if a particular bacterial strain produced a colicin, I used the method of Fredericq (38). The cells were grown in Hershey broth for 48 hours. Then, chloroform (1%) was added, and the suspension was agitated for 10 seconds. Thereafter, the suspension was centrifuged at 10,000 rpm for 10 minutes on an SS-34 rotor in a Sorvall (RC-5). The supernatant (0.005 ml) was spotted on sensitive indicator lawns of E. coli B and E. coli C. If a zone of clearing appeared after overnight incubation of the plates, the strain was called a colicinproducer. Results are indicated in Table 1.

Phage Growth
Phage stocks were prepared in one of two ways: by the confluent lysis technique of Adams (1), or by recovering phage from Hershey broth by polyethylene glycol precipitation (105). No differences in results were obtained using the different types of phage preparation.





12



Phage Infections

Bacterial hosts were grown, as outlined above, to a concentration of approximately 6 x 108 cells per, ml. The bacteria were then centrifuged at 4% for 10 minutes at 10,000 rpm in an SS-34 rotor on Sorvall (RC-5), and resuspended in 1/10th volume of fresh growth medium. Phage were added at a multiplicity of infection of 10 (except where indicated otherwise), and after 1 minute, the bacteriavirus mixture was rediluted in prewarmed growth medium to 6 x 108 cells per ml. The time of redilution was called time zero.

Macromolecular Synthesis

RNA synthesis was monitored by measuring incorporation of [3 H]uridine into acid-insoluble material at different times after infection. Bacteria were grown in basic growth medium, with 50 pg of uridine added per ml of medium. Bacteria were centrifuged, diluted, infected, and rediluted as outlined above. At time zero, [ 3H] uridine (l pCi per ml, 5 pCi per pmole) was added and, at indicated intervals thereafter,

0.9 ml aliquots were removed and added to 0.1 ml of cold 50% trichloroacetic acid (TCA). Of this mixture, 0.4 ml was filtered through Whatman glass fiber filters (GF/F) and washed with 15 ml of cold 5% TCA. The filters were dried in an oven at 60% for about 1 hour, and counted in a toluene-based liquid scintillation fluid using a Beckman LS200 counter. The results represent total (cumulative) counts per minute incorporated into samples of approximately 2.2 x 108 bacteria.

Protein synthesis was measured in the same way, except that amino acids were used. When tyrosine was employed, cold tyrosine (25 pg per sil) was added to basic growth medium, and [3H] tyrosine




13



(1 pCi per mnil, 7.2 pCi per mole) was added at time zero. When proline incorporation was measured, cold proline (25 pg per ml) was added to basic growth medium, and [3H] proline (1 pCi per ml, 4.8 pCi per pmole) was added at time zero. Experiments to measure incorporation of glucose were also performed as above, adding [14C] glucose (0.5 pCi per ml, 0.1 pCi per pmole) at time zero.

Scintillation fluid consisted to toluene (1 gallon), 19 g of PPO (2,5-diphenyloxazole), and 1.14 g of POPOP [l,4-bis-(5-phenyloxazsyl) benzene]. A magnetic stirring bar agitated a newly made mixture overnight.

Dye-Buoyant Density Equilibrium
Centrifugation of Bacterial DNA

Bacterial DNA was labeled, extracted, and banded on ethidium bromide-cesium chloride gradients according to a modification of the method of Clewell and Helinski (20,21).

Tris-buffered growth medium was used to grow bacteria from a 1:100 dilution of an overnight culture until the turbidity reached 115 Klett units (660 nm filter). RM 42 and RM 39 were grown in 15 ml of medium, whereas 30 ml of an RH 43 culture was grown. RM 42 and RM 39 were incubated in the presence of [ 14C] thymidine (50 pCi per ml, 1 pCi per 4 pg of thymidine); [3H] thymidine (50 pCi per ml, 1 pCi per 4 pg of thymidine) was used to label RM 43 DNA. After cells had reached the desired concentration, 15 ml of the RM 42 culture was mixed with 15 ml of the RM 43 culture, and 15 ml of the RM 34 culture was mixed with 15 ml of the RM 43 culture.

Each tube, containing a total of 30 ml of bacterial suspension, was spun at 10,000 rpm in an SS-34 rotor in a Sorvall (RC-5). Each





14



pellet was resuspended in 1 ml of cold 25% sucrose in Tris (0.05 M, pH 8). Lysozyme (0.2 ml of a solution, 5 mg per ml in Tris [0.25 M, pH 8]) was added, and the suspension was maintained at OC for 5 minutes. Thereafter, ethylenediamine tetraacetate (EDTA) (0.4 ml of an aqueous soluti(cn, 0.25 M adjusted to pH 8) was added subsequently and, with occasional swirling, maintained for another 5 minutes at OC. Then, 1.6 ml of a detergent solution (1% Brij 58, 0.4% sodium deoxycholate, 0.0625 M EDTA, 0.05 M Tris, pH 8) was added to the suspension, and the mixture was maintained at OC until cell lysis, 3 to 5 minutes later. The lysate was spun at 2C for 25 minutes at 20,000 rpm in an SS-34 rotor on a Sorvall (RC-5). The supernatant should only contain about 5% of the original quantity of chromosomal DNA and is called the cleared lysate.

Each of the cleared lysates was diluted to 12 ml in TES buffer (0.05 M NaCl, 0.005 M EDTA, 0.03 M Tris, pH 8), and 3 ml of an ethidium bromide solution (1 mg of ethidium bromide in 1 ml of TES buffer) was added. Then, CsCl was added in a quantity sufficient to bring the refractive index to 1.3886. Each mixture was spun in a Ti-60 fixed angle rotor at 32,000 rpm at 4C for 60 hours.

An 18 guage needle was inserted into the bottom of each centrifuge tube, and 0.25 ml samples were recovered. The refractive index of representative samples was determined. Each sample was then mixed with 0.05 ml of 50% trichloroacetic acid, and 0.15 ml of this mixture was filtered over Whatman glass fiber filters (GF/F). Each filter was washed with 15 ml of 5% TCA, placed in a glass vial, and dried in an oven at 60% for 1 hour. A toluene-based scintillation fluor was added (5 ml in each vial), and the scintillations per minute were





15


determined on a Beckman LS200 counter using the narrow [ 3H]-window and the [14C]-window purchased from Beckman.

Host-Cell DNA Breakdown
Bacteria were grown as previously described in basic growth medium (defined above) modified by having thymidine (25 pg per ml) in place of thyrine. The medium also contained [3H] thymidine (0.4 pCi per ml; 1 pCi per 50 pg of thymidine). When the cells reached the appropriate concentration, they were spun, washed twice in medium without labeled thymidine, concentrated, infected, and rediluted as described previously. Thereafter, 0.9 ml samples were removed at the indicated intervals and were added to 0.1 ml of 50% cold TCA. Of this,

0.4 ml samples were filtered over Whatman glass fiber filters (GF/C) and washed with 15 ml of 5% TCA. The filters were placed in glass vials and dried for 1 hour in an oven at 60'C. Scintillations per minute in a toluene-based liquid scintillation fluid were counted on a Beckman LS200 counter.

Proline and Glutamine Uptake

In these experiments, the amount of amino acid taken up by the cell in a 30-, 60-, or 90-second pulse is measured at various times after infection. Bacteria were grown in basic growth medium, spun, concentrated, infected, and rediluted as above. At the indicated times, 2 ml samples were removed, and chloramphenicol was added, yielding a final concentration of 100 pg per ml. One minute later,

0.9 ml of this mixture was added to 0.1 ml of the labeled amino acid solution. In the proline assay, 1 pCi [ 3H] proline (25 pCi per pmole) was present, while in the glutamine experiments, 0.5 lICi of




16


[14C] glutamine (5 pCi per pmole) was present. At indicated intervals after infected bacteria were added to the labeled amino acid, 6.4 ml samples were removed, filtered on Whatman glass fiber filters (GF/F), and washed with 8 ml of Mg. A positive control was done using uninfected cells; a negative control was done using cells treated for 15 minutes with NaN3 (1%) prior to addition of chloramphenicol. The results are presented here as percentage of uptake in infected cells, relative to that of uninfected controls.

c-Methllglucoside (aMG) Uptake

s-Methylglucoside uptake experiments utilized bacteria grown and infected in basic growth medium, as outlined above. At times indicated, 1 ml of the sample was removed and spun in an Eppendorf 3200 Centrifuge for approximately 30 seconds, resuspended in an equal volume of M9, and spun again. After the second spin, the bacteria were resuspended in basic growth medium, modified by having only 18 jg glucose per ml of medium. Of this suspension, 0.9 ml was added to 0.1 ml of [14C] MG solution (I pCi per ml, 184 uCi per mole). At indicated intervals,

0.4 ml aliquots were removed and filtered over Whatman glass fiber filters (GF/F) and washed with 8 ml of M9. The filters were dried and counted. A positive uninfected control was done; a negative control was done using cells treated for 15 minutes prior to the assay with NaF (0.07 M) and NaN3 (1%). The results are presented as percentage of uptake, relative to uninfected controls.

Fluorescence Experiments

Fluorescence intensity was measured using a Perkin-Elmer MPF-2A scanning fluorimeter equipped with a temperature-controlled chamber.





17


The instrument's output was corrected for wavelength variable response by means of a rhodamine B quantum counter, and its monochromators were calibrated against the xenon line emission spectrum.

From recrystallized N-phenyl-l-naphthylamine (NPN) a working

stock (20 mM in methanol) was prepared. Cells were grown, concentrated, and infected as described previously, except that the multiplicity of infection was 5. At time zero, the infected cells were resuspended in prewarmed (37C) growth medium containing 10 pM NPN. Three milliliter of the infected-cell samples was placed in quartz cuvettes and inserted into the 37C chamber.

Fluorescence intensity was measured in arbitrary units and recorded over time after infection. Excitation wavelength was 352nm with a bandwidth of 5nm. The emission wavelength was 41Onm with a bandwidth of lOnm.

Potassium Efflux

Dilute tryptone broth was used as growth medium. This medium contains a low concentration of patassium, making it easier to load cells with radiolabeled potassium (94,101).

Bacteria were grown for several generations in the presence of

0.1-0.2 mCi of 42K per nil (42K in the form of KCI in aqueous solution, 0.18 mCi per mg K). When the cells reached a concentration of 6 x 108 bacteria per ml, they were spun, concentrated, and infected as usual (a portion of cells was not infected). At intervals, aliquots of uninfected and infected cells were removed, filtered over glass fiber filters (GF/F), and washed with 8 ml of dilute tryptone broth. Cerenkov emissions were counted using the [ 3H] channel on the Beckman




18



LS200 counter. The results represent the amount of residual 42K remaining inside infected cells, relative to the amount remaining inside uninfected cells taken at the same time point (expressed as a percentage).

Attempts to Prevent Abortion of T5 Infection in ColIb+ Hosts

Cells were grown and infected, in 1% tryptone broth with Tris (5 mM, pH 7.2), as outlined above. Various additives were present as indicated in Table 7. The final concentrations of the additives were as follows: (a) potassium, 100 mM (b) magnesium, 80 mM (c) sodium, 80 mM (d) sucrose, 300 mM (e) polyamines, 30 al. Others were added in varying amounts, also as indicated in Table 7. When N,N'-dicyclohexylcarbodiimide (DCCD) was present, it was added 30 minutes prior to phage infection at a concentration of 0.1 mM. Polyamines, when used, were added at the time of infection. All other additives were present from the time when a 5% inoculum was added to fresh medium.

To determine the efficacy of the treatments, a phage titer was determined. Chloroform (1%) was added to the treated cultures 4 to 7 hours after infection, and samples were plated on lawns of E. coli B. This was designed as a screening procedure, so no attempt was made to remove unadsorbed phage.

Gel Analysis

To see what proteins were labeled with radioactive amino acids after infection, infected cells were pulse-labeled for 5 minutes with

1 pCi (14C) of an amino acid mixture per ml of culture. The fiveminute pulses were initiated at 1, 6, and 11 minutes after infection,





19


and tenninated by the addition of 100 pg of chloramphenicol per ml of medium and by chilling. The cells were then centrifuged, washed two times, and resuspended in 1/10 volume Laenmmli electrophoresis buffer

(58). The samples were then boiled for 5 minutes. Twenty microliters of each sample was loaded onto a 15% acrylamide slab gel and electrophoresed for 14 hours at 75 volts. The electrode buffer consisted of

0.192 M glycine and 0.1% SDS in 0.025 M Tris, pH 8.3. The dried gel was autoradiographed by exposing it to Kodak XR1 film for 10 days.

The gel was prepared by layering a stacking gel over a previously solidified 15% running gel. A stock acrylamide solution contained 30% acrylamide, 0.8% bisacrylamide. The recipe for the stacking gel was as follows: 1.0 ml of the stock acrylamide solution, 2.5 ml of Tris-HCl (0.5 M, pH 6.8), 6.4 ml of distilled water, 0.1 ml of 10% SDS, 0.04 ml of 20% ammonium persulfate (freshly made), and 0.02 ml of N,N,N',N'-tetra-methyl-ethylenediamine (TEMED). The running gel consisted of the following: 20 ml of stock acrylamide solution, 10 ml of Tris-HCl (1.5 M, pH 8.8), 9.6 ml of distilled water, 0.4 ml of 10% SDS, 0.035 ml of 20% ammonium persulfate (freshly made),

0.01 ml of TEMED. Each type of gel was made by first mixing the acrylamide solution, Tris-HCl, distilled water, and ammonium persulfate. These ingredients were placed in a vacuum flask and held under vacuum for 3 to 5 minutes to degas thoroughly. Thereafter, the SDS and TEMED were added, the solution was swirled gently, and the gel was poured immediately.

Statistical Methods
The means of two sets of data were compared using Student's t-test, according to the method of Snedecor and Cochran (96). When





20


an equal number of data points were included in each set, t values were calculated using the formula: t I- Y2 I S ~2 whr

Y= the mean of data points in set 1, n the number of data points in each set, S I = the standard deviation of the mean for data collected in set 1. P values were derived from a standard table. Application of the t-test presupposes that the data fit a normal t distribution.

Materials
42Kand the [ 14 C]-labeled amino acid mixture were purchased from New England Nuclear, Boston; [ 1 C] aMG was Purchased From Amersham, Arlington Heights, Illinois. All other radiolabeled products were obtained from Schwarz/Mann, Orangeburg, New York.

NPN was the gift of W. A. Cramer of West Lafayette, Indiana. DCCII was purchased from Sigma Chemical Company, St. Louis, Missouri. All other chemicals used were analytical reagent grade, and are readily available from many producers.













RESULTS

Macromolecular Synthesis in T5 and T5hl2- Infections of RM 42, RM 43, and RM 39

It has been previously reported that transcription and translation cease at 6 to 10 minutes after T5 infection of ColIb+ E. coli (79). To determine when these changes occurred in our system, I measured the cumulative incorporation of an RNA and a protein precursor into acid-insoluble macromolecules at various times after infection. Uridine was used as an indicator of RNA synthesis, while tyrosine or proline was used to monitor protein synthesis. The cells I have used are described in Table 1, as are the infecting phage.

Typical results of experiments measuring incorporation of [ 3H]uridine can be seen in Figure 1. It can be seen that RNA synthesis, measured in this way, continued for at least 30 minutes in productive infections, albeit at a slower rate than in uninfected cells (Fig. la). In infections of RM 43 (Collb), however, uridine incorporation stopped at some time approximately 9 to 12 minutes after initiation of infections (Fig. lb). Protein synthesis, as indicated by tyrosine or proline incorporation (Fig. 2 and Fig. 3) also stopped about 9 to 12 minutes after infection of Collb-containing cells. RNA and protein synthesis proceeded at least 30 minutes in T5 wild-type infections of RM 39 (ColIbh-) (Figs. Ic and 2c),and protein synthesis continued for at least 30 minutes in T5h12- infections of RM 43 (Collb) (Fig. 3b).






21












Figure 1. Uridine Incorporation into Acid- 55
insoluble Macromolecules in
Uninfected and Infected RM 42,
RM 43, and RM 39.
Cells were grown and infected in '10
synthetic medium. [31-1] Uridine
(1 wCi per ml, 5 uCi per limole) was
added at time zero. Samples of < 2510.9 ml were removed at the indicated I
tines and mixed with 0.1 ml of cold
50% TCA. Acid-insoluble material was 0
collected on glass fiber filters, and 20
the filters were washed. The amount x
of incorporated radioactivity was ,
determined by liquid-scintillation .1 o

counting. In Fig. la, the bacteria 15
used were RM 42, which contain no
plasmid. Fig. lb represents results 0
obtained with RM 43 (Collb), and in L
Fig. 1c, RM 39 (ColIb h-) was used. ,

uninfected cells
(9 T5-infected cells 0





0 6 12 3 24 30
TIME (MI NUTS)

Figure la







3






25



I2 20 2 /L



0



S5- 0 Sr

.51




0 6 12 l 24 30 0 6 12 18 24 30
TIME (MINUTES) TIME (MINUTES)

Figure lb Figure ic

















Figure 2. Tyrosine Incorporation into Acidinsoluble Macromolecules in 6
Uninfected and Infected RM 42,
RM 43, and RM 39.
Cells were grown and infected in<
synthetic medium. [3H] Tyrosine (1 Ci per ml, 7.2 PCi per Pmole)
was added at time zero. Samples of 4
0.9 ml were removed at the indicated X
times and mixed with 0.1 ml of cold
50% TCA. Acid-insoluble material
was recovered on glass fiber filters, b 0
and the amount of incorporated 0 0 0
radioactivity was determined. in Fig. 2a, the host was RM 42; in 0
Fig. 2b, the host was RM 43 (ColIb); 0 0
in Fig. 2c, the host was RM 39
(Collb h-). I

*, uninfected cells
0, T5-infected cells 6 L2 3 24 Z0
FMigNUES)

Figure 2a

















4




0







00
I I
3 0 2 i 4 3 2 i 4 3
TIM (MNTS IM MNTS

Fiur 0bFgue2















Figure 3. Proline Incorporation into Acid-Insoluble Macromolecules
in Uninfected and Infected RM 42, RM 43, and RM 39.

Cells were grown in synthetic medium. [3 H] Proline (I pCi per ml, 4.8 pCi per mole) was added at time zero. Samples of 0.9 ml were
removed at the indicated times and mixed with 0.1 ml of cold 50%
TCA. Acid-insoluble material was recovered on glass fiber filters,
and the amount of incorporated radioactivity was determined. In
Fig. 3a, the host was RM 42; in Fig. 3b, the host was RM 43 (ColIb h-).

0, uninfected cells
O), T5-infected cells
A T5hl2--infected cells











i4 14 r



I2H 0, 12j








00









0 12 t 44 30 0 i 4

TIME(MIUTES TIM E(M IN U EA
0iue3 iue3





28


Confirmation that RI 39 Contains Plasmid DNA

RM 39 is a strain sent to our laboratory by Richard Moyer and is said to contain a mutant Collb factor which will allow T5 to replicate. I have used this strain to eliminate the likelihood that pathophysiological alterations observed during T5 infection of RM 43 are due to Collb-borne genetic determinants unrelated to the determinants necessary for abortive infection. There is a possibility, however, that our laboratory stock of what is identified as RM 39 no longer contains the plasmid. This would explain T5's ability to replicate in the strain, but would invalidate the interpretations of experiments involving RM 39.

As carl be seen in Table 1, RM 39 does indeed produce a substance, thought to be colicin lb, which inhibits growth of bacteria sensitive to colicin lb. This is presumptive evidence that the strain contains the Collb factor. Since the presence of colicin Ib has not been rigorously demonstrated, however, I thought it wise to demonstrate the plasmid on an ethidium bromide-cesium chloride gradient.

Labeled DNA was extracted from RM 42, RM 43, and RM 39. [14C]Labeled DNA from RN 42 was mixed with [3 H]-labeled DNA from RM 43. As can be seen in Figure 4a, DNA from RM 43 appeared in two bands, one in the position characteristic of covalently closed, circular DNA (plasmid DNA), the other in the position characteristic of nicked circular DNA (E. coli chromosome) (21). DNA extracted from RM 42, however, banded in a single position characteristic of the E. coli chromosome. A second tube, which was centrifuged concurrently, contained [ 3H]-labeled DNA from RM 43 and [14C]-labeled DNA from RM 39












Figure 4. Ethidium Bromide-Cesium Chloride Gradients of DNA Extracted
from RM 42, RM 43, and RM 39.

DNA in RM 43 was labeled by growing in the presence of [3HJ thymidine (1 pCi per ml, 1 pCi per 4 ig of thymidine), whereas DNA in RM 42 and
RN 39 was labeled with [14C] thymidine (1 pCi per ml, 1 PCi per 4 pg of
thymidine). The DNA was extracted from the bacteria, and DNA from RM 42 and RM 43 was mixed in one tube, DNA from RM 39 and RM 43 was mixed in another tube. To the latter tube was added 0.1 pg of [32p]
labeled DNA of SV40. DNA forms 1 and 2 were banded by dye-buoyant
density equilibrium centrifugation. Fractions of about 0.25 ml were
removed and mixed with TCA (10%-final concentration). From each
sample, 0.15 ml was filtered over glass fiber filters and washed. The
number of 3H, 14C, and 32p counts on each filter were determined by liquid-scintillation counting. These figures are plotted. Fig. 4a
represents the RM 42 RM 43 mixture, whereas Fig. 4b represents the
RM 42 RM 43 SV40 mixture.

*, RM 42 DNA (14C)
Q, RM 43 DNA (3H)
A, RM 39 DNA (14C)
V, sv40 DNA (32P)










4001 350t


g300~ )




~200F









5 0 L0 I0 5 0 3 0 5


F RA8CTI ON NUMBER FR1ACTI0ON NUMBER

Figure 4a Figure 4b





31


(Fig. 4b). Additionally, this tube contained [32 P-labeled SV40 DNA which is known to consist of both covalently closed, circular DNA and nicked, circular DNA (4). As before, two peaks appeared, each containing [3 H] counts of RM 43 DNA. These peaks were in positions identical to peaks containing [ 32P] counts of SV40 DNA and [14 C] counts of RM 39 DNA. The results conclusively show that both RM 39 and RM 43 contain plasmid DNA.

Confirmation that T5hl2 Infections Begin Promptly

Studies using T5hl2 mutants have been performed to eliminate the likelihood that pathophysiological alterations observed during T5 infections of RM 43 are due to phage genetic determinants unrelated to the A3 gene function. If the T5hl2 phage were very slow to initiate infections in RM 43, normal bacterial physiological processes would be expected to continue in the T5hl2 -RM 43 suspension for some time. This could invalidate interpretations of the experiments using these mutants.

To confirm that T5hl2 phage adsorb and initiate infections promptly, breakdown of host DNA, an event normally occurringin the first minutes of infection, was monitored. As can be seen in Figure 5, breakdown of prelabeled host DNA into acid-soluble fragments occurs promptly after T5 wild-type or TShl2 infection of RM 42, RM 43, or RM 39. From these results, it is evident that T5hl2- phage are able to normally adsorb to and initiate infection in RM 43.

Amino Acid Accumulation in T5 and T5hl2 Infections of RM 42, RM 43, and RM 39
In an attempt to clarify the relationship between the inhibition

of RNA and protein synthesis and the membrane changes previously observed







Figure 5. Host DNA Breakdown After T5hl2
Infection of RM 42, RM 43, and
RM 39.
3000
DNA was labeled by growing the cells
in the presence of [3H] thymidine
(0.4 uCi per ml, 1 pCi per 50 ug of 200 e
thymidine). The cells were spun, 1
concentrated, infected, and rediluted
in medium without radiolabeled thymidine. At times thereafter, 0.9 ml 200
samples were removed and placed in so
0.1 ml of 50% TCA. Of this, 0.4 ml
was filtered over glass fiber filters 100
and washed. The number of acid- 0
precipitable counts were determined by liquid-scintillation counting. In
Fig. Sa, the host is RM 42; in Fig. 5b,
the host is RM 43 (Collb),whereas RM 39
(Collb h-) is the host in Fig. 5c.
c00
9, uninfected cells
0, T5-infected cells __
A, T5hl2--infected cells 4 a 1 20
TIWE (MNIUTES)

Figure 5a






















4- 0




coo






Fiur 5bFCur 5





34



(19), and to more specifically define the membrane defects, I looked at accumulation of two amino acids. The transport systems of proline and glutamine are fairly well characterized (6,7), and both systems require an "energized" membrane. Proline requires membrane-bound transport proteins (still present when purified membrane vesicles are made), but does not require a high-energy phosphorylated intermediate. Glutdmine, on the other hand, relies upon periplasmic binding proteins and needs ATP to energize its active transport. I wanted to see when, if at all, these uptake systems were inhibited during abortive infection. If they were inhibited, I wanted to know if they remained functional during infections involving h mutants of the phage or the plasmid.

In these experiments, aliquots of cells were removed at various times after infection and incubated with chloramphenicol for one minute -- a time sufficient to totally stop protein synthesis -prior to adding the labeled amino acid. The results, therefore, reflect only net uptake of the amino acid, and are not indicative of incorporation into proteins.

When wild-type T5 infected any of our three bacterial strains, there was a slight drop in proline accumulation at 5 minutes after infection (Fig. 6a and Table 2). By 10 minutes into the infectious process, however, net uptake returned to uninfected levels in all cases, and continued to increase in both infected RM 42 and infected RM 39 (Collbh-). In abortively infected RM 43 (Collb), however, there was a drastic reduction in the ability to accumulate proline between 10 and 15 minutes. If T5hl2 phage infected RM 43 (Collb)















Figure 6. Proline Accumulation in 60 Seconds by Infected RM 42, RM 43, and RI 39.

Cells were grown and infected in synthetic medium. At the indicated
times after infection, samples were removed and mixed with [3H] proline
(1 1Ci per ml, 25 pCi per molee. Aliquots were removed at 30 seconds
and 60 seconds thereafter and filtered through glass fiber filters.
Here, the amount of radioactive proline accumulated in 60 seconds by
infected cells is expressed as a % of the amount taken up by uninfected
controls in the same period of time. Fig. 6a represents T5wt infections
of E. coli RM 42, RM 43 (Collb), and RM 39 (Collb h-). Fig. 6b represents
T5hT2- infections of the same three bacterial strains.

0, RM 42
Q, RM 43 (Col Ib)
A, RM 39 (ColIb h')












I5O0
A



150 A


lZ5 0







7501



i-C EE AZIDE EVa





0 3 10 Is 0 11 30 0 5 10 15 20 25 zo

TIME (MINUTES)

Figure 6a Figure 6b

C,





TABLE 2. Proline Accumulation (in 60 seconds) by Infected RM 42, RM 43, and RM 39

TIME AFTER PHAGE RM 42-T5wt vs RM 43-T5wt (3) RM 43-T5wt vs RM 39-T5wt (3)

5 minutes 83 2 78 7 78 + 7 74 t 6
10 minutes 113 15 106 24 106 t 24 109 t 21
15 minutes 123 27 46 7 46 t 7 114 26
20 minutes 105 19 37 6 37 6 127 t 15
25 minutes 133 32 30 7 30 7 138 48
30 minutes 132 51 27 5 27 t 5 163 55
NaN3 +uninfected 12 8 14 4 14 4 16 t 8
cells
TIME AFTER PHAGE RM 43-T5hl2" vs RM 43-T5wt (3) RM 42-T5hl2- vs RM 43-T5h12- vs RM 39-T5h12- (4)

5 minutes 79 10 78 14 79 16 80 t 6 83 11
10 minutes 96 6 82 27 104 t 26 112 27 103 19
15 minutes 92 16 55 I 119 t 31 97 5 107 11
20 minutes 83 12 53 7 103 25 85 29 132 36
25 minutes 85 9 49 5 102 t 25 79 t 12 130 t 26
30 minutes 83 9 46 9 124 26 78 12 135 30
NaN3+uninfected 32 2 32 2 32 t 23 25 12 44 9
cells
indicates a number of experiments done in each group
indicates a difference between the 2 samples with a confidence level, p< 0.05
[3H] Proline accumulation by infected cells was determined as outlined in MATERIALS AND METHODS.
The results represent %'s of uptake, relative to that of uninfected control samples, in 60 seconds.





38


(Fig. 6b), there was only a slight reduction in net uptake as compared to that seen during infections of RM 42 or RM 39 (ColIbh-). in comparing results observed during T5hl2- infections of RM 42 and RM 43 (ColIb) by Student's t-test, only the difference in values at 30 minutes was significant. This slight loss in net uptake in colicinogenic hosts was perhaps due to the 50% plating efficiency of the mutant phage on RM 43 (ColIb). When T5 wild-type and T5hl2- infections of RM 43 (Collb) were compared directly, statistically significant differences were observed at all times after 10 minutes. In summary, the ability to accumulate proline was drastically reduced between 10 and 15 minutes in nonpermissive hosts. This inhibition was not observed when h mutations on either the phage or plasmid allowed productive infection to occur.

Similar results are seen when glutamine uptake is measured

(Fig. 7 and Table 3). After observing a reduction in uptake ability at five minutes after T5 wild-type infections of any one of the three strains, we saw a gradual recovery in uptake ability during productive infections. When RM 43 (Collb) was the host, uptake ability fell progressively, and there was a statistically significant difference, as compared to infections of RM 42, by 15 minutes. Although the 10 minute values were not significantly different, in some experiments there was indeed a large disparity. The mean 10-minute value of glutamine uptake in infected RM 43 (ColIb) was lower than the value of infected RM 39 (ColIbh-). Infection of RM 43 (ColIb) with T5hl2- or T5 wild-type showed that the h mutation prevented the fall in glutamine uptake. The cell's ability to accumulate glutamine, then, as was the


















Figure 7. Glutamine Accumulation in 90 Seconds by Infected RM 42, RM 43, and RM 39.

Cells were grown and infected in synthetic medium. At the indicated
times, samples were removed and mixed with [14C] glutamine (0.5 pCi per
ml, 5 pCi per molee. At 30, 60, and 90 seconds thereafter, aliquots
were removed and filtered through glass fiber filters. The results
here are for the 90-second pulses, expressed as a % of the net amount
taken up by uninfected controls in the same period of time. Fig. 7a
represents T5wt infections of RM 42, RM 43 (ColIb), and RM 39 (Collb h-).
Fig. 7b represents T5hl2- infections of the same three strains.

0, RM 42
0) RM 43 (ColIb)
A, RM 39 (ColIb h-)









120 A




I loo


80~






o L'


~' 0
u 20I 0
AZIDELEVEL 2h AZIDE LEVM..



0 10 20 30 0 10 20 3
TI ME(MINUTES) -n ME(?RJNUTES)

Figure 7a Figure 7b





TABLE 3. Glutamine Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39

TIME AFTER PHAGE RM 42-T5wt vs RM 43-T5wt (4) RM 43-T5wt vs RM 39-T5wt (3)

5 minutes 54 25 50 11 57 3 64 6
10 minutes 61 33 35 13 37 19 80 11
15 minutes 60 30 21 11 21 12 100 48
20 minutes 69 31 18 10 25 21 112 1 39
25 minutes 68 45 17 10 14 7 108 38
30 minutes 75 48 16 1 7 12 4 125 1 55
NaN3 +uninfected 17 17 15 3 15 3 22 12
cells
TIME AFTER PHAGE RM 43-T5hl2- vs RM 43-T5wt (4) RM 42-T5hl2- vs RM 43-T5h12- vs RM 39-T5h12- (1)

5 minutes 65 14 56 + 3 55 60 88
10 minutes 66 21 38 16 64 74 71
15 minutes 75 16 24 12 73 76 73
20 minutes 71 19 25 15 68 85 74
25 minutes 73 28 18 10 77 93 78
30 minutes 74 28 16 8 68 104 96
NaN3 +uninfected 12 5 12 5 2 5
cells
indicates the number of experiments done in each group
indicates a difference between the 2 samples with confidence level, p< 0.05
[14C] Glutamine accumulation by infected cells was determined as outlined in MATERIALS AND METHODS.
The results represent %'s of uptake, relative to that of uninfected control samples, in 90 seconds.





42



case with proline uptake, was drastically reduced during abortive infection. This dysfunction was prevented by phage or plasmid mutations which also allow productive infection to proceed. There was a tendency for glutamine uptake to decline a bit earlier than proline uptake but the two uptake systems were not compared in the same experiment.

Confirmation that Inhibited Proline and Glutamine
Accumulation is Not Due Solely to Inhibited Protein Synthesis

One possibility is that the decreased net amino acid uptake is

due solely to cessation of protein synthesis. There could, for example, be an accumulation of amino acids in T5-infected RM 43 after protein synthesis stops at 9 to 12 minutes after infection. This might limit net uptake of proline or glutamine from the medium.

I therefore treated uninfected or T5-infected cells with chloramphenicol 1 minute or 15 minutes prior to determining net uptake of proline or glutamine from the medium. Even 15 minutes after inhibition of protein synthesis with the antibiotic, there was no decrease in accumulation of either amino acid from the medium when glucose was the carbon source (Table 4). These results eliminate the likelihood that the decrease in net uptake of amino acids during abortive infection is due only to cessation of protein synthesis.

a-Methylglucoside Accumulation in T5 and T5hl2
Infection of RM 42, RM 43, and RM 39

Since accumulation of either proline or glutamine requires an energized membrane, I decided to measure net uptake of a substance which is transproted via a group translocation reaction, and does not require membrane polarization. AMG is a nonmetabolizable analogue of glucose taken into the cell using the phosphotransferase system







TABLE 4. Proline and Glutamine Accumulation in Uninfected and Infected RM 43 After 1 Minute or 15 Minutes of Incubation with Chloramphenicol

Proline (3) Glutamine (3)

Uninfected 5 Minutes Uninfected 5 Minutes
Cells After T5 Cells After T5

Chloramphenicol
for 1 Minute 970 t 166 875 52 1331 t 98 793 94

Chloramphenicol
for 15 Minutes 940 198 1033 t 190 1719 t 49 1047 118

( ) indicates the number of experiments done in each group
[3H] Proline or [14C] glutamine accumulation was determined as outlined in MATERIALS AND METHODS,
except that, where indicated, chloramphenicol was allowed to inhibit protein synthesis for
15 minutes prior to measuring accumulation of the labeled amino acid. Results represent raw counts accumulated by approximately 2.2 x 108 cells in 60 seconds (proline) or in 90 seconds
(glutamine).




44


and energy derived from phosphoenolpyruvate (57). I wanted to see if this transport system was functional in abortively infected cells.

Phage infection, whether productive or abortive, increased rather than decreased oMG accumulation (Fig. 8 and Table 5). By 5 minutes after infection, however, the increase was much greater in the abortive infection than it was in the productive infection. As was seen for proline or glutamine uptake, the absence of the normal phage or plasmid h gene product eliminated the effect. The augmented accumulation during abortive infection was inhibited by NaF, as is normal for transport systems energized by phosphoenolpyruvate (57), showing that the increased uptake was via the phosphotransferase system and not a new uptake system. After 10 minutes of infection in a non-permissive host, the increase in oMG accumulation began to decline, although the total uptake was still much greater than that during productive infection.

Glucose Incorporation into Macromolecules
in T5 and T5hl2- Infection of RM 42, RM 43, and RM 39

The rate of protein synthesis or nucleic acid synthesis measured by incorporation of radiolabeled precursors depends first upon the cells' ability to take up the precursor from the medium. Since the uptake of amino acids into the soluble pools of T5-infected cells was severely depressed during abortive infection, it cannot be said that protein synthesis was necessarily inhibited. Since the glucose transport system appeared to remain functional, as measured by aMG uptake, we have looked for the incorporation of label from [14CIglucose into acid-insoluble material (Fig. 9).



















Figure 8. a-Methylglucoside Accumulation in 90 Seconds by Infected RM 42, RM 43,
and RM 39.

Cells were grown and infected in synthetic medium. At the indicated
times after infection, samples were removed, spun, and washed with Mg.
After a second wash, the cells yre resuspended in M9 with 18 Pg of glucose per ml, and 0.1 ml of [ C] aMG (10 wCi per ml, 184 iCi per
mole) was added to 0.9 ml of the cell suspension. Aliquots were
removed at 45 and 90 seconds, thereafter, and were filtered through glass fiber filters. The results here are for the 90-second pulses,
expressed as a % of the amount taken up by uninfected controls in the same period of time. In Fig. 8a, the infecting phage is T5wt, whereas
in Fig. 8b, it is T5hl2-.

*, RM 42
0, RM 43 (ColIb)
A, RM 39 (ColIb h-)











500j500


400- -1,00



-00







i0o

R."

0 10 20 30 0 10 20 3
THAIE (MiNU-.-SS)
T;!.'E 7S) 41
Figure 8a C
Figure 8b





TABLE 5. m-Methylglucoside Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39

TIME AFTER PHAGE RM 42-TSwt vs RM 43-T5wt (4) RM 43-T5wt vs RM 39-T5wt (3)
5 minutes 177 40 270 t 41 254 f 18 140 4
10 minutes 140 12 426 72 439 74 104 31
15 minutes 108 19 384 t 70 392 85 131 t 12
20 minutes 107 20 343 26 323 9 108 15
25 minutes 114 18 296 + 52 282 t 48 113 t 27
30 minutes 100 28 261 t 33 257 30 141 46
NaN3 and NaF + 25 7 24 14 25 10 18 7
uninfected cells
TIME AFTER PHAGE RM 43-T5h12- vs RM 43-T5wt (1) RM 42-T5hi2- vs RM 43-T5h12- vs RM 39-T5h12- (2)
5 minutes 202 245 219 6 6 177 59 137 37
10 minutes 225 354 137 19 167 17 191 1 37
15 minutes 169 313 112 14 211 130 124 15
20 minutes 164 313 134 24 180 36 108 1 2
25 minutes 153 228 118 4 65 13 99 12
30 minutes 134 260 148 15 157 t 61 154 20
NaN3 and NaF + 20 37 18 8 25 t 6 17 t 1
uninfected cells
indicates the number of experiments done in each group
indicates a difference between the 2 samples with a confidence level, p< 0.05
14C] aMG accumulation by infected cells was determined as outlined in MATERIALS AND METHODS. The
results represent %'s of uptake, relative to that of uninfected control samples, in 90 seconds.








Figure 9. Glucose Incorporation into AcidInsoluble Macromolecules in
Uninfected and Infected RM 42, 10
RM 43, and RM 39.

Cells were grown and infected in synthetic medium. [14C] Glucose
(0.5 1iCi per ml, 0.1 uCi per mole)
was added at time zero. Samples
of 0.9 ml were removed at the 0
indicated times and mixed with 0.1 ml
of cold 50% TCA. Acid-insoluble
material was collected on glass fiber
filters and the filters washed. The amount of incorporated radioactivity
was determined by liquid scintillation 0
counting. In Fig. 9a, the bacteria
used were RM 42; in Fig. 9b, the host
was RM 43 (ColIb); in Fig. 9c, the
host cells were RM 39 (ColIb h-).,Q

0, uninfected cells
o), T5-infected cells 2
A T5hl2--infected cells



10 20 30
Ti (gINUTE )

Figure 9a














Q -j L1






4'4

Qm


2-2



[ -I I- I [ 1 ,
0 iC 20 30 0 10 20 .30
TIME (NiNUTES) !~M: (MINUTES)
Figure 9b Figure 9c





50



Cumulative incorporation of glucose into an insoluble form was

inhibited in the abortively infected M.M 43 (ColIb) at about 10 minutes after infection (Fig. 9b). This was not the case during T5 infections of RM 42 or RM 39 (Collbhr) (Fig. 9a end 9c), nor was it true of T5hl2_ infections of RM 43 (Collb) (Fig. 9b). Since the glucose transport system remained operative for at least 30 minutes during abortive infection, it appeared that macromolecular synthesis per se was inhibited at about 10 minutes. This conclusion must still be regarded as tentative, however, since the results could be explained by leakage of intermediary metabolites of glucose. Further investigation of this problem may be warranted.

Fluorescence Intensity of NPN During
T5 and T~h12- Infection of RM 42, RM 43, and RM 39

The results of the uptake studies suggested that membrane depolarization was occurring at about 10-12 minutes after T5 infection of Colb-containing hosts. If true, when cells are infected in the presence of NPN, the fluorescence intensity emitted should increase at about this time, since chemicals known to depolarize the bacterial membrane have this effect (44).

As can be seen in Fig. 10, when T5 infects any of the 3 strains

in the presence of NPN, there is an increase in fluorescence intensity at about 3 minutes after infection and another at about 6 minutes after infection. This biphasic effect has been observed previously

(41) and is thought to reflect changes in the cell membrane at the times of first- and second-step DNA transfer. Data presented below

areconsistent with this interpretation.






















Figure 10. Fluorescence Intensity of N-phenyl-lnaphthylamine during T5 and T5hl2Infections of RM 42, RM 43, and RM 39.

Cells were grown, concentrated, and
infected in synthetic medium. At time zero, the infected cells were put into prewarmed growth medium containing the
fluorescent probe. Relative fluorescence intensity was recorded in arbitrary
units using a scanning fluorimeter. In Fig. 10a, the infecting phage was T5wt;
in Fig. O10b, the phage was T5h12-.

0, RM 42
O, RM 43 (Collb)
A, RM 39 (ColIlb h-)





52












50 /





22 0
L,
10

D 0









00



4 C)





0

2 4~ 6 a 0C 2 16 IS 20 22

TIME (MINUTES) Figure 10b





53



Only in Tb-infected RMl 43 (ColIb) is a third rise noted. The final increase in the fluorescence signal begins about 12 minutes after infection of the colicinogenic hosts, coinciding with the time when macromolecular synthesis ceases and uptake systems fail. Only the first two rises are seen after infection of RM 42 or RM 43 (Collb) by the T5 host range mutant, Tbhl2. The latter finding is consistent with the idea that the third rise in fluorescence yield is related to the abortive process.

Potassium Efflux During T5 Infection of RM 42, RM 43, and RM 39

The third increase in fluorescence intensity during abortive infection suggests that membrane depolarization occurs. To collect further evidence of this, 1 decided to measure efflux of potassium ions during T5 infection.

After preloading cells with 42K, a portion was infected with T5, whereas another portion remained uninfected. Samples of each were taken at intervals. As can be seen in Table 6 and Figure 11, labeled potassium ions pass to the medium during the first 4 to 8 minutes of productive or abortive infection. Thereafter, however, efflux stops in infected RM 42 or RM 39. Efflux continues in the abortive infection, though, so that by 10 minutes after infection there is a marked difference between the cells which are productively or abortively infected as regards labeled potassium content.

Attempts to Prevent Abortion of T5 Infection of Col~b-Containing Cells
In several other systems where abrupt cessation of energyrequiring events occurs in conjunction with membrane alterations,


















Figure 11. Potassium Efflux from T5-Infected RM 42,
RH 43, and RM 39.

Cells were grown in the presence of radiolabeled potassium (0.1 mCi per ml, 0.18 mCi per mg of K), spun, washed, and infected or
mock-infected. At time zero, the infected or mock-infected cells were resuspended in
medium without the radiolabel. At the indicated intervals thereafter, 0.9 ml
samples were removed, filtered over glass
fiber filters, end the filters were washed.
The amount of residual, intracellular
labeled potassium was determined by liquid
scintillation counting. 4 he results
represent the amount of K remaining inside
infected cells, expressed as a % of the
amount remaining in mock-infected cells taken
at the same time point.

0 RH 42
CJ RM 43 (Collb)
A ,RM 39 (Collb h-)





55













I0







0





G o
5




"" A
40


66 30




20






08 12 G 20

T I M E (MINUTES)

Figure 11




56







TABLE 6. Potassium Efflux From 15-Infected RM 42, RM 43, and RM 39




Minutes
After RM 42 (3) RM 43 (3) RM 39 (1)
Infecti on


2 56 +-2 46 +-27 68

4 43 +l0 39 +-28 56

6 32 7 36 -126 45

8 34 8 23t12 37

10 *35 +7 *20 +8 36

15 *31 + 9 *12 9 39

20 *35 t 11 *10 3 47


indicates the number of experiments done in each group

*indicates a statistically significant difference, p <0.05,
between mean values for infected RM 42 and infected RM 43.
Since efflux from infected kM 39 was only measured in one
experiment, statistical analysis was not used to compare
these results with those obtained with infected RM 42 and
kM 43.

RM 42, kM 43, and kM 39 were preloaded with 42K. At intervals,
uninfected and infected cells were taken to determine the
amount of potassium efflux. The results represent the amount
of 42K remaining inside infected cells, expressed as a % of
the amount remaining in uninfected cells taken at the same
time point.




57



means have been devised to prevent interruption of normal processes. I decided to screen a variety of these treatments to determine if any would be effective in allowing T5 to replicate in Collb-containing cells.

DCCD is an inhibitor of membrane-bound ATP'ase (43), and has been used effectively in conjunction with high concentrations of magnesium and potassium to prevent colicin K-mediated cell death (56). Sucrose or polyamines, or high concentrations of either magnesium or calcium with low sodium concentrations have been effective in preventing abortion of T4-11 mutant infections of E. coli k-12 (X) (14).

Table 7 outlines my results. In no case was a treatment effective in circumventing abortive T5 infection, as determined by titering phage present 4 to 7 hours after infection. Since calcium is required for successful T5 replication (59, 77, 78), high calcium concentrations were also tested. These trials were similarly unsuccessful.

Macromolecular Synthesis in T5 Wild-Type
and T5AI- Infection of RM 42, RM 43, and RM 39

It was my opinion that a previous report (45) suggesting that early protein synthesis was necessary for abortive T5 infection was inconclusive. Therefore, I used mutants of T5 to restudy this question. The T5AI phage mutants are eminently suited for this purpose. As discussed in the INTRODUCTION, these mutants cannot produce any class II or class III proteins, since the Al function is required for second-step DNA transfer. Al mutants are also unable to shut off class I gene expression, another function controlled by the Al gene

(71). I have used two mutants, T5aml6d and T5amH27, each of which has an amber mutation in the Al region (5).





TABLE 7. Attempts to Prevent Abortion of T5 Infection of Collb+ Hosts


Bacteria Potassium Calcium Magnesium Sodium Sucrose DCCD Polyamine Titer

RM 42 0.5 mM + 1 x 1011/ml
RM 42 0.5 mM + + 3 x 1010/ml
RM 43 0.5 mM + 6 x 108/ml
RM 43 0.5 mM + + 1 x 108/ml
RM 43 0.5 mM + + 1 x 108/ml
RM 43 + 0.5 mM + 7 x 108/ml
RM 43 + 0.5 mM + + 1 x 109/ml
RM 43 0.5 mM + 6 x 108/ml
RM 43 0.5 mM + + 6 x 108/ml
RM 43 + 0.5 mM + + -2 x 108/ml
RM 43 0.5 mM + cadaverine 7 x 108/mi
RM 43 0.5 mM + cadaverine 1 x 109/ml
RM 43 0.5 mM + arginine 6 x 108/ml
RM 43 0.5 mM + arginine 1 x 109/ml
RM 43 0.5 mM + spermidine 1 x 108/ml
RM 43 0.5 mM + spermidine 3 x 108/ml
RM 43 4 mM + 3 x 108/ml
RM 43 -4 mM + + 6 x 108/ml
RM 43 4 mM + + + 1 x 108/ml
RM 43 4 mM + + + 1 x 108/ml


Cells were grown and infected as outlined in MATERIALS AND METHODS.
Where indicated, additives were present at concentrations also indicated in MATERIALS AND
METHODS. At 4 to 7 hours after infection, the phage were plated on a lawn of E. coli B
to determine the titer.




59



Figure 12 represents cumulative [3 H] uridine incorporation into TCA-precipitable material in infected bacteria, while Figure 13 shows the results of the analogous experiments with [3HJ tyrosine. Uptake of the labeled precursors and incorporation into macromolecules continue for at least 24 minutes after RM 42 is infected with either wild-type T5 or with an Al mutant. In the corresponding infections of RM 43 (Collb), however, incorporation of both uridine and tyrosine into macromolecules stops at 6 to 12 minutes after either mutant or wild-type phage is added. The possibility that this shut-off in R1 43 (ColIb) is due to a property of the colicinogenic host unrelated to abortive infection is ruled out by the fact that incorporation of both substances in infected RM 39 (ColIb h-) continues for the duration of the experiments. From these experiments, it is evident that only class I (pre-early) gene products are necessary to induce the abortive cessation of RNA and protein synthesis.

Proline, Glutamine, and aMG Accumulation
in T5 Wild-Type and T5aml6d-Infected RM 42, RM 43, and RM 39
Previously, it was shown that decreased ability of host cells to accumulate proline and glutamine is characteristic of abortive infections, while enhancement of net uptake of aMG occurs. Experiments comparing the ability of T5aml6d- or wild-type T5-infected RM 43 (Collb) and RM 39 (Collb h-) to transport these three substances are shown (Fig. 14, 15, 16; Tables 8, 9, 10). The inhibition of proline and glutamine net uptake is the same whether a wild-type or an Almutant infect a non-permissive host. Likewise, the stimulation of OMG transport occurs when either Al mutants or wild-type phage are used.










Figure 12. Uridine Incorporation into Acidinsoluble Macromolecules in
Uninfected and T5Al--Infected 25
RM 42, RM 43, and RM 39.
Cells were grown and infected in 24
synthetic medium. [ H] Uridine 20
(1 pCi per ml, 5 pCi per mole)
was added at time zero. Samples
of 0.9 ml were removed at the
indicated times and mixed withi- 1
0.1 ml of cold 50% TCA. Acid- 10
insoluble material was collected X
on glass fiber filters, and the
filters were washed. The amount C
of incorporated radioactivity 1I0
was determined by liquid scintillation counting. In Fig. 12a,
the bacteria used were RM 42; |
Fig. 12b represents results 5obtained with RM 43 (Collb), and
in Fig. 12c, RM 39 (Collb h-) was I
used.

0, control, no infection 0 16 9 2 5 8 21 24
U, T5wt infection TIME (MINUTES)
*, T5aml6d infection Figure 12a
A, T5amH27 infection













254 1



20



151

x !0



5




0
1 2 I I'S P-4 0 I 1'5 I'L t i4
T!ME MINUTES) TIME MINUTES)

Figure 12b Figure 12c









Figure 13. Tyrosine Incorporation into Acidinsoluble Macromolecules in
Uninfected and T5Al--Infected
RM 42, RM 43, and RM 39.

Cells were grown and infected in synthetic medium. [3H] Tyrosine (0 pCi per ml, 7.2 iiCi per pmole) was added at time zero. Samples
of 0.9 ml were removed at the 4indicated times and mixed with A
0.1 ml of cold 50% TCA. Acidinsoluble material was recovered x 3
on glass fiber filters, and the
amount of incorporated radioactivity was determined. In Fig. 13a, the host was RM 42;
in Fig. 13b, the host was RM 43 (ColIb); in Fig. 13c, the host
Ii/
was RM 39 (ColIb h-).

0, control, no infection
*,T~tinetin0 3 j 9 J IS 5 Z3 4 2
T5wt infection(MINUTES)
T5aml6d infection
A, T5amH27 infection Figure 13a














:.











0z z






0 3 6 9 21 IS '$2i 24 27 30 0 3 6 9 12 ,5 :32'12427 30
TIME (MINUTES) TIME (MINUTES)


Figure 13b Figure 13c



















Figure 14. Proline Accumulation in 60 Seconds
by T5Al--Infected RM 42, RM 43,
and RM 39.

Cells were grown and infected in
synthetic medium. At the indicated times after infection, samples were removed and mixed with [3H] proline
(1 uCi per ml, 25 pCi per pmole).
Aliquots were removed at 30 seconds
and 60 seconds thereafter and filtered
through glass fiber filters. Here,
the amount of radioactive proline
accumulated in 60 seconds by infected
cells is expressed as a % of the
amount taken up by uninfected controls
in the same period of time.

0 T5aml6d-infected RM 42
O, T5aml6d-infected RM 43 (Collb)
A T5aml6d-infected RM 39 (Collb h-)
M T5wt-infected RM 43 (Collb)




65















14012010



eo 60



40 20

-AZIDE LOVEL

0 10 20 30
TIME(PAINUTES)

Figure 14







TABLE 8. Proline Accumulation (in 60 seconds) by Infected RM 42, RM 43, and RM 39

CONDITION RM 42-T5aml6d vs RM 43-T5aml6d vs RM 39-T5aml6d(3) RM 43-T5aml6d vs RM 43-T5wt(1)

5 minutes after phage 97 + 40 89 1 15 64 t 10 88 70

10 minutes after phage 96 t 24 91 4 107 + 26 87 115
15 minutes after phage 110 t 15 46 2 112 t 33 45 54

20 minutes after phage 99 8 37 t 13 109 t 25 40 31
25 minutes after phage 115 t 9 37 10 121 t 29 41 24
30 minutes after phage 142 26 27 t 9 113 t 22 32 21

NaN3 + uninfected cells 10 t 5 6 t 1 11 t 4 8

indicates the number of experiments done in each group
indicates a difference between the 2 samples with a confidence level, p <0.05

[3H] Proline accumulation by infected cells was determined as outlined in MATERIALS AND METHODS.
The results represent %'s of uptake, relative to that of uninfected control samples, in 60 seconds.





67








Figure 15. Glutamine Accumulation in 90 Seconds by T5AI -Infected
RM 42, RM 43, and RM 39.

Cells were grown and infected in synthetic medium. At
the indicated times, samples were removed and mixed
with [14C] glutamine (0.5 pCi per ml, 5 pCi per pmole).
At 30, 60, and 90 seconds thereafter, aliquots were
removed and filtered through glass fiber filters. The
results here are for the 90-second pulses, expressed as a % of the amount taken up by uninfected controls
in the same period of time.

0, T5aml6d-infected RM 42
O, T5aml6d-infected RM 43 (Collb)
A, TSaml6d-infected RM 39 (Collb h-)
T5wt-infected RM 43 (Collb)






p A

A
680






40



2.0

-AZIDE LEVEL

0 10 20 30
TU E( MI NUTES)





TABLE 9. Glutamine Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39

CONDITION RM 42-T5aml6d vs RM 43-T5aml6d (3) RM 43-T5aml6d vs RM 43-T5wt (3)

5 minutes after phage 61 17 70 24 70 t 24 52 20
10 minutes after phage 55 12 33 10 33 t 10 35 2
15 minutes after phage 54 t 11 21 2 21 2 24 7
20 minutes after phage 65 24 16 5 16 5 18 5
25 minutes after phage 71 31 16 5 16 5 17 4
30 minutes after phage 74 36 14 5 14 5 17 t 6
NaN3 + uninfected cells 7 2 5 4 5 4

CONDITION RM 39-T5am16d vs RM 43-T5 16d (2)

5 minutes after phage 90 8 82 15
10 minutes after phage 90 6 31 13
15 minutes after phage 86 1 20 2
20 minutes after phage 75 17 13 2
25 minutes after phage 77 2 19 0
30 minutes after phage 60 8 16 5
NaN3 + uninfected cells 5 4 2 2

indicates the number of experiments done in each group
indicates a difference between the 2 samples with a confidence level, p< 0.05
co
[14C] Glutamine accumulation by infected cells was determined as outlined in MATERIALS AND METHODS.
The results represent percentages of uptake, relative to that of uninfected control samples, in 90
seconds.





69




Figure 16. m-Methylglucoside Accumulation in 90 Seconds
by T5Al--Infected RM 42, RM 43, and RM 39.

Cells were grown and infected in synthetic medium. At
the indicated times after infection, samples were removed, spun, and washed with M9. After a second wash, the cells were resuspended in M9 with 18 pg of glucose per ml, and
0.1 ml of [14C] caMG (10 pCi per ml, 184 pCi per mole) was added to 0.9 ml of the cell suspension. Aliquots
were removed at 45 and 90 seconds, thereafter, and were filtered through glass fiber filters. The results here
are for the 90-second pulses, expressed as a % of the
amount taken up by uninfected controls in the same period
of time.

0, T5aml6d-infected RM 42
0, T5aml6d-infected RM 43 (Collb)
A, T5aml6d-infected RM 39 (Collb h-)
T5wt-infected RM 43 (Collb)






400

350





o s a







POISONED LEVEL
30 15-O 3
o.5 5 0 i toAs o





0I
450- 0





TABLE 10. a-Methylglucoside Accumulation (in 90 seconds) by Infected RM 42, RM 43, and RM 39

CONDITION RM 42-T5aml6d vs RM 43-T5aml6d (4) RM 43-T5aml6d vs RM 43-T5wt (1)
5 minutes after phage 180 + 23 296 t 67 328
10 minutes after phage 165 41 429 89 393 331
15 minutes after phage 212 t 27 361 t 50 225
20 minutes after phage 181 60 348 54 300
25 minutes after phage 157 45 320 69 213 212
30 minutes after phage 198 54 256 t 77 165
NaN3 + NaF + uninfected cells 19 7 19 10

CONDITION RM 39-T5aml6d vs RM 43-T5aml6d (3)
5 minutes after phage 143 8 267 42
10 minutes after phage 165 21 405 92
15 minutes after phage 194 1 37 393 49
20 minutes after phage 259 72 352 66
25 minutes after phage 239 38 320 1 85
30 minutes after phage 234 21 283 70
NaN3 + NaF + uninfected cells 10 8 17 1 10
( ) indicates the number of experiments done in each group
indicates a difference between the 2 samples with a confidence level, p< 0.05
[14C] aMG accumulation by infected cells was determined as outlined in MATERIALS AND METHODS. The a
results represent %'s of uptake, relative to that of uninfected control samples, in 90 seconds.





71


Fluorescence of Membrane-Bound NPN During T5AI- Infections

When T5aml6d infects RM 42, RM 43, or RM 39 in the presence of NPN, there is an increase in fluorescence intensity at about three minutes after infection but no increase at about 6 minutes (Fig.17). Since the mutant is deficient in second-step DNA transfer, this is consistent with Hantke and Braun's idea that the second increase in fluorescence intensity reflects membrane alterations related to second-step DNA transfer (41).

What is evident, however, is the marked increase in fluorescence intensity at about 10 to 12 minutes only occurring after infection of RM 43. This is a pattern similar to that found after T5 wild-type infection of the ColIb+ cells.

Potassium Efflux During T5aml6d Infectionsof RM 43

Cells were preloaded with radiolabeled potassium ions as described above. As can be seen in Table 11 and Fig. 18, the pattern of ionic efflux in T5aml6d-infected RM 43 is similar to that during T5 wild-type infection of R1 43. After plateauing early after infection, the numbers of residual intracellular counts falls rapidly beginning at about 8 to 10 minutes in each case. The reason for the greater early retention of preloaded ions in the T5 wild-type-infected cells is unknown. These figures are much higher than those obtained in other experiments (Table 6 ). Since the amount of initial efflux is very dependent upon multiplicity of infection (94) and since the data in Tables6 and 11 were derived on different days, it might be that the multiplicities of infection were not comparable. If so, however, this was inadvertent. Nonetheless, if comparing the patterns of efflux seen





72







Figure 17. Fluorescence Intensity of N-phenyl-lnaphthylamine During T5A1- Infections
of RM 42, RM 43, and RM 39.

Cells were grown, concentrated, and
infected in synthetic medium. At
time zero, the infected cells were put into prewarmed growth medium containing
the fluorescent probe. Relative fluorescence intensity was recorded in arbitrary
units using a scanning fluorimeter.

0 RM 42
0, RM 43 (Collb)
A, RM 39 (Collb h-)









so
40



20




2 4 6 0 to 12 H14 Is IB 0 22 TIME (MINUTES)




73







TABLE 11. Potassium Efflux from T5Al--Infected RM 42 and RM 43



Minutes
After RM 42-T5aml6d RM 43-T5aml6d RM 43-T5wt
Infection


4 65 67-55 99

8 43 52-48 95

10 42 40-33 79

12 46 27-29 62

15 51 21-17 51

20 55 11-9 37


RM 42, RM 43, and RM 39 were preloaded with 42K. At intervals,
uninfected cells were taken to determine the amount of
potassium efflux. The results represent the amount of 42K
remaining inside infected cells, expressed as a % of the
amount remaining in uninfected cells taken at the same time point. Leakage from infected RM 43 was measured during two
trials.




74




Figure 18. Potassium Efflux from T5Al -Infected RM 42 and RM 43.

Cells were grown in the presence of radiolabeled
potassium, spun, washed, and infected or mock-infected.
At time zero, the infected or mock-infected cells were
resuspended in medium without the radiolabel. At the
indicated intervals thereafter, 0.9 ml samples were removed, filtered over glass fiber filters, and the filters were washed. The amount of residual, intracellular labeled potassium was determined by liquid
scintillation counting. The results represent the
amount of 42K remaining inside infected cells,
expressed as a % of the amount remaining in mock-infected
cells taken at the same time point.

V, T5aml6d-infected RM 42
O, T5aml6d-infected RM 43 (Collb)
T5wt-infected RM 43 (Collb)






100
90i 80

S0

0 e

40
z





20
0




4 8 12 16 20
TIME (MINUTES)





75


during T5 wild-type or T5aml6d infection of RM 43 to the efflux seen after T5anl6d infection of RM 42, it is apparent that the initial decline in residual counts during intection of RM 42 plateaus at about 8 minutes after infection. This is not true during infection of RM 43, wherein ionic efflux is marked beyond 10 minutes after infection.

Absence of Host DNA Breakdown During T5aml6d or T5amH27 Infection
To confirm the identity of T5aml6d and T5amH27 mutants, a number of studies were performed. As can be seen in Table 1, these mutants do not plate efficiently unless the strain has suppressor activity. I have also tested these mutants for their ability to break down host DNA into acid-soluble fragments. This is the function of the Al polypeptide, and should be lacking in Al- mutants (62).

RM 42, RM 43, and RM 39 were grown in the presence of [3 H]thymidine as described in MATERIALS AND METHODS. As seen in Fig. 19, host DNA is not degraded to acid-soluble material after infection by T5AI mutants, confirming the lack of production of a functional Al polypeptide.

Gel Analysis of Phage Proteins

It was suggested that RM 43 (ColIb) could have an amber suppressor present on the plasmid, invalidating any conclusions regarding infection of these cells with amber mutants. To show that this was not the case, by confirming that class II (early) or class III (late) proteins are not made in Al--infected, ColIb+ cells, we pulse labeled proteins synthesized from 1 to 6 minutes, 6 to 11 minutes, or 11 to 16 minutes after TSaml6d infections of the colicinogenic hosts (RM 43). On SDSpolyacrylamide gels, we compared these samples with those of T5aml6d









Figure 19. Absence of Host DNA Breakdown During
T5Al -Infections of RM 42, RM 43, 3500
and RM 39.

DNA was labeled by growing the cells 3000 o
in the presence of [3H] thymidine
(0.4 NCi per ml, 1 jiCi per 50 pg of <
thymidine). The cells were spun, concentrated, infected, and rediluted in 2500 LL< __ __ -M---medium lacking radiolabeled thymidine. i
At times thereafter, 0.9 ml samples
were removed and placed in 0.1 ml of 0 2000 I
50% TCA. Of this, 0.4 ml was filtered
over glass fiber filters and washed.
The numbers of acid-precipitable counts N
were determined by liquid-scintillation 1500
counting. In Fig. 19a, the host is
RI 42; in Fig. 19b, the host is RM 43 0
(Collb), whereas in Fig. 19c, the host = 000
is RM 39 (Colib h-).

uninfected cells 500
T5-infected cells
T5aml6d-infected cells
A, T5amH27-infected cells 4 8 2 16 20

TIME(MINUTES)

Figure 19a
















30 5 Cc






4 8



x










Fiur Fi1ure6 20





78



infections of RM 42, and with T5 wild-type infections of both strains. Only class I (pre-early) proteins were seen during Al- infections of either host strain (Fig. 20).




















Figure 20. Gel Analysis of Phage-Induced Proteins During
T5 and T5AI- Infections of RM 42 and RM 43.

Cells were grown and infected in a synthetic
medium. At the times indicated, RM 42 or RIM 43 (Collb) infected with either T5 or T5aml6d were labeled with 1 pCi per ml of a [14C] amino acid
mixture. Chloramphenicol was added 5 minutes
later, and the cells were chilled. Then, they
were centrifuged, washed and resuspended in 1/10th volume of electrophoresis buffer and
boiled for 5 minutes. Electrophoresis of 20 pl
samples was on a 15% slab gel for 14 hours at 75 volts. The dried gel was autoradiographed for 10 days by exposing it to Kodak XRl film.

The numbers across the bottom indicate the
time after infection when the pulse began. The proteins were called pre-early or early according to their time of appearance (70).





80




















Pre-early Early
Early--
Pre-early -Early -------Pre-early -Pre-early -Pre-early -U 1 6111 6_11jU 1 611 1 611
T5 A1- T5 AlRM 42 Wis 43













DISCUSSION

It has been known for more than 10 years that T5 is unable to replicate in hosts containing the colicin lb factor (80, 97). Very little is known, however, about the mechanism whereby phage development is inhibited. Originally, no class II (early) or class III (late) proteins or RNA was seen on polyacrylamide gels. Because of this, it was hypothesized that a class-specific transcriptional block occurred (79). Later, though, it was found that some class II (early) proteins do actually appear. Additionally, functional RNA polymerase can be recovered from abortively infected cells (99), and no changes in the DNA platee are found that explain the abortive infection (45, 89). Thus, there seems to be no evidence to implicate a primary transcriptional or translational dysfunction.

It seems more plausible to suggest that a generalized cellular dysfunction is the cause. During abortive infection, there is abrupt, simultaneous cessation of both transcription and translation, and there are numerous other concurrent physiological alterations not easily explained by specific-site inhibition. As membrane defects are thought to play a key role in other abortive systems, I decided to see if changes in membrane function during T5 infections of Collb + hosts could underlie the abortive process.

Previously, others in Donna Duckworth's lab have reported that uptake of thio-5-methyl-D-galactoside (1MG), a nonmetabolizable lactose analogue taken up by an ATP-dependent, active transport



81





82



mechanism (104) is inhibited during abortive infection (19). In the present study, I sought to further define the membrane's functional defects by looking at uptake of three additional substances -- proline, glutamine, and aMG -- each of which is taken up by a different mechanism (reviewed in 102).

Initially, however, I wanted to define the abortive system in

our laboratory. In agreement with the results reported by others (79), incorporation of radiolabeled uridine into TCA-insoluble macromolecules was found to stop 9 to 12 minutes after T5 infection of cells containing the ColIb factor. This was the same time at which cessation of tyrosine and proline incorporation into acid-insoluble macromolecules occurred during abortive infection. Since I wished to define physiological alterations directly related to this abrupt interruption of the infectious process, I looked at changes occurring within the first 30 minutes after infection.

During abortive infection glutamine accumulation is inhibited, beginning to decline by 10 minutes after infection. Since the active transport of glutamine is an ATP-dependent process requiring periplasmic binding proteins (6, 7), decreased uptake ability might be due to decreased available ATP, loss of periplasmic binding proteins, or marked permeability changes which prevent concentration of the substance. The last of these explanations probably cannot totally explain the data, since at a time when glutamine accumulation is markedly depressed, aMG accumulation is three- to four-fold greater than that in uninfected cells. Regarding the loss of binding proteins, the inner membranes of abortively infected cells do not become sensitive




83



to SOS-induced lysis until more than 20 minutes after infection (19), so it is unlikely that the outer membrane integrity is destroyed to an extent necessary for periplasmic proteins to leak by 10 minutes after infection. The most likely explanation for the inhibition of glutamine uptake is that adequate energy is no longer available.

Net uptake of proline decreases during abortive infection at about the same time, or slightly later than the decline of glutamine accumulation. Proline uptake, unlike that of glutamine, does not require periplasmic binding proteins (6, 7). Even isolated membrane vesicles are capable of proline uptake, indicating that components of the uptake system are bound firmly to the membrane (reviewed in 102). Nonetheless, proline uptake is similar to glutamine uptake in that it requires an"energized membrane." Invoking the same argument against marked membrane permeability changes by 10 to 15 minutes after infection, I think the most likely reason for decreased proline accumulation is also membrane deenergization.

By membrane deenergization, I refer to loss of the transmembranal electrochemical gradient normally present (73). Since ions at low concentration obey the gas laws, this gradient can be expressed as: A V =AT' 2. 3RT AH
F AH
where A-H+ equals the electrochemical proton gradient, AT represents electrical potential difference, R is the gas constant (8.3 joules per Kelvin degree), T equals the temperature in Kelvin degrees, F is the faraday (96,500 coulombs), and ApH means the proton gradient. It is thought that a normal bacterium has a transmembranal electrical potential of 75 mV (84) to 140 mV (40), but at pH's near 7, ApH is




84



very small (34, 88). Therefore, under conditions used in the proline and glutamine uptake experiments, loss of membrane energization would be equivalent to loss of ionic polarization.

If decreases of proline and glutamine accumulation do indeed result from membrane deenergization during abortive infection, the prediction is that loss of ionic polarization should occur at the same time. This, in fact, has been shown herein, at least for the case of a major intracellular cation, potassium. Efflux of preloaded potassium from abortively infected cells becomes significantly greater than efflux from productively infected cells at about 10 minutes after infection.

Another prediction which is based on the assumption that the

membrane is deenergized is that the fluorescence intensity omitted by NPN should increase dramatically during abortive infection. This change in fluorescence signal is observed when bacteria are treated with substances, such as derivatives of carbonyl cyanide phenylhydrazone, known to dissipate the high energy state of membranes (44). Since this lipophilic probe is neutral at physiological pH, it should not respond merely to changes in transmembranal potential (81). Rather, the rise in intensity is thought to occur due to structural changes in the outer membrane that are secondary to membranal deenergization

(44). The structural changes in the outer membrane allow more NPN to bind to the inner membrane. Since the fluorescence intensity emitted by NPN increases directly as its environment becomes more hydrophobic (Leonard Rosenberg, personal communication), the observed increase is consistent with the interpretation that more of the probe moves into the lipid phase of the membrane. How or why it happens





85


was not investigated in the current study, but the predicted fluorescence changes did, in fact, coincide with the time when membrane depolarization is thought to occur in abortive infection.

Since binding-protein transport, represented by glutamine uptake, and membrane-bound transport, exemplified by proline uptake (reviewed in 102) were both inhibited, I decided to see if a substance taken up by group translocation was similarly affected. aMG is a nonmetabolizable glucose analogue taken into the cell by the phosphotransferase system (57). As the sugar is taken into the cell it is phosphorylated using phosphoenolpyruvate as its primary source of the phosphate group. At first, it seemed surprising that I found aMG accumulation was stimulated during abortive infection. However, this result is to be expected if the bacterial membrane becomes deenergized (Hans Kornberg, personal communication). In the absence of an energy-requiring dephosphorylating reaction, olG becomes trapped within the cell as its phosphorylated derivative (49, 52). In fact, within a wide range of concentrations, dinitrophenol or azide causesa marked stimulation of cMG uptake (32).

In all cases, the pathophysiological alterations indicative of membrane depolarization occur only during abortive infection. When the infection is productive because of plasmid-borne or phage-borne mutations, uptake systems function as in normal T5 infections of ColIbhosts, and neither the pronounced potassium leakage nor the abnormal fluorescence pattern is seen. It appears likely, therefore, that the membrane alterations are due to genetic determinants on the phage genome or plasmid DNA at those loci which also produce cessation of the infectious process.




86



The abortive pattern of pathophysiological alterations occurs, however, when Collb + cells are infected with mutant phage deficient in second-step DNA transfer. The identity of the Al mutants used in our laboratory has been confirmed previously (29). 1 have also confirmed the mutant's inability to destroy the host genome, a known Al function (62). Furthermore, gel analysis of proteins synthesized during Al infections confirms the absence of early and late proteins. These data are strong evidence that only pre-early phage genes are required for the abortive response. They also indicate that the shutdown of phage gene expression is not the result of a class-specific transcriptional or translational defect, since the cessation of macromolecular synthesis during abortive infection usually occurs after early gene expression has begun (45, also see Fig 20 of this dissertation). Stated another way, the mechanism operable in shutting off the T5 replicative cycle does so at 10 to 12 minutes after infection, regardless of whether pre-early or, early proteins are being synthesized at that time.

The evidence against a specific-site block and the data indicative of membrane depolarization strongly suggest that the mechanism leading to the abortive response is quite similar to the killing action of phage ghosts and to the bactericidal action of colicin proteins El, K, Ia, and lb (66). Osmotically shocked T-even phage lose their DNA and their infectivity, but the empty protein shells retain the capacity to kill the host cell (47,48). Within two minutes, these "ghosts" inhibit DNA, RNA, and protein synthesis (27). Also quite early, uptake of DL-leucine and nucleic acid precursors (27), as well as the





87


uptake of 0-galactosides (103), is diminished. Phosphorylated compounds including ATP, UTP, UDP, UMP, a-MG phosphate, and thio-B-methylgalactoside phosphate leak from ghost-treated cells (30,48). Yet o-nitrophenylgalactoside (ONPG), carbamyl phosphate, or ATP entry is not enhanced (30) indicating a peculiar sort of one-way permeability defect. Additionally, ghost preparations contain some factor which causes a variable amount of cell lysis (47), but this lysis can be prevented by 0.05 M spermidine without influencing cell killing (D. Duckworth, unpublished data, cited in 28). Thus, phage ghosts inhibit macromolecular synthesis, inhibit active transport systems, cause "excretion" of at least some phosphorylated compounds, and cause a certain amount of cell lysis.
Similar actions are observed when sensitive bacteria are treated with colicin El, K, la, or Ib -- collectively representing a group of proteins which are produced by plasmid-containing Enterobacteria and which seem to kill bacteria by the same (or similar) mechanism(s) (reviewed in 64). These proteins, for example, inhibit DNA, RNA, and protein synthesis (63,82). They also inhibit uptake of several amino acids (Gilchrist & Konisky, unpublished data, cited in 55,65; J. P. Kabat, cited in 86), of B-galactosides (36, Gilchrist and Konisky, unpublished data cited in 55), and of potassium (Gilchrist & Konisky, unpublished data, cited in 55,101), rubidium (101), magnesium, and cobaltous ions (67). Colicins accelerate loss of preaccumulated potassium (Gilchrist & Konisky, unpublished data, cited in 55,101), magnesium, and cobalt ions (67) -- a change unlike that due to energy poisons (93) -- probably indicating a permeability defect. Also,





88



they cause cells to lose phosphorylated compounds (37), sugars, and amino acids (30, Gilchrist & Konisky, unpublished data, cited in 55). The membrane lipid and protein composition of treated cells is altered (16,17,18,53,90) and, under some conditions, the cells lyse (17). The lysis does not occur in minimal medium (67). Further, the rate of ONPG hydrolysis is unchanged (36), ATP is not found free in the medium

(35), and a-MG uptake is augmented (52). Therefore, permeability to, or excretion of, various substances seems to be selective. In summary, physiological changes induced by ghosts and by colicins El, K, la, and Ib are quite similar.

This group of colicins is thought to act by forming ion channels

(91). The evidence for this is derived from in vitro studies with phospholipid bilayers. Voltage-dependent, ion-permeable channels are introduced into bilayers by purified colicin preparations. Other data are consistent with this proposal. (1) Fluorescent dye probes indicate membrane deenergization (44,85), and the electrical transmembrane gradient is dissipated (100). (2) The decreased ATP level, characteristic after colicin treatment, is prevented in colicin Elor colicin K-treated cells by chemically or genetically inducing loss of membrane-bound ATP'ase activity (86). The inference is that the ATP'ase-deficient cells are unable to exhaust ATP supplies in what would be a futile effort to reestablish membrane polarity. (3) When ATP supplies are maintained in this way, macromolecular synthesis continues at a substantial rate (75), but transport activities are not improved. The idea arising from these observations is that cessation of macromolecular synthesis is secondary to the ATP deficit. However,




89


the membrane depolarization is not corrected, and therefore, active transport systems are still impaired. (4) ATP'ase-deficient strains, when grown in glucose medium with high Mg++ and K+ concentrations, remain viable in the presence of colicin K or El ( 56). Under these conditions, essential intracellular cation concentrations are maintained, and the cell can continue metabolic functions. Since physiological studies completed thus far indicate numerous similarities among abortive T5 infections, phage ghost action, and colicin action, it might be that they all act by a mechanism somewhat similar to this.

Recently it has been proposed that T5 and BF23 infectious

processes halted by the colicin Ib protein itself (D. J. McCorquodale, et al., submitted to J. Virol.; D. J. McCorquodale, personal communication). According to the model, the pre-early A3 gene product inactivates an immunity protein, which normally protects the bacterium from the colicin it produces. Abortive infection should not occur if the ColIb plasmid is mutated such that the colicin protein is not produced, nor should it occur if the immunity protein is altered in such a way that it cannot be bound by the A3 protein but that it can still prevent the bactericidal action of the colicin protein. The infectious process should likewise proceed unhindered if there are alterations at the target site of the colicin protein or in biochemical pathways required for expression of the colicin'slethality. Furthermore, according to the model, other abortive systems such as T7 infection of F-containing E. coli and T4rll mutant infection of X lysogens can be similarly explained. In each case, the hypothesis is that a potentially lethal protein encoded on the extrachromosomal




9O



element (42) is activated by the infection -- leading to the cessation of the infectious process before progeny appear.

This model, though promising, is currently based only on circumstantial evidence. Although an immunity protein for colicin E3 has been isolated (51), no direct evidence for the existence of a colicin Ib immunity protein has been described. Secondly, some permissive hosts bearing plasmid mutations have been found to lack the ability to produce the colicin protein (S. S. Tung, cited by D. J. McCorquodale), while other permissive strains with mutant plasmids do produce colicin (D. J. McCorquodale, personal communication). This does suggest that both the colicin and an immunity protein are involved in abortive infections, but the mutant strains have not been characterized well enough to say this with any certainty. Thirdly, there is only a weak suggestion that the chromosomal loci, cmrA and cmrB, involved in abortive infection are also the loci coding for the colicin Ib target. The evidence cited is that cmrA and cmrB map close to trkA and trkB, loci involved in potassium transport (33). Also, the toll gene, which confers tolerance to colicin Ib (15), maps close to another locus, trkC, involved in potassium transport (33). From these data, the hypothesis is that the cmrA, cmrB, and toll loci are identical to trkA, trkB, and trkC, respectively. Beyond this, it must be assumed that the potassium transport system is the target of colicin lb. Fourthly, the comparison of the ColIb system with other abortive infections cannot be as direct as is suggested by the model. For example, in the T4rIl mutant system, the lack of a functional rll protein contributes to the abortive infection, whereas the presence of the A3 gene product is necessary for abortive T5 infection.