JOURNAL OF BACTERIOLOGY, Dec. 1984, p. 1191-1195 Vol. 160, No. 30021-9193/84/121191-05$02.00/0Copyright © 1984. American Society for Microbiology

Bacteriophage T5 Gene A2 Protein Alters the Outer Membrane ofEscherichia coli

CLIFFORD E. SNYDER, JR.t

Department of Biology, University of Virginia, Charlottesville, Virginia 22901

Received 19 March 1984/Accepted 10 September 1984

Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 proteinwas obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis,and in vitro binding studies with purified gene A2 protein. The results suggested that the T5 gene A2 proteinperturbs the host envelope as it functions to promote DNA transfer.

The DNA comprising the genome of bacteriophage T5 istransferred to Escherichia coli in an unusual two-step fash-ion (12). After bacteriophage attachment and penetration ofthe envelope by the tip of the phage tail, the T5 first-step-transfer (FST) DNA, amounting to 8% of the length of the T5DNA molecule, becomes accessible to the synthetic machin-ery of the host. The entry of FST DNA is apparently notdependent on the metabolic energy of the host (17). Themechanism responsible for arrest of DNA transfer remainsunknown. All of the T5 DNA is ejected from the capsid whenthe phage is incubated with the isolated receptor (29),suggesting that arrest results from interaction of T5 DNAwith other components of the host envelope; this proposalwas supported by the results of experiments done by Labe-dan (10) involving envelope components immobilized incolumn bed material. In these experiments, an analog ofarrest was observed, and treatment with the detergent TritonX-100, an agent known to solubilize components of the hostinner membrane (25), released T5 DNA from the analog ofarrest. It has been suggested that arrest ofDNA transfer maybe due to an unusual structure of T5 DNA (27).The mechanism of second-step transfer of T5 DNA is

unknown. The polypeptides whose production is encoded bygenes Al and A2 are among ca. 10 polypeptides encoded bythe T5 FST DNA (20). Although the only function known forT5 gene A2 is that of DNA transfer, gene Al is implicated inthe additional functions of degradation of host DNA andtermination of expression of those T5 genes located on theT5 FST DNA (1, 12). It appears that host cell metabolicenergy, after synthesis of the T5 gene Al and A2 proteins, isnot required for second-step transfer of bacteriophage T5DNA (17). This investigation provides evidence that the T5gene A2 protein perturbs the structure of the host envelope.Given the postulated role of envelope components in thearrest of transfer of T5 DNA, perturbation of envelopestructure by the gene A2 protein may help to explain its rolein DNA transfer.

E. (oli B Leu- was used as the nonpermissive host forinfection by T5 amber mutants; it was also the host forgrowth of and infection by T5b1. T5b1 is a mutant phagelacking much of the T5 DNA coding for tRNA (9, 23); thesetRNA genes are apparently not required for phage growth.E. coli CR63 siupD was used to grow amber mutants of T5.T5amHl6d, defective in gene Al (20), and T5amH231,defective in gene A2 (20), were originally provided by D. J.

t Present address: Aerobiology Division, USAMRIID. Fort De-trick, Frederick, Maryland 21701.

McCorquodale. Cells were grown in 100-liter batches at 37°Cin a Bio-Tec fermentor as previously described (28), harvest-ed by centrifugation, frozen in liquid nitrogen, and stored at-70°C. Phage infections, initiated in a culture at an opticaldensity at 550 nm of 0.7, were done at a multiplicity of 5PFU/CFU. Cells infected with T5b1 or T5amH231 wereharvested 15 min after addition of phage; T5amH16d infec-tions were allowed to continue for 60 min. Although phage-infected cells did not lyse during incubation in the fermentor,infected cells were inviable (less than 10% survival of CFU).

Cells were disrupted by passage through a French pres-sure cell, and a crude envelope fraction was separated fromcytoplasm essentially as described by Schnaitman (24).Envelope material was treated successively with 2% TritonX-100 (25), 2% sodium dodecyl sulfate (SDS) in the presenceof 0.05 mM magnesium chloride (SDS-Mg2+) (22), and 2%SDS in the presence of 5 mM EDTA (SDS-EDTA), andcentrifugation was used to collect insoluble material aftereach treatment. Envelope material rendered soluble bydetergent treatment was precipitated with acetone and col-lected by centrifugation.The T5 gene A2 protein was purified as previously de-

scribed (28) from E. coli B infected with T5amHl6d. On thebasis of SDS-polyacrylamide gel electrophoresis and Coo-massie blue staining, the A2 polypeptide (molecular weight,15,000) represented about 95% of the protein in the prepara-tion. lodination (16) resulted in the incorporation of 1 mol of1251 per 500 mol of A2 protein.SDS-polyacrylamide gel electrophoresis was based on the

discontinuous system described by Laemmli (11). A 0.1-mlportion of cytoplasm was added to 0.4 ml of Laemmli samplebuffer. Each acetone pellet was dissolved in 0.5 ml of samplebuffer. The SDS-EDTA-insoluble material was dissolved in0.5 ml of sample buffer. Samples, with the exception ofportions of SDS-EDTA-soluble fractions, were placed inglass tubes and held in boiling water for 1 min beforeapplication to the gel. Whole cells (50 mg of frozen cells)were treated with 0.5 ml of sample buffer for 20 min in a bathof boiling water; soluble material was separated from insolu-ble material by centrifugation. The volumes of samplesapplied to gels ranged from 0.010 to 0.060 ml. Electrophore-sis was done at constant voltage and terminated when thefastest-moving component of the tracking dye (bromophenolblue) reached a position 0.5 cm from the bottom of theseparating gel. The procedure for visualization of binding ofradiodinated A2 protein to components on denaturing gelsfollowed that developed for calmodulin (4). Gels were fixed,washed in distilled water, and then washed with 50 mM Tris

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1 2 3 4 5 patterns from uninfected cells or T5amH231-infected cells,the T5b1 (A1+ A2') pattern (lane 3) showed reduced stainingat 37K and increased staining at 28K, whereas theT5amHl6d (Al- A2+) pattern (lane 4) showed reducedstaining at 37K and increased staining at 38K and 15K.

Figure 2 shows the Coomassie blue-stained patterns de-

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FIG. 1. Electrophoretic analysis of whole cell extracts. Cellswere prepared for electrophoresis by solubilization in Laemmlisample buffer and electrophoresed on a 7.5 to 15% polyacrylamidegel. The gel was stained with Coomassie blue. Lanes: 1, molecularweight standards (Bio-Rad), 0.002 mg of protein per band; 2,uninfected cells; 3, T5b1 (A1+ A2+)-infected cells; 4, T5amHl6d(Al- A2+)-infected cells; 5, T5amH231 (A1l A2-)-infected cells.Lanes 2 through 5 were loaded with 0.010 ml of the product ofsolubilization of cells in Laemmli sample buffer. The molecularweights (in thousands) of the polypeptide standards are indicated.The diamond symbols indicate the following polypeptides cited inthe text: lane 2, a 37K polypeptide; lane 3, a 28K polypeptide; lane4, 38K and 15K polypeptides; lane 5, a 37K polypeptide.

hydrochloride (pH 7.5) containing 1 mM sodium EDTA and0.15 M NaCl (buffer A). The gels were further washed withbuffer A containing 1 mg of bovine serum albumin per ml.Each gel (containing about 10 lanes) was then incubated in 50ml of buffer A containing 0.1 ml of radioiodinated A2 protein(0.05 mg). The gels were then washed with buffer A. Gelswere stained with Coomassie brilliant blue R-250, destained,dried onto paper by heating under vacuum, and exposed at20°C to Kodak NS-5T film. Films and gels were photo-graphed on Polaroid type 55 film. The mobilities of Coomas-sie blue-stained bands were measured relative to that of thetracking dye. The molecular weights of protein standards(Bio-Rad Laboratories) were plotted against their relativemobilities; such plots were then used to estimate the molecu-lar weights of unknown polypeptides.The electrophoretic patterns indicated differences be-

tween uninfected cells and cells infected with T5b, (A1+A2+) or T5amHl6d (Al- A2+). The pattern from T5amH231(A1+ A2-)-infected cells was indistinguishable from that ofuninfected cells. The differences between A2- (T5b1 andT5amH16d) and A2- (TamH231) infections were evident atpositions indicating polypeptide sizes of 38,000 molecularweight (38K), 37K, 28K, and 15K (Fig. 1). Compared to the

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FIG. 2. Electrophoretic analysis of cell fractions. Samples ob-tained by the fractionation procedure described in the text wereelectrophoresed on 7.5 to 15% polyacrylamide gels. The gels werestained with Coomassie blue. (A) Fractions from uninfected cells.(B) Fractions from T5b1-infected cells. (C) Fractions fromT5amHl6d-infected cells. (D) Fractions from T5amH231-infectedcells. Lanes: 1, cells solubilized by Laemmli sample buffer; 2,cytoplasmic fraction; 3, Triton-soluble fraction; 4, SDS-Mg2 -solu-ble fraction; 5, SDS-EDTA-soluble fraction; 6, SDS-EDTA-insolu-ble fraction; 7, SDS-EDTA-soluble fraction, not boiled beforeelectrophoresis; 8, molecular weight standards (Bio-Rad), 0.002 mgof protein per band. (A) Loading (in microliters) for lanes 1 through7: 10, 60, 30, 30, 20, 10, and 20. (B) Loading (in microliters) for lanes1 through 7: 10, 60, 30, 30, 60, 60, and 60. (C) Loading (inmicroliters) for lanes 1 through 7: 10, 60, 30, 30, 60, 60, and 60. (D)Loadings (in microliters) for lanes 1 through 7: 10, 60, 30, 30, 40, 20,and 40.

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rived from the envelope fractionation scheme. Lanes 1through 4 of each pattern were heavily loaded with cellularmaterial to provide potential substrate for binding of radioio-dinated A2 protein, resulting in heavy staining along thelength of these lanes and thus making comparisons on thebasis of staining difficult. The striking differences betweenthe electrophoretic patterns of boiled and nonboiled samplesof the SDS-EDTA-soluble fraction (lanes 5 and 7, respec-tively) were of particular interest since they aided in theidentification of outer membrane proteins. The 37K band ofthe SDS-EDTA-soluble and -insoluble fractions representsthe OmpF protein. This identification is consistent with thefollowing characteristics of the OmpF protein: its presencein strain B cells (13, 22), the molecular weight of the OmpFprotein (22), the contrast in electrophoretic mobilities be-tween heated and unheated samples (8), and tight associationwith the peptidoglycan in the presence of magnesium (13, 15,22). Judged on the basis of staining, the amount of peptido-glycan-associated OmpF protein in cells infected with T5b1or T5amH16d (both A2' ) was only a small fraction (about10%) of the amount found in uninfected cells or cells infectedwith T5amH231 (A2-). Radioiodinated T5 gene A2 proteinbound to the OmpF polypeptide (Fig. 3) and, in nonboiledsamples, to bands of decreased electrophoretic mobility (the60K bands), in a manner that was disrupted by increasing theconcentration of sodium chloride in the incubation mediumfrom 0.15 to 0.50 M, suggesting that binding was electro-static in nature.

Figure 3 shows that the A2 protein bound to three differentregions of the gels: (i) at the top of the gel, principally to thehigh-molecular-weight components in the lanes containingLaemmli sample buffer-soluble, Triton X-100-soluble, andSDS-Mg2+-soluble fractions; (ii) in the middle of the gel(corresponding to the range 60K to 12K), principally to thelanes containing the SDS-EDTA-soluble and -insoluble frac-tions; and (iii) at the bottom of the gel, principally to thelanes containing the SDS-EDTA-soluble and SDS-EDTA-insoluble fractions. Staining showed that binding to region (i)was to both Coomassie blue-stained and unstained parts ofthe gel. The binding to region (ii) was correlated with thepresence of distinct Coomassie blue-stained bands. Amongthese bands was the OmpF protein. Region (iii) binding waslargely to an area of the gel not stained by Coomassie blueand almost obscured binding to a Coomassie blue-stainedarea just within region (iii). Binding to regions (i) and (ii) wasdiminished when the concentration of salt in buffer A wasincreased from 0.15 M to 0.5 M. Binding to the lanescontaining SDS-EDTA-soluble and -insoluble fractions wasenhanced in region (iii) by increasing the salt concentration.Thus, high salt (0.5 M versus 0.15 M) enhanced the bindingto species not stained by Coomassie blue but decreased thebinding of 125I-A2 to species stained by Coomassie blue.The A2 protein did not bind to the polypeptide that I

identified as the OmpA protein, also an outer membraneprotein. The OmpA protein, treated in SDS at 100°C, mi-grates with an electrophoretic mobility corresponding to33.5K; treated in SDS at 20°C it migrates as a 28K compo-nent (2). Unlike the pore proteins, treatment with SDS in thepresence of magnesium at 56°C releases the OmpA proteinfrom association with the peptidoglycan (2). In my experi-ments, putative OmpA protein was found in the SDS-Mg2+-insoluble fraction, since electrophoretic analysis of boiledportions of the SDS-EDTA-soluble fractions and samplesnot boiled before electrophoresis showed the characteristicmobility difference (34K and 29K, respectively). I attributethe incomplete solubilization of the OmpA protein by treat-

ment with SDS-Mg2+ to the temperature of incubation withthe detergent, 37°C, a temperature lower than the 56°Ctemperature used by some other workers (2).Although I was able to identify the OmpF protein as one of

the host envelope components involved in changes associat-ed with infection by bacteriophage T5 specifying productionof full-length gene A2 protein, the identity of other suchcomponents remains unknown; the changes in the amountsof 21K and 12K polypeptides found in SDS-EDTA-insolublefractions paralleled the changes in OmpF protein distribu-tion. I believe that the 38K component in the Triton X-100-soluble fraction of T5amH16d-infected cells represents the

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FIG. 3. Autoradiographs of the gels in Fig. 2 after incubationwith radioiodinated A2 protein. Radioiodinated T5 gene A2 proteinwas incubated with the gels as described in the text. Kodak NS-5Tfilm was used to visualize the binding of A2 protein. (A) through (D)as in Fig. 2. The indications of polypeptide molecular weight (inthousands) were made possible by overlaying the stained gel, driedon paper, with the exposed and developed autoradiography film.The blackening of the film could thus be carefully compared with theCoomassie blue-stained gel to see whether autoradiography bandscoincided with Coomassie blue-stained bands.

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amber fragment of the T5 gene Al protein. This identifica-tion is consistent with the known overproduction of T5 pre-early polypeptides, including the Al protein amber frag-ment, in cells infected with T5 defective in gene Al (1, 19)and with the finding that the Al protein is associated with theinner and outer membranes of T5-infected cells (7). I believethat the intensely stained 15K band of the SDS-Mg2+-solublefraction of T5amHl6d-infected cells represents the T5 geneA2 protein. This identification is consistent with the poly-peptide molecular weight of the A2 protein (28) and theoverproduction of the A2 protein in cells infected with T5defective in gene Al. It is possible that the binding of theradioiodinated A2 protein to this band on the gel reflectedthe previously demonstrated self-association of the A2 poly-peptide (28).The bulk of the binding was to the gel near the bottom of

the lanes containing Triton X-100-insoluble material. Thisarea of the gel was not stained by Coomassie blue; it mayhave contained lipopolysaccharide, since about half of thecell wall lipopolysaccharide remains associated with the cellwall after Triton X-100 extraction in the presence of magne-sium (26). Binding of A2 protein to this area was enhancedby a high salt concentration, suggesting that this binding ofA2 protein was mediated by hydrophobic interactions. TheA2 protein also bound, at the bottom of the gel, to lanes notloaded with samples. This presumably represented bindingof the basic A2 protein (28) to a portion of the gel containingbuffer components that migrated in a broad band along withthe tracking dye; it is remarkable that these putative buffercomponents remained associated with the gel even after theextensive washing procedures that preceded incubation withthe iodinated A2 protein. Since I found binding of the A2protein to both ovalbumin (one of the molecular weightstandards) and the OmpF protein, I searched for evidence ofsimilarity of these proteins. Ovalbumin is a secreted protein,and it has been postulated that it interacts with membranesvia an internal signal sequence during synthesis and secre-tion (14). The amino acid sequence of ovalbumin (21) wascompared with that of OmpF protein (5) and the followinghomology was detected. The sequence of ovalbumin resi-dues 60 to 66 is Asp-Lys-Leu-Pro-Gly-Phe-Gly, whereas thatof OmpF residues 113 to 119 is Asp-Met-Leu-Pro-Gly-Phe-Gly. It is possible that this sequence homology (six of sevenresidues match) is related to the common property of bindingto the A2 protein.The genetic evidence implicating the T5 gene A2 protein in

perturbation of host envelope structure consists of thecorrelation between the presence of full-length A2 protein inthe infected cells (in the case of T5b1 and T5amH16dinfections) and altered electrophoretic patterns. Thesechanges did not take place in cells infected with T5amH231,a mutant specifying production of an incomplete A2 poly-peptide. T5amH231-infected cells were not viable as colonyformers; extracts of these cells, as characterized by two-dimensional electrophoretic analysis, differed from those ofuninfected cells (28). Previous investigations have shown(20, 28) that cells infected with T5amH231 fail to produce aT5 pre-early polypeptide of 30K in addition to production ofan incomplete A2 polypeptide. Thus, the genetic evidencefor a role of the A2 protein in envelope changes is notconclusive.Although it is known that the gene Al and gene A2

proteins are required for second-step transfer, their mecha-nism of action remains obscure. The evidence for physicalassociation of the Al and A2 proteins (1) suggests that theyact in concert, the presence of Al protein in envelope

fractions (7) suggests that the site of action is the envelope,and association of the Al protein with host RNA polymerase(18) suggests a role for host components. I purified the A2protein from the water-soluble fraction of T5amHl6d-infect-ed cells lysed by sonication. In this study, I believe that Ihave detected the A2 protein in the SDS-Mg2+-solubleenvelope fraction. Although the Al protein has been detect-ed in envelope fractions, the same investigation did notdetect the A2 protein in envelope fractions (3, 7). Takentogether, these results suggest that the A2 protein is relative-ly loosely associated with the envelope; perhaps it is aperipheral membrane protein whose association with themembrane is quite sensitive to ionic strength.Given the observation that host membrane protein synthe-

sis was affected only slightly by T5 infection (6), I wassurprised that I obtained evidence of virtual disappearanceof the OmpF protein from the envelope of T5b1-infectedcells. In an effort to determine whether constituents of theperiplasm leaked from infected cells, the distribution of theperiplasmic enzyme alkaline phosphatase was studied. Ifound that only 1% of the alkaline phosphatase activityreleased into the medium by osmotic shock of uninfectedcells was released (without osmotic shock) when cells wereinfected by either T5amHl6d or T5amH231. Cells prelabeledwith tritiated leucine were washed and then infected witheither T5amHl6d or T5amH231; I found no difference be-tween the two infections in the amount of trichloroaceticacid-insoluble radioactivity in the culture medium. I did findthat T5amHl6d-infected cells released into the mediumtwice as much material that absorbed light of wavelength 280nm as did T5amH231-infected cells. Relative to infection byT5amH231, T5amHl6d-infected cells released an equivalentamount of OmpF protein into the culture medium, usingelectrophoresis and silver staining as a criterion. The evi-dence I gathered of in vitro interaction between the T5 geneA2 protein and the OmpF protein proves neither that such aninteraction occurs in the bacteriophage-infected cell nor thatsuch an interaction is relevant to the infection process; Ifind, nonetheless, that a connection between binding of theA2 protein and the depletion of OmpF protein is logicallysatisfying. Although the apparent effect of the T5 gene A2protein on the OmpF protein is remarkable, the observationsare consistent with the previously proposed role of envelopecomponents in the arrest of T5 DNA transfer (10) and thedependence of completion of DNA transfer on the functionof the gene A2 protein (12).

I thank Carl Schnaitman, Ulf Henning, and Ronald Bauerle foradvice and criticism and Wilson Burgess for the introduction to thegel binding techniques. This work was done in the laboratory of RolfBenzinger, whose advice and assistance were invaluable.C.E.S. was the recipient of National Research Service Award

T32GM07082-07. Work in the laboratory was supported by NationalScience Foundation grant PCM-83-02811.

LITERATURE CITED

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2. Beher, M. G., C. A. Schnaitman, and A. P. Pugsley. 1980. Majorheat-modifiable outer membrane protein in gram-negative bac-teria: comparison with the ompA protein of Escherichia coli. J.Bacteriol. 143:906-913.

3. Billmire, E. W., and D. H. Duckworth. 1976. Membrane proteinbiosynthesis in bacteriophage BF23-infected Escherichia coli. J.Virol. 14:475-489.

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9. Hunt, C., S. M. Desai, J. Vaughan, and S. B. Weiss. 1980.Bacteriophage T5 transfer RNA. Isolation and characterizationof the tRNA species and refinement of the tRNA gene map. J.Biol. Chem. 255:3164-3173.

10. Labedan, B. 1978. In vitro study of the phage T5 DNA injectionprocess: use of columns of Escherichia coli membranes immobi-lized on kieselguhr. Virology 85:487-493.

11. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

12. Lanni, Y. T. 1968. First-step-transfer deoxyribonucleic acid ofbacteriophage T5. Bacteriol. Rev. 32:227-242.

13. Lee, D. R., C. A. Schnaitman, and A. P. Pugsley. 1979. Chemicalheterogeneity of major outer membrane pore proteins of Esche-richia coli. J. Bacteriol. 138:861-870.

14. Lingappa, V., J. R. Lingappa, and G. Blobel. 1979. Chickenovalbumin contains an internal signal sequence. Nature (Lon-don) 281:117-121.

15. Lugtenberg, B., R. Peters, H. Bernheimer, and W. Berendsen.1976. Influence of cultural conditions and mutations on thecomposition of the outer membrane proteins of Escherichia coli.Mol. Gen. Genet. 147:251-262.

16. Marchalonis, J. J. 1969. An enzymic method for the traceiodination of immunoglobulins and other proteins. Biochem. J.113:299-305.

17. Maltouf, A. F., and B. Labedan. 1983. Host cell metabolicenergy is not required for injection of bacteriophage T5 DNA. J.Bacteriol. 153:124-133.

18. McCorquodale, D. J., C. W. Chen, M. K. Joseph, and R.Woychik. 1981. Modification of RNA polymerase from Esc/he-richia coli by pre-early gene products of bacteriophage T5. J.Virol. 40:958-962.

19. McCorquodale, D. J., and Y. T. Lanni. 1970. Patterns of proteinsynthesis in Escherichia coli infected by amber mutants in thefirst-step-transfer DNA of T5. J. Mol. Biol. 48:133-143.

20. McCorquodale, D. J., A. R. Shaw, P. K. Shaw, and G. Chinna-durai. 1977. Pre-early polypeptides of bacteriophage T5 andBF23. J. Virol. 22:480-488.

21. McReynolds, L., B. W. O'Malley, A. D. Nisbet, J. E. Fothergill,D. Givol, S. Fields, M. Robertson, and G. G. Brownlee. 1978.Sequence of chicken ovalbumin mRNA. Nature (London)273:723-728.

22. Rosenbusch, J. P. 1974. Characterization of the major envelopeprotein from Escherichia coli. J. Biol. Chem. 249:8019-8029.

23. Rubenstein, I. 1968. Heat-stable mutants of T5 phage. I. Thephysical properties of the phage and their DNA molecules.Virology 36:356-376.

24. Schnaitman, C. A. 1970. Examination of the protein composi-tion of the cell envelope of Escherichia coli by polyacrylamidegel electrophoresis. J. Bacteriol. 104:882-889.

25. Schnaitman, C. A. 1971. Solubilization of the cytoplasmicmembrane of Escherichia coli by Triton X-100. J. Bacteriol.108:545-552.

26. Schnaitman, C. A. 1971. Effect of ethylenediaminetetraaceticacid, Triton X-100, and lysozyme on the morphology andchemical composition of isolated cell walls of Escherichia coli.J. Bacteriol. 108:553-563.

27. Shaw, A. R., D. Lang, and D. J. McCorquodale. 1979. Terminal-ly redundant deletion mutants of bacteriophage BF23. J. Virol.29:220-231.

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29. Zarybnicky, V., A. Zarybnicka, and H. Frank. 1973. Infectionprocess of T5 phages. I. Ejection of T5 DNA on isolated T5receptors. Virology 54:318-329.

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