Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Escherichia coli K1's Capsule Is a Barrier to Bacteriophage T7

Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.     

The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

Unlike other characterized phages, the lytic coliphage N4 must inject the 360-kDa virion RNA polymerase (vRNAP), in addition to its 72-kbp genome, into the host for successful infection. The process of adsorption to the host sets up and elicits the necessary conformational changes in the virion to allow genome and vRNAP injection. Infection of suppressor and nonsuppressor strains, Escherichia coli W3350 supF and E. coli W3350, with a mutant N4 isolate (N4am229) harboring an amber mutation in Orf65 yielded virions containing (N4gp65⁺) and lacking (N4gp65⁻) gp65, respectively. N4gp65⁺ but not N4gp65⁻ phage was able to adsorb to the host. Recombinant gp65 with a hexahistidine tag at the N terminus or hexahistidine and c-myc tags at the C terminus was able to complement N4gp65⁻ virions in vivo and in vitro. Immunogold detection of gp65 in vivo complemented virions revealed its localization at the N4 tail. Finally, we show both in vitro and in vivo that gp65 interacts with the previously determined N4 outer membrane receptor, NfrA.     

Bacteriophage phiX174 growth in an Escherichia coli dnaIts mutant, KS810.

A bacteriophage phiX174-sensitive Escherichia coli dnaIts mutant, KS810, was constructed and growth of phiX174 in the cells was investigated. phiX174 and phiX174am3trD could grow normally at 43 degrees C as well as 27 degrees C, therefore we conclude that the growth of bacteriophage phiX174 is not dependent upon the host dnaI gene product.     

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.     

Membranous Structure of Purified Escherichia coli Lipopolysaccharide

The ultrastructure of the purified and lyophilized endotoxin from Escherichia coli O111 was observed by ultrathin sectioning. Onion-like globular membrane structures were observed in addition to rod-like and ribbon-like structures, indicating the existence of a globular membrane structure even in the dried state.     

A New Locus of Escherichia coli That Determines Sensitivity to Bacteriophage φX174

A new gene designated phxB, necessary for adsorption of phiX174 to the cell surface of Escherichia coli, is located between gal and aroG on the E. coli chromosome.     

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.     

Bacteriophage T4D Receptor and the Escherichia coli Cell Wall Structure: Binding of Endotoxin-Like Particles to the Cell Wall

A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70°C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20°C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55°C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100°C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed.     

Phi X174 protein E-mediated lysis of Escherichia coli

Bacteriophage PhiX174 encodes a single lysis gene, E, the function of which is necessary and sufficient to induce lysis of Escherichia coli. Here we present a novel model for E-lysis: physiological, genetic and biochemical data are presented which suggest that a transmembrane tunnel penetrating the inner and outer membrane is formed during the lytic action of protein E. Moreover, using high magnification scanning and transmission electron microscopy in this study, it was possible to visualize the transmembrane lysis structure directly.     

Breakdown and Exclusion of Superinfecting T-Even Bacteriophage in Escherichia coli

In bacterial strains containing the deoxyribonuclease endonuclease I (endonuclease I(+) strains), 70 to 80% of the injected superinfecting T-even phage deoxyribonucleic acid (DNA) is rapidly degraded to oligonucleotides having an average chain length of 8, the same value as that obtained by endonuclease I digestion of purified T-even phage DNA in vitro. In endonuclease I(-) strains, less than 5% of the injected superinfecting T-even phage DNA is degraded to acid-soluble components. The superinfecting phage DNA is, however, fragmented into a large segment having a molecular weight of about 90 x 10(6) and 30 or more small acid-insoluble segments having molecular weights of less than 10(6). In both endonuclease I(+) and endonuclease I(-) strains, over 80% of the DNA from adsorbed primary T2 or T4 phage, but only 50% of the DNA from adsorbed superinfecting T2 or T4 phage, is injected. Superinfecting T4 are genetically excluded as efficiently from endonuclease I(-) strains as they are from endonuclease I(+) strains. The excluded phage cannot complement defects in either early or late gene functions carried by the primary phage. The induction of both superinfection breakdown and superinfection exclusion requires a period of protein synthesis between primary infection and addition of the superinfecting phage. These observations seem best explained by failure of superinfecting DNA to enter the host cell cytoplasm, presumably as a result of changes in the cell envelope induced by the primary phage.     

Bacteriophage conversion of heat-labile enterotoxin in Escherichia coli.

A temperate phage designated obeta1 (omicron beta) was mitomycin C induced and isolated from heat-labile enterotoxin (LT)-producing Escherichia coli E2631-C2. Phage obeta1 infected the nonlysogenic, nontoxigenic, mitomycin C-sensitive strain of E. coli K-12 (CSH38) and converted it to lysogeny and enterotoxigenicity. After the establishment of lysogeny, E. coli CSH38(obeta1) produced produced LT and phage particles at maximal levels following mitomycin C induction. The LT Tox+ character is carried by the temperate phage obeta1.     

Bacteriophage Mu DNA circularizes following infection of Escherichia coli

Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms. Circularization does not require protein synthesis in the infected cells. A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients. Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

Mutation Preventing Capsular Polysaccharide Synthesis in Escherichia coli K-12 and Its Effect on Bacteriophage Resistance

A mutant strain of Escherichia coli K-12 was found in which spontaneous mutation to phage T7 resistance occurred at a very low frequency. T7 resistance in the parental strain from which this mutant was derived resulted from a mutation to excess capsular polysaccharide synthesis. The mutation preventing T7 resistance, non-9, inhibited capsule formation when transduced into capsulated strains. The non-9 mutation was cotransducible with his, the gene order in this region being non-9 his Su-1.