Template Properties of Glucose-Deficient T-Even Bacteriophage DNA

The vegetative DNA isolated from T4-infected Escherichia coli W4597 (UDPG PPase(-)) was about two to six times more active in stimulating protein synthesis in cell-free extracts than that isolated from T4-infected E. coli B06. This suggested that nonglucosylated vegetative DNA may be a better template than the glucosylated form. This view was supported by experiments measuring RNA synthesis on mature T-even DNAs with a range of glucose contents. The extent of (14)C-GTP polymerization was inversely proportional to the glucose content of the DNA. Differences were also observed in both the kind and quantity of polypeptides produced in response to these DNAs.     

T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B II. Nucleic Acid Metabolism

T-even bacteriophage-tolerant mutants are strains of Escherichia coli which can adsorb T-even phages but cannot support the growth of infective virus. Under some conditions, the infected cells are not killed. Mutant cells infected by phage T6 are able to carry out several metabolic functions associated with normal virus development, including arrest of bacterial nucleic acid and protein synthesis, incorporation of isotopic precursors into viral nucleic acids and proteins, synthesis of early enzymes of deoxyribonucleic acid (DNA) metabolism, formation of rapidly sedimenting DNA intermediates, and formation of normal levels of early and late messenger ribonucleic acid species. Phage are unable to mutate to forms capable of growth on these mutants. The nature of the biochemical alteration leading to tolerance is still unknown.     

Inhibition of T4 Bacteriophage Multiplication by Superinfecting Ghosts and the Development of Tolerance After Bacteriophage Infection

Simultaneous addition of T4 phage and ghosts to host cells prevents infective center formation. Cells which have been infected with phage for less than 2 min are also inhibited by superinfecting ghosts. After this time, a chloramphenicol-inhibitable reaction occurs which causes the phage-infected cells to become increasingly tolerant of added ghosts.     

Characterization of T-Even Bacteriophage Substructures: I. Tail Fibers and Tail Tubes

T-even bacteriophages were grown and purified in bulk quantities. The protein coats were disrupted into their component substructures by treatment with 67% dimethyl sulfoxide (DMSO). Tail fibers and tubes were purified on glycerol-CsCl-D(2)O gradients and examined with respect to sedimentation properties, subunit molecular weights, amino acid composition, isoelectric points, and morphology. It was found that intact tail fibers had a sedimentation coefficient of 12 to 13S and that dissociated fibers consisted of three classes of proteins having molecular weights of 150 K +/- 10, 42 K +/- 4, and 28 K +/- 3 daltons. A model was constructed in which the 150-K subunit folded back on itself twice to give a three-stranded rope. Each 150-K subunit then represented a half-fiber and it was proposed that the role of the 42- and 28-K subunits was to hold each half-fiber together as well as serve as a possible link with other substructures. Isoelectric point studies also indicated that there were three different proteins with pI values of 3.5, 5.7, and 8.0. Amino acid analyses indicated that fibers had a composition distinct from other phage substructures. In addition, a striking difference was noted in the content of tryptophan among the phages examined. T4B had three to five times more tryptophan than did T2L, T2H, T4D, and T6. Intact tail tubes had an S(20,w) of 31 to 38S and dissociated tubes consisted of three proteins of molecular weights 57 K +/- 5, 38 K +/- 4, and 25 K +/- 3 daltons. Based on degradation studies with DMSO, it was proposed that these three proteins were arranged in a helical array yielding the tube structure. Isoelectric point studies indicated that there were three major proteins in the tube whose pI values were 5.1, 5.7, and 8.5. No significant differences were observed in the amino acid content of tubes obtained from all the T-even bacteriophages.     

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.     

Molecular Studies on Entry Exclusion in Escherichia coli Minicells

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).     

Bacteriophage Tail Components: I. Pteroyl Polyglutamates in T-Even Bacteriophages

A pteroylpolyglutamate has been found to be a constituent of all Escherichia coli T-even bacteriophages and has been characterized with regard to its oxidation state, molecular weight, origin, and location on the phage particle. The phage compound has been shown to be a dihydropteroyl penta- or hexaglutamate on the basis of its chemical and physical properties. Analyses of extracts of uninfected and T2L-infected E. coli have indicated that the phage dihydropteroyl polyglutamate was present only in infected cells. Its synthesis was sensitive to the addition of chloramphenicol before infection, and the compound appeared to be specifically induced by phage infection. Analyses of isolated phage ghosts and tail substructures have shown that each phage particle contains between two and six phage-specific pteroyl derivatives and that the juncture of the phage tail plate with the tail tube is the most likely site of binding of the phage-induced pteroyl compound.     

Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.     

Structural Aberrations in T-Even Bacteriophage IV. Parameters of Induction and Formation of Lollipops

Previous results from our laboratory have shown that when a T-even bacteriophage-infected bacterial cell was exposed to l-canavanine followed by an l-arginine chase, a monster phage particle, termed a lollipop, was formed. We now describe certain parameters concerning (i) the induction and (ii) the formation of T4 lollipops. The induction step involves a T4 late function, and can require only a 3-min exposure to l-canavanine. Short pulses of l-canavanine result in the formation of shorter lollipops indicating the presence of a possible "precursor substance" which is influenced by l-canavanine. DNA synthesis is inhibited by l-canavanine but is stimulated 20 to 40 min after the addition of l-arginine. Chloramphenicol prevents both responses indicating a possible protein involvement. The appearance of lollipops and phage was noted only after 25 min after the addition of l-arginine.     

A Remeasurement of the Molecular Weights of T-Even Bacteriophage Substructural Proteins

T-even bacteriophage substructural proteins were studied by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that tail fibers are composed of two major proteins of 155,000 and 120,000 daltons molecular weight and four minor proteins of 51,000, 38,000, 27,000, and 23,000 daltons. Tail tubes were composed of one predominant protein of 18,500 daltons and one minor protein of 35,000 daltons molecular weight. Tubular polyheads obtained from a T4D amber mutant and by treatment of T4B-infected cells with L-canavanine were also examined, and no significant differences were noted in the molecular weight of the P23 protein.     

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.     

Lysis Inhibition in Escherichia coli Infected with Bacteriophage T4

A technique of continuous filtration of T4-infected Escherichia coli has been devised to study the phenomenon of lysis inhibition. Studies using this technique revealed that the length of the lysis delay caused by superinfection can attain only certain discrete values, which for low average multiplicity of superinfection is thought to be a reflection of the actual number of superinfecting particles per cell. The time interval between primary and superinfection had little effect on the length of lysis delay. With increasing rate of superinfection, the length of lysis delay decreased. In superinfected cells, the concentration of endolysin exceeded the final concentration in nonsuperinfected cells. Superinfection of a lysing culture induced lysis inhibition immediately. Temperature-shift experiments, with cells primarily infected by a temperature-sensitive endolysin mutant, revealed that after the normal latent period superinfection was unable to induce lysis inhibition. Amber-restrictive cells, which were primarily infected by an endolysin negative amber mutant, released adenosine triphosphate (ATP) at the end of the normal latent period although lysis did not occur. Superinfection reduced the loss of ATP markedly. The hypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.     

Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm

An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h(-1) for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology.     

Bacteriophage Types and O Antigen Groups of Escherichia coli from Urine

Urinary strains of Escherichia coli from seven geographical regions were typed serologically for O-specific antigens and with phages capable of lysing the majority of urinary isolated. The O antigen groups 4, 6, 75, 1, 50, 7, and 25 were the common ones found. Of the 454 cultures tested, 66.1% were phage typable and 65.2% were serotypable with the 48 antisera employed. Also, 71.6% of the cultures for which an O group could be determined were phage typable. Furthermore, of those seven O-antigen groups implicated in urinary tract infection, 80.2% exhibited a phage pattern. Various phage types were found within an O-antigen group, and, although one phage type associated a high percentage of the time with one O-antigen group, no correlation was observed between other O-antigen groups and phage types. Studies with bacteriuric patients by phage typing showed the presence of two strains of E. coli within an O-antigen group. Serogrouping and phage typing of fecal isolates of E. coli revealed the presence of some O-antigen groups and phage types also found as predominant types among urinary isolates. Phage typability correlated highly with hemolysis of human erythrocytes. Elevated temperatures of incubation and a chemical curing agent were used to enhance typability of cultures refractory to the typing phages. Phage typing, due to its rapidity, ease, and ability to distinguish strains of E. coli within an O-antigenic group, is suggested as a possible method by which a better insight into the epidemiology of urinary tract infections may be obtained.     

Infection of Actinomycin-Permeable Mutants of Escherichia coli with Urea-Disrupted Bacteriophage

Intact cells of actinomycin-permeable mutants of Escherichia coli could be infected with urea-disrupted phage T4 (designated as T4pi). The parental strains and the revertants, which are impermeable to actinomycin, were not susceptible to T4pi unless they had been treated with agents which altered their permeability. The permeable mutants developed competence for pi infection during the growth cycle; cells in the early stationary phase produced 10- to 100-fold more plaques on plating with T4pi than did exponentially growing cells. Colistin (polymyxin E) was effective in converting noncompetent cells of either permeable or nonpermeable strains to the competent state. Treatment with lysozyme resulted in a considerable increase in susceptibility to T4pi of permeable mutants but not of nonpermeable cells. It appears that development of competence for pi infection is mainly due to alterations in the permeability barriers of the cell.     

Cation Fluxes and Permeability Changes Accompanying Bacteriophage Infection of Escherichia coli

Infection of Escherichia coli by bacteriophage T2 was accompanied by a rapid but transient increase in the rate of loss of small molecules from the bacterial cells. This transient leakage was studied with radioactive labels such as (42)K and (28)Mg. Bacteriophage-induced leakage was dependent on the ratio of phage to bacteria: the higher the multiplicity of infection, the greater the leakage. No leakage occurred at 4 C [when adsorption proceeds but injection of phage deoxyribonucleic acid (DNA) is blocked]. Leakage was caused by heavily irradiated phage as well as by normal phage; therefore, the intracellular functioning of the bacteriophage DNA was not required. This conclusion was supported by experiments which showed phage-induced leakage in the presence of chloramphenicol or sodium cyanide. Leakage could be prevented by infecting the bacteria with phage in the presence of high magnesium concentrations. Phage-induced leakage was terminated by a "sealing" reaction, after which potassium turnover by infected and uninfected cells was very similar. The sealing reaction occurred even in the presence of chloramphenicol, suggesting that the sealing is controlled by bacterial and not bacteriophage genes. We were not able to detect any effect of normal bacteriophage infection on the influx (active transport) of potassium and magnesium into the cells.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Polyamine Synthesis and Accumulation in Escherichia coli Infected with Bacteriophage R17

We have studied the biosynthesis of polyamines during the multiplication of the RNA bacteriophage R17. R17-sensitive strains of Escherichia coli were derived from the stringent CP78 and the relaxed mutant derivative CP79. The cells were infected with R17 in the presence or absence of arginine, a required amino acid, and both the RNA and polyamine contents of the bacteria were determined before and after the infection. The uninfected CP79 rel derivative accumulated RNA and spermidine in the absence of arginine, unlike the stringent organism that accumulated neither under these conditions. After R17 infection, the stringent strain accumulated RNA and spermidine in the presence or absence of arginine. The data indicate a close correlation between the synthesis of RNA and spermidine, suggesting a significant role for this polyamine in the multiplication of phage R17.