Repression of lambda-Associated Enzyme Synthesis After lambda(vir) Superinfection of Lysogenic Hosts.

Lisio, Arnold L. (National Institutes of Health, Bethesda, Md.), and Arthur Weissbach. Repression of lambda-associated enzyme synthesis after lambda(vir) superinfection of lysogenic hosts. J. Bacteriol. 90:661-666. 1965.-Phage lambda(vir) is a multiple mutant of lambda which is capable of overcoming the immunity of a host lysogenic for lambda, and initiating normal vegetative replication of the superinfecting phage genome. Superinfection of Escherichia coli K-112 (lambda(22)) with lambda(vir) results in a normal phage yield, lysis time, and H(3)-thymine incorporation compared with infection of the sensitive host, K-112 (S). However, the production of the lambda phage-specific early protein, lambda-exonuclease, after superinfection of E. coli K-112 (lambda(22)) with lambda(vir) is only 25 to 50% of that obtained from corresponding infection of a nonlysogenic host, E. coli K-112 (S). This repression of lambda-exonuclease synthesis is dependent on the C(1) cistron of the prophage and is overcome if the lysogenic host cells are induced prior to superinfection. The data are interpreted as evidence for partial repression of lambda(vir) by the host immunity.     

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Effect of Prophage W on the Propagation of Bacteriophages T2 and T4

Studies have been undertaken to determine whether the temperate phage ω present in Escherichia coli strain W is responsible for the inability of this strain to act as a host for T2 and T4. E. coli WS, cured of phage ω, was sensitive to T2 and T4. Lysogenation of E. coli C and WS with phage ω resulted in loss of ability to plate T2 and T4. However, E. coli K-12 lysogens still served as hosts for the T -even phage. Two of three WS lysogens studied resembled strain W at the biochemical level. They converted about 30% of infecting T2 deoxyribonucleic acid (DNA) to acid-soluble fragments and limited macromolecular synthesis to a few minutes after infection. The third lysogen did not degrade phage DNA, and nucleic acid and protein synthesis continued for some time, although no phage production occurred. It is concluded that phage ω plays a role in the restriction of virulent phage but that it is not the only factor involved. Since acid solubilization was not observed in all cases of phage ω-mediated restriction of T -even phage, a hypothesis for the restriction has been proposed which is based on an alteration in the cell envelope after lysogenation with phage ω.     

Roles of cell surface components of Escherichia coli K-12 in bacteriophage T4 infection: interaction of tail core with phospholipids.

The cell surface of Escherichia coli K-12, reconstituted from the OmpC protein, lipopolysaccharide, and the peptidoglycan layer, was active as a receptor for phage T4, resulting in the contraction of the tail sheath and the penetration of the core through the cell surface (Furukawa et al., J. Bacteriol. 140:1071--1080, 1979). In the present work the process of DNA ejection from the contracted T4 phage particle was studied. Contracted phage particles were adsorbed to phospholipid liposomes by the core tip. This adsorption resulted in ejection of phage DNA. Either phosphatidylglycerol or cardiolipin was active for the DNA ejection. A proton motive force across the liposome membrane was not required for these processes. The process of DNA ejection, however, was temperature dependent, whereas the adsorption of the core tip to liposomes took place at 4 degrees C. Based on these observations together with those in the previous paper, the process of T4 infection of E. coli K-12 cells is discussed with special reference to the roles of cell surface components.     

Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Effect of Bacteriophage R17 Infection on Hostdirected Synthesis of Ribosomal Ribonucleates

Studies were performed on the synthesis of ribosomal ribonucleates in cells of Escherichia coli K-12 infected by the ribonucleic acid (RNA) bacteriophage R17. Host-specific RNA was measured in the presence of phage RNA by in vitro hybridization of the purified ribonucleates with E. coli deoxyribonucleic acid. The results showed that, although the overall rate of RNA synthesis was only slightly affected by phage infection, the level of host RNA synthesis was decreased by 70 to 80%. Fractionation of the purified ribonucleates by sucrose gradient sedimentation, followed by hybridization of fractions sedimenting in the 23S and 16S regions, revealed that the level of ribosomal RNA synthesis was also decreased by 70 to 80%, and that this inhibition occurred during the first 15 to 20 min after infection. These findings are discussed in light of what is known about the inhibition of host RNA synthesis by other virus systems.     

Presence of rfe genes in Escherichia coli: their participation in biosynthesis of O antigen and enterobacterial common antigen.

In Salmonella, ilv-linked rfe genes participate in the biosynthesis of the enterobacterial common antigen (CA) as well as of certain types of O antigen (serogroups C1 and L). rff genes, probably in the same cluster with rfe, are required for CA synthesis (P.H. M?kel? et al., in preparation). Several Escherichia coli strains were studied to determine whether they also have rfe-rff genes that are involved in the synthesis of O antigen and CA, or of CA only. In a first approach, E, coli K-12 F-prime factors carrying the genes ilv and argH or argE and presumably rfe-rff genes were introduced into CA-negative Salmonella mutants that are blocked in CA synthesis because of mutated rfe or rff genes. All resulting ilv+ hybrids were CA positive. In recipients with group C1-derived rfb genes, the synthesis of O6,7-specific antigen was also restored. This result shows that E. coli K-12 has rfe and rff genes providing the functions required in the synthesis of CA and Salmonella 6,7-specific polysaccharide. By introduction of defective rfe regions from suitable Salmonella donors into E. coli O8, 09, and O100 strains, the synthesis of CA as well as of the O-specific polysaccharides was blocked. This indicates that in the E. coli strains tested the rfe genes are involved in the synthesis of both O antigen and CA. This suggestion was confirmed by the finding of E. coli rough mutants that had simultaneously become CA negative. In transduction experiments it could be shown that the appearance of the rough and CA- phenotype was due to a defect in the ilv-linked rfe region.     

Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.     

Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites.

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.     

Escherichia coli capsule bacteriophages. VII. Bacteriophage 29-host capsular polysaccharide interactions.

Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.     

Accelerated Adsorption of Bacteriophage T5 to Escherichia coli F, Resulting from Reversible Tail Fiber-Lipopolysaccharide Binding

A dual specificity for phage T5 adsorption to Escherichia coli cells is shown. The tail fiber-containing phages T5(+) and mutant hd-3 adsorbed rapidly to E. coli F (1.2 x 10(-9) ml min(-1)), whereas the adsorption rate of the tail fiber-less mutants hd-1, hd-2, and hd-4 was low (7 x 10(-11) ml min(-1)). The differences in adsorption rates were due to the particular lipopolysaccharide structure of E. coli F. Phage T4-resistant mutants of E. coli F with an altered lipopolysaccharide structure exhibited similar low adsorption for all phage strains with and without tail fibers. The same held true for E. coli K-12 and B which also differ from E. coli F in their lipopolysaccharide structures. Only the tail fiber-containing phages reversibly bound to isolated lipopolysaccharides of E. coli F. Infection by all phage strains strictly depended on the tonA-coded protein in the outer membrane of E. coli. We assume that the reversible preadsorption by the tail fibers to lipopolysaccharide accelerates infection which occurs via the highly specific irreversible binding of the phage tail to the tonA-coded protein receptor. The difference between rapid and slow adsorption was also revealed by the competition between ferrichrome and T5 for binding to their common tonA-coded receptor in tonB strains of E. coli. Whereas binding of T5(+) to E. coli K-12 and of the tail-fiber-less mutant hd-2 to E. coli F and K-12 was inhibited 50% by about 0.01 muM ferrichrome, adsorption of T5 to E. coli F was inhibited only 40% by even 1,000-fold higher ferrichrome concentrations.     

Effective, plasmid RP4-dependent replication-transposition of DNA of the transposable phage D3112 of Pseudomonas aeruginosa in a heterologous Escherichia coli system

The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells.     

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools

The gene 1.2 protein of bacteriophage T7, a protein required for phage T7 growth on Escherichia coli optA1 strains, has been purified to apparent homogeneity and shown to restore DNA packaging activity of extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants (Myers, J. A., Beauchamp, B. B., White, J. H., and Richardson, C. C. (1987) J. Biol. Chem. 262, 5280-5287). After infection of E. coli optA1 by T7 gene 1.2 mutant phage, under conditions where phage DNA synthesis is blocked, the intracellular pools of dATP, dTTP, and dCTP increase 10-40-fold, similar to the increase observed in an infection with wild-type T7. However, the pool of dGTP remains unchanged in the mutant-infected cells as opposed to a 200-fold increase in the wild-type phage-infected cells. Uninfected E. coli optA+ strains contain severalfold higher levels of dGTP compared to E. coli optA1 cells. In agreement with this observation, dGTP can fully substitute for purified gene 1.2 protein in restoring DNA packaging activity to extracts prepared from E. coli optA1 cells infected with T7 gene 1.2 mutants. dGMP or polymers containing deoxyguanosine can also restore packaging activity while dGDP is considerably less effective. dATP, dTTP, dCTP, and ribonucleotides have no significant effect. The addition of dGTP or dGMP to packaging extracts restores DNA synthesis. Gene 1.2 protein elevates the level of dGTP in these packaging extracts and restores DNA synthesis, thus suggesting that depletion of a guanine deoxynucleotide pool in E. coli optA1 cells infected with T7 gene 1.2 mutants may account for the observed defects.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Replication of T4rII Bacteriophage in Escherichia coli K-12 (λ)

The defect of T4rII replication in Escherichia coli K-12 (lambda) can be phenotypically reversed by various supplements to the growth medium. Arginine, lysine, spermidine, and a number of diamines allowed varying levels of rII replication. The best reversion was obtained with 0.4 m sucrose in 0.002 to 0.005 m Ca(++). Monovalent cations severely inhibited reversion. A cell surface site of polyamine action is consistent with the fact that spermidine inhibits phage ghost-induced cell lysis and with the finding that sufficient polyamine is available within the cells to allow normal patterns of neutralization of phage deoxyribonucleic acid, as detected by the polyamine content of progeny phage. In the absence of effective supplements, rII-infected cells swelled and lost refractility. The data indicate that a leaky cell envelop is involved. No difference in mucopeptides of uninfected K-12 (lambda) and K-12 was detected and, because the mucopeptide in r(+) infected cells was found to be at least partially hydrolyzed midway through the lytic cycle, it did not appear that the rII defect concerned mucopeptide synthesis. The pattern of cell phospholipid synthesis changes after phage infection, but no difference was detected between r(+) and rII with regard to biosynthesis of phosphatidylethanolamine and phosphatidylglycerol.     

Mutants of Escherichia coli O9:K30 with altered synthesis and expression of the capsular K30 antigen

Escherichia coli K30 produces a thermostable group I capsular polysaccharide. Two classes of mutants were isolated with defects in the synthesis or expression of capsule. The most common mutant phenotype was acapsular (K-), with no K-antigen synthesized. A second class of mutants, termed Ki or intermediate forms, produced colonies which were indistinguishable from those of acapsular forms yet K-antigenicity was expressed. Previous studies had demonstrated that E. coli strains that produce K30 antigen synthesize a lipopolysaccharide (LPS) fraction that is recognised by monoclonal antibodies against the K30 antigen. Synthesis of this LPS fraction was not affected in Ki forms. The results of morphological examination, LPS analysis and phage sensitivity studies are consistent with the interpretation that the defect in Ki strains results from an inability to polymerize the K30 antigen. Using plasmid pULB113 (RP4::mini-Mu), mutations resulting in both K- and Ki phenotypes were localized near the his region of the chromosome.     

Electrooptical analysis of the Escherichia coli-phage interaction

This article describes electrooptical (EO) characterization of biospecific binding between the bacterium Escherichia coli XL-1 and the phage M13K07. The electrooptical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for EO measurements. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with the phage M13K07 is based on registration of changes of optical parameters of bacterial suspensions. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of E. coli with phage M13K07 induces a strong and specific EO signal as a result of substantial changes of the EO properties of the E. coli XL-1 suspension infected by the phage M13K07. The signal was specific in the presence of foreign microflora (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the EO approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the infection of phage to bacteria is specific, (3) exogenous substrates and mediators are not required for detection, and (4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.