Locations and amounts of major structural proteins in bacteriophage lambda

The locations in the virion of the three major protein components of lambda, pE, pD and pV, have been determined by electron microscopic examination of complexes between virions and antisera specific for individual proteins. By this test both pE and pD are distributed over the entire surface of the phage head and pV is distributed along the length of the phage tail. Quantitation of sodium dodecyl sulfate-polyacrylamide gel patterns indicates that pE and pD are present in the head in equal numbers. The total amounts of pE and pD per phage are most consistent with a head structure of triangulation number 7, containing 420 subunits each of pE and pD.     

Isolation and characterization of deletion mutants affected in early genes of bacteriophage ?80

Summary When Escherichia coli cells that had been irradiated with ultraviolet light were infected with bacteriophage 80, five major (pE, pB, pA, pC and pD) and two minor (pU and pV) proteins were found to be synthesized during early stages of infection. The genss coding for the five major proteins were mapped on the 80 chromosome using various deletion mutants which lacked the capacity to synthesize some or all the major proteins. The size and positions of all the deletions were determined by gel electrophoresis of EcoRI digests of phage DNA and by electron microscopy of heteroduplexes between DNAs of the deletion and wild-type phage. The five major proteins designated pE(25K), pB(40K), pA(45K), pC(34K) and pD(31K) were shown to be encoded in this order presumably by a single operon that was located at 60.2–67.4% on the 80 genome. These proteins were found to be involved in phage recombination. The absence of pE or pB resulted in a Red^– phenotype and the absence of three proteins (pE, pB and pA) resulted in a Fec^– phenotype. The exact positions of the genes for the minor proteins pU(29K) and pV(26K) have not been determined.     

Genetically modified filamentous phage as bactericidal agents: a pilot study

AIMS: To evaluate the ability of a filamentous phage encoding lethal proteins to kill bacteria without host-cell lysis. METHODS AND RESULTS: Bacterial survival was determined after infection of a growing Escherichia coli culture with phage M13 encoding either the restriction endonuclease BglII gene or modified phage lambda S holin genes. The genetically engineered phage exerted a high killing efficiency while leaving the cells structurally intact. When compared with a lytic phage, the release of endotoxin was minimized after infection with the genetically modified phages. CONCLUSIONS: Genetically engineered phage can be used for efficient killing, concomitantly minimizing endotoxin release. SIGNIFICANCE AND IMPACT OF THE STUDY: This feasibility study provides a possible strategy for the use of genetically engineered phage as bactericidal agents by optimizing the advantages and minimizing potential risks such as release of pyrogenic cell wall components.     

A second function of the S gene of bacteriophage lambda

Infection of Escherichia coli by bacteriophage lambda caused an immediate inhibition of uptake by members of all three classes of E. coli active transport systems and made the inner membrane permeable to sucrose and glycine; however, infection stimulated alpha-methyl glucoside uptake. Phage infection caused a dramatic drop in the ATP pool of the cell, but the membrane did not become permeable to nucleotides. Infection by only one phage per cell was sufficient to cause transport inhibition. However, adsorption of phage to the lambda receptor did not cause transport inhibition; DNA injection was required. The inhibition of transport caused by lambda phage infection was transient, and by 20 min after infection, transport had returned to its initial level. The recovery of transport activity appeared to require a lambda structural protein with a molecular weight of 5,500. This protein was present in wild-type phage and at a reduced level in S7 mutant phage but was missing in S2 and S4 mutant phage. Cells infected with S7 phage had a partial recovery of active transport, whereas cells infected with S2 or S4 phage did not recover active transport. Neither the inhibition of transport caused by phage infection nor its recovery were affected by the protein synthesis inhibitors chloramphenicol and rifampin.     

Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7

Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.     

A novel biotinylated degradable polymer for cell-interactive applications

We previously demonstrated that the lambda system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant lambda was only twice as high as that of the single copy. To increase the expression in the lambda system, we constructed a Q- mutant lambda vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage lambda is deficient in the synthesis of proteins involved in cell lysis and lambda DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The beta-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of beta-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.     

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.     

Abortive replication of choleraphage phi 149 in Vibrio cholerae biotype el tor.

Choleraphage phi 149 adsorbed irreversibly to Vibrio cholerae biotype el tor cells, and 50% of the injected phage DNA bound to the cell membrane. Although no infectious centers were produced at any time during infection, the host macromolecular syntheses were shut off and the host DNA underwent chloramphenicol-inhibitable degradation. Synthesis of monomeric phage DNA continued similar to that observed in the permissive host. However, the concatemeric DNA intermediates produced were unstable and could not be chased to mature phage DNA. Pulse-labeling of UV-irradiated infected cells at different times during infection allowed identification of phage-specific proteins made in this nonpermissive host. Although most of the early proteins were made, only some of the late proteins were transiently synthesized.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Characterization of a temperate phage isolated fromLeuconostoc oenos strain 1002

A temperate phage was induced by mitomycin C fromLeuconostoc oenos strain 1002, a New Zealand isolate. The phage has an isometric head (52 ± 3 nm) with a tail (210 ± 10 nm) and gives a lytic burst size of 25 when grown on a sensitive strain. Three major structural proteins of 43, 30 and 14 kDa are visible by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A restriction map of the 36-kb genome was determined. Commercially availableL. oenos strains PSU-1, ML34 and Lco23 and 46.6% of New Zealand isolates were sensitive to the phage.     

Recruitment of host ATP-dependent proteases by bacteriophage lambda

Upon infection of a bacterial cell, the temperate bacteriophage lambda executes a regulated temporal program with two possible outcomes: (1) Cell lysis and virion production or (2) establishment of a dormant state, lysogeny, in which the phage genome (prophage) is integrated into the host chromosome. The prophage is replicated passively as part of the host chromosome until it is induced to resume the lytic cycle. In this review, we summarize the evidence that implicates every known ATP-dependent protease in the regulation of specific steps in the phage life cycle. The proteolysis of specific regulatory proteins appears to fine-tune phage gene expression. The bacteriophage utilizes multiple proteases to irreversibly inactivate specific regulators resulting in a temporally regulated program of gene expression. Evolutionary forces may have favored the utilization of overlapping protease specificities for differential proteolysis of phage regulators according to different phage life styles.     

Lactobacillus plantarum bacteriophage LP65: a new member of the SPO1-like genus of the family Myoviridae

The virulent Lactobacillus plantarum myophage LP65 was isolated from industrial meat fermentation. Tail contraction led to reorganization of the tail sheath and the baseplate; a tail tube was extruded. In ultrathin section the phage adsorbed via its baseplate to the exterior of the cell, while the tail tube tunneled through the thick bacterial cell wall. Convoluted membrane structures were induced in the infected cell. Progeny phage was detected 100 min postinfection, and lysis occurred after extensive digestion of the cell wall. Sequence analysis revealed a genome of 131,573 bp of nonredundant DNA. Four major genome regions and a large tRNA gene cluster were observed. One module corresponded to DNA replication genes. Helicase/primase and two replication/recombination enzymes represented the only links to T4-like Myoviridae from gram-negative bacteria. Another module corresponded to the structural genes. Sequence relatedness identified links with Listeria phage A511, Staphylococcus phage K, and Bacillus phage SPO1. LP65 structural proteins were identified by two-dimensional proteome analysis and mass spectrometry. The putative tail sheath protein showed a shear-induced change in electrophoretic migration behavior. The genome organization of the structural module in LP65 resembled that of Siphoviridae from the lambda supergroup.     

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.     

Analysis of productivity in lysis-deficient lambda expression systems

The integrated state of lambda in the host chromosome in lysogeny can be combined with its extrachromosomal replication in the lytic state to achieve high cloned gene productivities. Our previous studies on lambda expression systems(21,22) have shown 100% segregational stability of the cloned gene in lysogeny and cloned gene product levels up to 15% of total cell protein in a mutant lytic state. However, the expression phase of systems based on Escherichia coli JM109 and JM105 showed partial lysis of the productive culture despite a mutation in the lysis gene S of the lambda vector resulting in extracellular release of the cloned gene product. In the current study, we have eliminated partial lysis in the expression phase of lambda systems and conducted a detailed comparative analysis of these systems in relation to maximization of cloned gene productivity. The elimination of partial cell lysis by using a nonpermissive strain Y1089 did not enhance product yields vs. earlier systems that exhibited partial lysis. The elimination of nonessential lambda protein production by construction of a new vector NP326 did not yield higher product yields presumably because of the small fraction of these proteins in the lytic state. Temperature induction of the lysogen Y1089(NM1070) resulted in higher product levels than direct infection of Y1089 by the phage vector at a high multiplicity. Using infection experiments, we found the promoter lacUV5 in the vector lambdaZEQS to yield threefold higher product levels than lac in NM1070, suggesting possible further enhancement of productivity with stronger promoters. The occurrence or absence of partial lysis in lambda systems could be used beneficially to achieve extracellular or intracellular product as desired. The large capacity of lambda vectors for insert DNA suggests potential applications in obtaining highly amplified levels of operons and multienzyme systems. (c) 1992 John Wiley & Sons, Inc.     

Functional domains of bacteriophage Mu transposase: properties of C-terminal deletions

We have generated a series of 3' deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the lambda PI promoter, were analysed in vivo for their capacity to complement a super-infecting MuAam phage, both for lytic growth and lysogeny, and for their effect on growth of wild-type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.     

Ecophysiology of Bacteriophage S5100 Infecting Halobacterium cutirubrum

Increasing salinity reduces burst size and increases the latent period of infection of Halobacterium cutirubrum by lytic bacteriophage S5100. Cells become reversibly and persistently infected at saturation-level concentrations of NaCl. We propose that high salinity provides a natural refuge for sensitive host bacteria and that phage S5100 acts as a scavenger, proliferating when host viability is threatened by dilution of the environment.     

New rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.