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PubMed2000

Characterization of a shiga toxin 2e-converting bacteriophage from an Escherichia coli strain of human origin.

An infectious Shiga toxin (Stx) 2e-converting bacteriophage (phiP27) was isolated from Stx2e-producing Escherichia coli ONT:H(-) isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to E. coli laboratory strain DH5alpha, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner. By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of phiP27, we demonstrated that the stx(2e) gene was located directly downstream of ileZ and argO tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage phiCTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.

Bacterial Toxins↗
PubMed1970

Radiation sensitivity of Haemophilus influenzae: a composite response.

The survival of ultraviolet (UV)-irradiated cultures of Haemophilus influenzae Rd is determined by at least two responses: (i) excision-repair ability and (ii) UV-induced cell lysis. An UV-resistant mutant, BC200, has the same capabilities as the wild type, Rd, for excising dimers but does not exhibit lysis. Lytic response is dose-dependent. Relative to the wild type, a lower dose of UV causes lysis of a UV-sensitive mutant, BC100, which is incapable of excising thymine dimers. A lytic protein is present in cultures undergoing lysis. Synthesis of this protein is initiated 45 to 60 min after irradiation. Lysis appears to be due to derepression of a defective prophage which codes for an endolysin-like lytic enzyme.

Bacterial Proteins↗
PubMed1982

Effect of the lambda S gene product on properties of the Escherichia coli inner membrane.

The S gene of bacteriophage lambda is a late gene required for cell lysis, but unlike the other two lysis genes, R and Rz, it does not code for an endolysin. Earlier studies have shown that the S gene product inhibits respiration and macromolecular synthesis and makes the inner membrane permeable to sucrose. In this study, the effect of the S gene product on a number of Escherichia coli membrane functions (active transport, permeability, respiration, and transhydrogenase and ATPase activity) were measured, and a product of the lambda S gene was identified in the inner membrane fraction by two-dimensional polyacrylamide gel electrophoresis. The results of these experiments indicate that the lambda S product is present in the inner membrane, that it increased the permeability of the membrane for all of the small molecules that were tested, and that its action is reversible. The simplest explanation of these results is that the S gene product forms a hydrophilic pore through the inner membrane, allowing small molecules and lambda lysozyme to pass through.

Adenosine Triphosphatases↗
PubMed1986

Mutational analysis of bacteriophage lambda lysis gene S.

A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.

Alleles↗
PubMed1993

Phi X174 E complements lambda S and R dysfunction for host cell lysis.

Hybrid lambda phages which have the E lysis gene of the bacteriophage phi X174 in cis to defective nonsense and deletion alleles of the normal lambda lysis genes S and R have been constructed and shown to be fully competent for plaque-forming ability, which demonstrates that the single-gene, lysozyme-independent lysis system of phi X174 and related phages can serve the lytic function for large complex phages. These hybrid phages are unable to form plaques on a slyD host. Moreover, plaque morphology indicates that in E-mediated lysis the soluble lambda R endolysin can participate in lysis, indicating that the protein E-mediated lesions are not completely sealed off from the periplasm.

Bacteriophage lambda↗
PubMed1993

The missing link in phage lysis of gram-positive bacteria: gene 14 of Bacillus subtilis phage phi 29 encodes the functional homolog of lambda S protein.

In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. The lesion is brought about by a second phage-encoded lysis function. For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. Production of protein 14 in E. coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed1994

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.

Amino Acid Sequence↗
PubMed1995

S gene expression and the timing of lysis by bacteriophage lambda.

The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2... N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

Amino Acid Sequence↗
PubMed1997

The Serratia marcescens NucE protein functions as a holin in Escherichia coli.

The recently discovered nucC locus of Serratia marcescens encodes the cryptic prophage genes nucE, nucD, and nucC. NucC is required for expression of the S. marcescens nuclease and functions as a transcriptional activator of the nuclease gene, nucA. NucE and NucD are dispensable for nuclease expression but were proposed to allow for secretion of the nuclease by Escherichia coli. Here, we show (i) that the NucE protein is membrane bound, (ii) that it can complement the lambda S holin, (iii) that it can be triggered by potassium cyanide, (iv) that it is detrimental to cell viability, and (v) that the concomitant expression of nucE and nucD results in cell lysis. Apparently NucE and NucD function as a holin and an endolysin, respectively. This suggests that their roles in nuclease secretion by E. coli are indirect, possibly through directed cell lysis.

Amino Acid Sequence↗
PubMed1998

Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells.

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.

Amino Acid Sequence↗
PubMed1999

Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene.

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition. We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal. The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes. tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function.

Amino Acid Sequence↗
PubMed2000

Dimerization between the holin and holin inhibitor of phage lambda.

Holins are integral membrane proteins that control the access of phage-encoded muralytic enzymes, or endolysins, to the cell wall by the sudden formation of an uncharacterized homo-oligomeric lesion, or hole, in the membrane, at a precisely defined time. The timing of lambda-infected cell lysis depends solely on the 107 codon S gene, which encodes two proteins, S105 and S107, which are the holin and holin inhibitor, respectively. Here we report the results of biochemical and genetic studies on the interaction between the holin and the holin inhibitor. A unique cysteine at position 51, in the middle of the second transmembrane domain, is shown to cause the formation of disulfide-linked dimers during detergent membrane extraction. Forced oxidation of membranes containing S molecules also results in the formation of covalently linked dimers. This technique is used to demonstrate efficient dimeric interactions between S105 and S107. These results, coupled with the previous finding that the timing of lysis depends on the excess of the amount of S105 over S107, suggest a model in which the inhibitor functions by titrating out the effector in a stoichiometric fashion. This provides a basis for understanding two evolutionary advantages provided by the inhibitor system, in which the production of the inhibitor not only causes a delay in the timing of lysis, allowing the assembly of more virions, but also increases effective hole formation after triggering.

Bacteriolysis↗
PubMed2000

Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system.

The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308-7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in B. burgdorferi dramatically stimulated blyAB expression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Amino Acid Sequence↗
PubMed2005

Identification of genes of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae.

VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

Cloning, Molecular↗
PubMed2005

Periplasmic domains define holin-antiholin interactions in t4 lysis inhibition.

Bacteriophage T4 effects host lysis with a holin, T, and an endolysin, E. T and E accumulate in the membrane and cytoplasm, respectively, throughout the period of late gene expression. At an allele-specific time, T triggers to disrupt the membrane, allowing E to enter the periplasm and attack the peptidoglycan. T triggering can be blocked by secondary infections, leading to the state of lysis inhibition (LIN). LIN requires the T4 antiholin, RI, and is sensitive to the addition of energy poisons. T is unusual among holins in having a large C-terminal periplasmic domain. The rI gene encodes a polypeptide of 97 residues, of which 72 are predicted to be a periplasmic domain. Here, we show that the periplasmic domain of RI is necessary and sufficient to block T-mediated lysis. Moreover, when overexpressed, the periplasmic domain of T (T(CTD)) was found to abolish LIN in T4 infections and to convert wild-type (wt) T4 plaques from small and fuzzy edged to the classic "r" large, sharp-edged plaque morphology. Although RI could be detected in whole cells, attempts to monitor it during subcellular fractionation were unsuccessful, presumably because RI is a highly unstable protein. However, fusing green fluorescence protein (GFP) to the N terminus of RI created a more stable chimera that could be demonstrated to form complexes with wild-type T(CTD) and also with its LIN-defective T75I variant. These results suggest that the function of the unusual periplasmic domain of T is to transduce environmental information for the real-time control of lysis timing.

Amino Acid Sequence↗
PubMed1966

Inhibition of coliphage reproduction after superinfection of induced lysogens.

Brenner, Don J. (University of Washington, Seattle), and Neal B. Groman. Inhibition of coliphage reproduction after superinfection of induced lysogens. J. Bacteriol. 92:1727-1734. 1966.-Purified preparations of phages lambda and lambda112 inhibited lysis, phage reproduction, and endolysin synthesis by ultraviolet-induced strains of K-12 (lambda) and K-12 (lambda112). Structural and partial functional integrity of phage was required for inhibition, and the role of phage deoxyribonucleic acid (DNA) in inhibition was demonstrated. Both ultraviolet-irradiated and host-modified lysates of lambda were inhibitory, suggesting that replication of superinfecting phage DNA was not essential for inhibition. Using various combinations of superinfecting and resident phage, including lambda, 434, 434hy, and C-mutant derivatives, we observed that all three C region cistrons played some role in inhibition. Of the three, the C(1) cistron was the most critical. With certain phage combinations, inhibition was observed even though the resident and superinfecting phage differed in immune specificity. Both the medium and the method of superinfection determined whether lysis or lysis inhibition would occur. These and other observations with K-12(lambda)thy(-) indicated that many factors influence the outcome of superinfection. The data are compatible with the view that superinfection inhibition is due to the establishment or re-establishment of phage repressor activity, with the result that replication of both superinfecting and resident phage is blocked.

Antibodies↗
PubMed1975

Host-phage interaction on Agrobacterium tumefaciens. IV. Phage-directed protein synthesis.

Gel electrophoretic and autoradiographic techniques were used to detect the temporal sequence of protein synthesis after infection of the sensitive strain Agrobacterium tumefaciens with phage LV-1. Three classes of protein were detected: early proteins, class I, which include a protein capable of shutting off host protein synthesis; class II, proteins which are detected after 30 min; and late proteins, class III, which include the phage-directed endolysin and five additional proteins that appear 45 min after infection.

Amino Acids↗
PubMed1969

Phospholipid metabolism in T4 bacteriophage infected Escherichia coli K-12 (lambda).

Infection of Escherichia coli K-12 (lambda) by bacteriophage results in an altered labeling pattern of phospholipids in the host cell. Although the overall incorporation of (32)P(i) into phospholipids is decreased by infection, the relative amounts of phosphatidylglycerol and cardiolipin are increased. Phospholipid changes occurring at later stages in the lytic cycle of infected bacteria are more prominent than those at earlier time intervals. The uptake of (32)P(i) into phospholipids of cells infected with T4Bs and endolysin-negative mutants was similar to that observed with the wild-type phage, suggesting that the development of resistance to lysis from without and the repair of mucopeptides are not responsible for the phospholipid changes. The metabolism of phospholipids in uninfected cells treated with cyanide was similar to that of infected cells, indicating that part of the phage-induced alterations may be a consequence of impaired respiration.

Chromatography, Thin Layer↗