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PubMed1981

Genetic analysis of prophage effects on heteroimmune superinfection in Serratia marcescens.

Plaque formation of phage kappa on Serratia marcescens strain HY normally depends on the presence of either a psi or y prophage in the indicator bacteria. Bacterial ink mutants allowing kappa growth in the absence of either prophage were isolated from the doubly cured strain HY (psi, y)--. By means of a kappa mutant, named gdy, an active participation of kappa in antagonizing inhibition of its own growth on HY (psi, y)-- was demonstrated. The gdy mutation is closely linked to gene cIII coding for the kappa repressor. The prophages psi and y enable kappa to grow undisturbed probably by modifying the kappa DNA during replication in such a way that it is not susceptible to the ink+ effect. Whereas kappa grown on HY (psi, y)--ink-- gave only rare productive infections of HY (kappa, y)--, psi and y grown on the same strain were fully infective. The interference exerted by a kappa prophage on vegetative propagation of y is based upon a multi-component mechanism, the interference being removed, or diminished by mutations residing either inside or outside of the kappa prophage. The responsive phage gene iny+ is dominant over its mutant allele iny--; hence it codes for a diffusible product. Both on the vegetative and the prophage genome iny is located near gene lI responsible for lysogenic conversion of bacteria to non-adsorption of kappa. The restriction-modification system of HY is not involved in the growth inhibition of kappa by HY (psi, y)--. Contrary to the other phages used in this work y is refractory to restriction.

Bacteriophages↗

PubMed2006

Multilocus sequence typing (MLST) analysis of Vibrio cholerae O1 El Tor isolates from Mozambique that harbour the classical CTX prophage.

Vibrio cholerae O1 isolates belonging to the Ogawa serotype, El Tor biotype, harbouring the classical CTX prophage were first isolated in Mozambique in 2004. Multilocus sequence typing (MLST) analysis using nine genetic loci showed that the Mozambique isolates have the same sequence type (ST) as O1 El Tor N16961, a representative of the current seventh cholera pandemic. Analysis of the CTX prophage in the Mozambique isolates indicated that there is one type of rstR in these isolates: the classical CTX prophage. It was also found that the ctxB-rstR-rstA-rstB-phs-cep fragment was PCR-amplified from these isolates, which indicates the presence of a tandem repeat of the classical CTX prophage in the genome of the Mozambique isolates. The possible origin of these isolates and the presence of the tandem repeat of the classical prophage in them implicate the presence of the classical CTX phage.

Bacterial Proteins↗

PubMed2005

Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1.

The sequence of 50 625 bp of chromosomal DNA derived from Shiga-toxin (Stx)-producing Escherichia coli (STEC) O111: H- strain 1639/77 was determined. This DNA fragment contains the cryptic Stx1-encoding prophage CP-1639 and its flanking chromosomal regions. The genome of CP-1639 basically resembles that of lambdoid phages in structure, but contains three IS629 elements, one of which disrupts the gene of a tail fibre component. The prophage genome lacks parts of the recombination region including integrase and excisionase genes. Moreover, a capsid protein gene is absent. CP-1639 is closely associated with an integrase gene of an ancient integrative element. This element consists of three ORFs of unknown origin and a truncated integrase gene homologous to intA of CP4-57. By PCR analysis and sequencing, it was shown that this integrative element is present in a number of non-O157 STEC serotypes and in non-STEC strains, where it is located at the 3'-end of the chromosomal ssrA gene. Whereas in most E. coli O111: H- strains, prophages are inserted in this site, E. coli O26 strains contain the integrative element not connected to a prophage. In E. coli O103 strains, the genetic structure of this region is variable. Comparison of DNA sequences of this particular site in E. coli O157: H7 strain EDL933, E. coli O111: H- strain 1639/77 and E. coli K-12 strain MG1655 showed that the ssrA gene is associated in all cases with the presence of foreign DNA. The results of this study have shown that the cryptic prophage CP-1639 is associated with an integrative element at a particular site in the E. coli chromosome that possesses high genetic variability.

Chromosome Mapping↗

PubMed2006

Prophages of Staphylococcus aureus Newman and their contribution to virulence.

Four prophages (phiNM1-4) were identified in the genome of Staphylococcus aureus Newman, a human clinical isolate. phiNM1, phiNM2 and phiNM4, members of the siphoviridae family, insert at different sites (poiA, downstream of isdB and geh) in the staphylococcal chromosome. phiNM3, a beta-haemolysin (hlb) converting phage, encodes modulators of innate immune responses (sea, sak, chp and scn) in addition to other virulence genes. Replication of phiNM1, phiNM2 and phiNM4 occurs in culture and during animal infection, whereas phiNM3 prophage replication was not observed. Prophages were excised from the chromosome and S. aureus variants lacking phiNM3 or phiNM1, phiNM2 and phiNM4 displayed organ specific virulence defects in a murine model of abscess formation. S. aureus Newman lacking all four prophages was unable to cause disease, thereby revealing essential contributions of prophages to the pathogenesis of staphylococcal infections.

Animals↗

PubMed2003

Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci.

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.

Attachment Sites, Microbiological↗

PubMed2005

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes.

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.

Amino Acid Sequence↗

PubMedDate not supplied

[Functional organization of prophage and lysogeny in Erwinia carotovora with participation of a temperate bacteriophage ZF40].

Functional organization of a prophage of the temperate bacteriophage ZF40 of Erwinia carotovora subsp. carotovora which includes its immunity and inducibility as well as its effect on the host phenotype. It was established that the prophage ZF40 forms several different states in E. carotovora which are distinguished by the indices of spontaneous and lysogenic induction. In contrast to other prophages, including the lambdoid ones, the prophage ZF40 is capable to establish cytoplasmic overimmunity which protects the lysogenic system from superinfection by virulent mutants or other homoimmune bacteriophages. An increase of sensitivity of ZF40-lysogens to killing activity of colicino-like carotovoricin (CCTV) and destabilization of defective lysogeny, or resistant MCTV-prophages are related to the phenomenon of the phage lysogenic conversion of E. carotovora.

Bacteriocins↗

PubMed1991

Genetic studies of the ends of a locked-in kappa prophage in Serratia marcescens by transductional and vegetative crosses.

In temperate phage kappa of Serratia marcescens several special features of different phages are combined. The unessential genes lI, iny, cII and, at least to some extent, even the integrase gene int are not subject to negative control by the repressor, the product of gene cIII. A genetic map of the prophage was established using defective, heat-induced lysates of int- lysogens both in vegetative crosses with sus mutants of essential genes and in transduction of the four unessential genes to lysogenic recipients. Results from reciprocal four factor-crosses concerning the order of the four genes had to be included. The four genes are located near the right end of the prophage, whereas cIII lies near its left end. In vegetative phage all five genes lie in an interval between the essential genes T and U, comprising 10% of kappa's genetic map. The right prophage end appears to face at least two trp cistrons, among them the gene encoding anthranilate synthetase. lI encodes a product that masks the phage receptors in the cell wall. The gene product of iny interferes with the growth of infecting phage y. The natural function of cII is still unknown, but some of its mutants display a cold-sensitive phenotype, their plaques being clear at 30 degrees C and turbid at 37 degrees C. Bacteria with such prophages stop producing viable progeny when the cultures are shifted from 37 degrees C to 30 degrees C. These cold-sensitive mutants are partly dominant and partly recessive. Analysing a virulent mutant, a gene ant encoding an antirepressor was discovered, but so far there is no evidence that it is regulated by an extra repressor. The gene is located relatively near the left prophage end. Evidence is presented that the exogenotes in transduction with the defective lysates continue to exist for some time after a first recombinational event.

Anthranilate Synthase↗

PubMed1993

Anti-SOS effects induced in Bacillus subtilis by a phi 105 mutant prophage.

The presence of the mutant prophage phi 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.

Bacillus Phages↗

PubMed1980

Induction of lambdoid prophages by amino acid deprivation: differential inducibility; role of recA.

Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and lambda i21 are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of lambda and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of lambda repressor with a cI carrying-plasmid prevented amino acidless induction of lambda as did the lambda ind- mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of lambda and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. lambda i80 was inducible both by amino acid and thymine deprivation.

Amino Acids↗

PubMed1977

Escape synthesis of RNA polymerase subunits and termination factor rho following induction of prophage lambda in Escherichia coli.

Synthesis of RNA polymerase subunits and of transcription termination factor p was studied after thermoinduction of prophage lambdac1857 located at several unusual sites on the chromosome of Escherichia coli. When a lysogen carrying the prophage at the bfe gene was induced at 42 degrees C, the rate of synthesis of core polymerase subunits (alpha, beta and beta') rapidly decreased, followed by a marked increase after about 10 min. The latter increase was observed specifically in the "bfe lysogen" and not in any of the other lysogens tested. Similarly, the rate of synthesis of p factor increased appreciably in the induced ilv lysogen carrying the prophage at the ilv gene, and possibly in the bfe lysogen as well, but not in other lysogens examined. Taken together with other evidence, these results suggest that the enhanced syntheses of beta and beta' subunits of RNA polymerase and of p factor observerd represent "escape synthesis", resulting from the close linkage of the prophage genome to the respective structural genes. In contrast, omega factor synthesis was stimulated upon induction of any of the lysogens used without respect to the site of prophage location, suggesting the involvement of an entirely different mechanism.

Coliphages↗

PubMed1979

A new pleiotropic bacteriophage P1 mutation, bof, affecting c1 repression activity, the expression of plasmid incompatibility and the expression of certain constitutive prophage genes.

In bacteriophage P1 an amber mutation in a new gene, bof, has been isolated. The bof-1 phage mutant exhibits a pleiotropic phenotype; bof product is non-essential, and acts as a positive modulator. In P1 bac-1 mutants, in which a dnaB analog product, ban, is expressed constitutively, the bof product activates ban expression both in the prophage state and in lytic growth: P1 bof bac prophages have a reduced ban activity and in lytic growth P1 bof bac phages show a lower ban activity than P1 wild type. This effect on ban activity is observed specifically in P1 bac-1 mutants; it is not mediated by the c1 repressor of the lytic functions (repressor of the ban operon) since this effect occurs even if the phage carries a heat sensitive c1 repressor. Thus we concluded that the bac mutation put the ban operon under an abnormal, unknown control, modulated by the bof product. P1 bof lysogens show an increased immunity to superinfecting P1 phage and are affected in their inducibility properties; in the presence of the altered c1-100 repressor, bof product is required for maintenance of lysogeny, as shown by the induction of P1 c1-100 bof-1 lysogens at 30 degrees. P1 bof superinfecting phage can be established together with a resident P1 bof prophage in a recA host, unlike P1 wild type which cannot form double lysogens. P1 bof double lysogens are unstable and segregate one or the other prophage. P1 Cm bof and P1 Km bof lysogens show higher levels of antibiotic resistance than the corresponding bof+ lysogens. The bof gene has been mapped, in an interval defined by P1 prophage deletion end points, far from both ban and c1. All bof phenotypes are reversed by single mutations.

Coliphages↗

PubMed1984

Prophage lambda induction caused by mini-F plasmid genes.

When bacterial cells harboring a temperature-sensitive replication plasmid, which carries the particular ccd segment (coupled cell division) containing the ccdA and ccdB genes of a mini-F plasmid, are transferred to 42 degrees C, cell division is inhibited after incubation for an appropriate time. The inhibition occurs, when the copy number of the plasmid decreases to become critically low, about one per cell (Ogura and Hiraga 1983b). In lambda phage lysogens carrying this type of plasmid, the prophage is induced in a small portion of the cell population under the same conditions, in addition to the inhibition of cell division in most of cells. The prophage induction, but not the inhibition of normal cell division, depends on normal recA function. Both induction of prophage and inhibition of cell division are suppressed by the simultaneous presence of a replication proficient plasmid carrying the ccdA gene. We discuss molecular mechanisms of the ccd function that couples host cell division to plasmid proliferation and induces the prophage. Additionally, we propose a hypothesis that the ccd mechanism of F plasmid contributes to indirect induction of prophage lambda by an F plasmid damaged by UV-irradiation and then introduced into a lysogen via conjugation.

Bacteriophage lambda↗

PubMed1975

Indirect induction of a Staphylococcus aureus prophage by P11de a plasmid phage hybrid.

The ability to mediate indirect induction of staphylococcal prophages was found to be a property of the cryptic high frequency transducing phage P11de but not of three other phages tested. P11de is the product of a recombination between a P11 phage and a gamma plasmid. Irradiated P11de preparations could not induce prophage development in strains which contained either a P11 prophage or a gamma plasmid. The establishment of P11de in a strain was not, however, inhibited by the presence of a P11 prophage. It is inferred that the inhibition of indirect induction exerted by the resident P11 prophage occurs at a stage other than the establishment of the P11de replicon.

Plasmids↗

PubMed1976

Regional replication of the bacterial chromosome induced by derepression of prophage lambda. II. Direction and origin.

We have demonstrated previously by DNA-DNA hybridization that induction of lambda phage with wild type O and P genes results in an increase of bacterial DNA in the chromosomal region adjacent to the left of the prophage; is a segment between gal and attlambda (gal DNA) (Imae and Fukasawa, 1970). Evidence is presented in this report that such an increase of bacterial DNA is also seen in the region to the right of the prophage; a segment between bio and attlambda (bio DNA). We postulate therefore that the bidirectional replication of lambda DNA extends beyond the prophage and copies the neighboring host DNA until the prophage is excised. The model is verified by making use of excision-defective lambda phages. The synthesis of gal DNA (or bio DNA) slows down to a halt within 40 min after the induction in the normal lysogens. The results are attributed to the prophage excision: (1) In lysogens for lambdaint, synthesis of the bacterial DNA continues for longer times. (2) The synthesis of the bacterial DNA slows down to a halt in lysogens for lambdaxis or lambdab2 as in the control. However lambda DNA synthesis also slows down in parallel so that the amount of the bacterial DNA relative to that of lambda DNA synthesized by a given time stays constant from 20 min to 80 min. During that time the relative amount of the bacterial DNA rapidly decreases in the normal lysogen.

Chromosome Mapping↗

PubMed1980

Properties of Bacillus subtilis 168 derivatives freed of their natural prophages.

An isogenic set of Bacillus subtilis 168 strains which are non-inducible for prophage PBSX and are cured of prophage SP beta has been constructed. By utilizing these strains, prophage SP beta has been shown to control the inducible DNA modification system which exists in this bacterium. However, neither the PBSX nor the SP beta prophages alter the ability of the bacterium to undergo genetic recombination, to repair damaged DNA or to sporulate. Prophageless B. subtilis would be a useful host for the phi 3T cloning vector, because of the absence of vector--prophage interactions.

Bacillus subtilis↗

PubMed2000

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Amino Acid Sequence↗

PubMed2001

Loss of lambda prophage recombinogenicity in UV-irradiated Escherichia coli: the role of host genes ruvA, ruvB, ruvC, and recG.

Earlier studies have revealed a radiation-induced process leading to the loss of lambda prophage recombinogenicity. The process takes place in UV-irradiated Escherichia coli cells, and renders the prophage incapable of site-specific recombination with the host chromosome, and of general recombination with an infecting homologous phage. It was found that the inhibition of prophage recombinogenicity depends on functional RecBCD enzyme of E. coli. In this work, the role of ruvABC and recG genes in the inhibitory process was assessed. The products of these genes are known to act at the last step of homologous recombination and recombinational DNA repair by catalyzing the resolution of recombination intermediates (the Holliday junctions). Irradiated prophage retained its ability to recombine in ruvA, ruvB, ruvC, and recG mutants. These results suggest that in addition to RecBCD enzyme, RuvABC and RecG proteins are also involved in the inhibition of prophage recombinogenicity. We infer that RuvABC and RecG act in this process before RecBCD, probably by processing the Holliday junctions formed upon replication arrest, and thereby providing double-stranded DNA breaks as substrate for RecBCD-mediated recombinational repair of UV-damaged bacterial chromosome.

Bacterial Proteins↗