Induction of prophage & absence of mutation induction by psoralen plus near UV light exposure in Haemophilus influenzae.
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Bacteriophages↗
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Relationship between prophage induction and transformation efficiencies in Haemophilus influenzae Rd9.
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Bacteriophages↗
Lambda prophage decreases Escherichia coli sensitivity to human serum bactericidal effect.
Escherichia coli C600 and C600(lambda) strains were tested for their susceptibility to the bactericidal action of 4% normal human serum. C600 survival was reduced to 30%, 23% and 16% after 60, 150 and 180 min of exposure to serum, respectively, whereas the percentage of survival of C600(lambda) was 199, 109 and 65% at the same times. The estimated exposition times for 50% killing showed an eight-fold difference, they were 23 and 202 min for C600 and C600(lambda), respectively. None of the two strains tested was killed when incubated with serum whose alternative complement pathway was inactivated by heating at 50 degrees C for 20 min, showing that this pathway, and not the classical one, was responsible of the bactericidal action, a conclusion further supported by the finding that both strains were differentially killed by the alternative complement pathway, C600 showing a 14X, 10X and 4X greater susceptibility than C600(lambda) at 60, 120 and 180 min of exposure to serum whose classical pathway was selectively inhibited by chelation with 10 mM EGTA plus 2 mM MgCl2. We feel that lambda phage may lower the serum sensitivity of its lysogen by altering the bacterial external surface, perhaps by the inclusion of some protein encoded by an accessory gene of the lambda genome, and thus interfering with either the formation, deposition or activity of the membrane attack complex.
Bacteriophage lambda↗
[Interaction between Pseudomonas aeruginosa phages-transposons: genetic analysis of the trait of inhibition by prophage D3112 of phage B39 development].
A novel, previously unknown cip gene of Pseudomonas aeruginosa phage D3112 was discovered. The cip gene is responsible for inhibiting the intracellular growth of the related heteroimmune phage B39 of P. aeruginosa. It was shown that the cip gene product inhibits replication of the B39 genome, interacting, apparently, with the definite s+ site in the phage B39 genome. Mutants cip- of phage D3112, incapable of inhibiting the growth of phage B39, and B39 mutants, insensitive to the action of the cip gene product of phage D3112, were isolated.
DNA Transposable Elements↗
[Role of prophages in the formation of antibiotic-resistant populations of Staphylococcus in the process of transformation, transduction and conjugation].
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Conjugation, Genetic↗
Transcription analysis of Streptococcus thermophilus phages in the lysogenic state.
The transcription of prophage genes was studied in two lysogenic Streptococcus thermophilus cells by Northern blot and primer-extension experiments. In the lysogen containing the cos-site phage Sfi21 only two gene regions of the prophage were transcribed. Within the lysogeny module an 1.6-kb-long mRNA started at the promoter of the phage repressor gene and covered also the next two genes, including a superinfection exclusion (sie) gene. A second, quantitatively more prominent 1-kb-long transcript was initiated at the promoter of the sie gene. Another prophage transcript of 1.6-kb length covered a group of genes without database matches that were located between the lysin gene and the right attachment site. The rest of the prophage genome was transcriptionally silent. A very similar transcription pattern was observed for a S. thermophilus lysogen containing the pac-site phage O1205 as a prophage. Prophages from pathogenic streptococci encode virulence genes downstream of the lysin gene. We speculate that temperate phages from lactic streptococci also encode nonessential phage genes ("lysogenic conversion genes") in this region that increase the ecological fitness of the lysogen to further their own evolutionary success. A comparative genome analysis revealed that many temperate phages from low GC content Gram-positive bacteria encode a variable number of genes in that region and none was linked to known phage-related function. Prophages from pathogenic streptococci encode toxin genes in this region. In accordance with theoretical predictions on prophage-host genome interactions a prophage remnant was detected in S. thermophilus that had lost most of the prophage DNA while transcribed prophage genes were spared from the deletion process.
Base Sequence↗
Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.
Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.
Bacteria↗
CTXphi contains a hybrid genome derived from tandemly integrated elements.
CTXphi is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5' prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3' element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXphi, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXphi DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXphi-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXphi does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXphi to new hosts.
Bacteriophages↗
The production of generalized transducing phage by bacteriophage lambda.
Generalized transduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage lambda to assess some of the factors that affect that process. The results of those experiments indicate: the production of generalized transducing particles by bacteriophage lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by lambda Exo is the production of lambda docR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of lambda docL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing particles are not detected in induced lysis-defective (S-) lambda lysogens until about 60-90 min after prophage induction. Since wild-type lambda would normally lyse cells by 60 min, the production of lambda-generalized transducing particles depends on the phage being lysis-defective; if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10-20-fold range. In contrast, if transducing lysates are prepared by the induction of a lambda lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers; if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA. These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by lambda, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather recognize some feature of the DNA tht is sensitive to lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED AT 400 WORDS)
Bacteriophage lambda↗
Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system.
Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.
Animals↗
Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.
HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.
Bacterial Proteins↗
Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of the CTXphi array and its position in the genome.
The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXphi. The genome of V. cholerae, the host for CTXphi, consists of two chromosomes, one large and one small. Here, it is shown that localization and array of CTX prophage DNA in either the large or small chromosome of V. cholerae is likely to be one of the reasons for the emergence of O1 biotype El Tor variants isolated just before and after the V. cholerae O139 cholera outbreak in 1992. Analyses of the organization of the CTX region of the genome of pre-O139 El Tor strains revealed that these strains carry two distinct CTX prophages integrated in the small chromosome in tandem: CTX(ET), the prophage having a conserved NotI site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CTX(calc)-like genome, the prophage found in recent O139 clinical isolates from Calcutta. In sharp contrast, in post-O139 El Tor strains only one copy of the CTX(ET) prophage was found to be integrated in the large chromosome. To the authors' knowledge, the presence of CTX prophage in the small chromosome of O1 El Tor strains has not been reported previously. It is also shown that the difference in the CTX copy number and the position of the bacteriophage on the genomes of pre- and post-O139 El Tor strains have an effect on cholera toxin production. While a pre-O139 strain produced maximum cholera toxin in yeast extract/peptone medium at 30 degrees C, a post-O139 El Tor strain showed maximal yield at 37 degrees C, indicating differential regulation of cholera toxin between the strains. It appears from this study that the variation in the integration site of the CTX prophage, its copy number and the presence of diverse phage genomes in V. cholerae O1 biotype El Tor may be strategically important for generating variants with subtle phenotypic modulations of virulence factor production in this longest-ruling seventh pandemic strain.
Bacteriophages↗
Nucleotide usage, synonymous substitution pattern, and past recombination in genomes of Streptococcus pyogenes.
We examined (1) five variables summarizing nucleotide usage at synonymous sites; (2) pairwise patterns of nucleotide substitution among five genomes of Streptococcus pyogenes in order to examine the extent to which these variables are associated with past recombination events. Predicted prophage genes of MGAS10394 were characterized by an average pattern of nucleotide usage at synonymous sites that was distinct from that seen at most other genes. Ribosomal protein genes also showed a distinctive pattern, but one that differed from prophage genes in several key respects. The six most anomalous genes with respect to synonymous substitution in pairwise comparisons among the genomes included four prophage genes; these six genes were characterized by unusually high values of d(S), suggesting that these genes had different evolutionary histories from those of the other shared genes in the genome. This hypothesis was supported by phylogenetic analyses, which revealed phylogenies inconsistent with that of the majority of genes. Eighty-nine genes outside prophage regions were found to resemble prophage genes with respect to nucleotide content and the pattern of synonymous substitution, suggesting that these genes may have been involved in horizontal gene transfers. Although prophage genes do show distinctive patterns of nucleotide composition, the results indicated that those patterns were not in themselves sufficient to distinguish all known prophage genes from other genes. On the other hand, nucleotide content, when suitably measured, can form one of a set of biological indicators that can be used to identify candidate genes for horizontal gene transfer.
Amino Acid Substitution↗
Starvation-induced Mucts62-mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp.
The formation of araB-lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage-encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB-lacZ fusion formation, confirming that derepression is a rate-limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild-type repressor. It depended upon the host ClpXP and Lon ATP-dependent proteases and the RpoS stationary phase-specific sigma factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner-regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase-specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.
ATP-Dependent Proteases↗
Control of bacteriophage lambda repressor establishment transcription: kinetics of l-strand transcription from the y-cII-oop-O-P region.
The kinetics of lambda l-strand repressor establishment RNA synthesis were measured from the y-cII region of induced tof- prophage. The activity of the repressor is epistatic to the expression of gene tof coding for the antirepressor (Tof). The activity of Tof, is epistatic to the expression of repressor gene cI transcription from Prm and the expression of repressor establishment transcription from a site 600 to 800 nucleotides upstream from Prm. Three modes of l-strand rex-cI-tof-y-cII-oop transcription occur: (a) Prm promoted cI-rex mRNA synthesis from noninduced prophage, (b) coordinate lit and oop synthesis from induced tof+ prophage and (c) establishment transcription from induced tof- prophage. The synthesis or stability of oop RNA is much reduced from induced tof-, compared with tof+ prophage. The oop transcription from tof- prophage is not coordinate with RNA synthesis from the y-cII interval. The y-cII-(oop) portion of the establishment transcript appears more unstable than the translated downstream copy of genes rex-cI. The initiation of any repressor establishment transcription requires the products of lambda genes cIII, cII, P and Escherichia coli genes dnaB, dnaG, but not actual lambda DNA synthesis. This result demonstrates that common factors, i.e. replication gene products, are required for the initiation of establishment transcription, lambda replication and lit, oop RNA synthesis; and explains why cIII+ cI+ cII+ replication defective phage lysogenize poorly at low multiplicities of infection. The cIII and cII products were shown to act after an earlier replication initiation or activation event. Repressor establishment transcription and repressor mRNA synthesis from Prm (from induced cI- tof-, cIII- cI- tof- or cI- tof- cii- prophage) are amplified by gene dosage. The extent of lysogenization of E. coli by lambda cIII-, cII- or replication minus mutants, defective for initiation of establishment synthesis, is attributed to gene dosage dependent transcription from Prm. The mechanism by which Tof inhibits the initiation of establishment transcription does not appear to require repression of RNA synthesis from PL and PR. RNA synthesis from these promoters is blocked by renaturation of the repressor 5 min after induction, before establishment transcription is detected; however, establishment RNA synthesis measured between 12-13 min after induction, i.e. 7 min after renaturation of the repressor, is only partially reduced.
Coliphages↗
Genetic diversity among type emm28 group A Streptococcus strains causing invasive infections and pharyngitis.
Genome sequencing of group A Streptococcus (GAS) has revealed that prophages account for the vast majority of gene content differences between strains. Serotype M28 strains are a leading cause of pharyngitis and invasive infections, but little is known about genetic diversity present in natural populations of these organisms. To study this issue, population-based samples of 568 strains from Ontario, Canada; Finland; and Houston, Texas, were analyzed. Special attention was given to analysis of variation in prophage-encoded virulence gene content by a PCR-based method. Thirty and 29 distinct prophage-encoded virulence gene profiles were identified among pharyngitis and invasive infection isolates. Thirteen profiles, representing the majority of the strains, were shared between these two classes of isolates. Significant differences were observed in the frequency of occurrence of certain prophage toxin gene profiles and infection type. M28 strains are highly diverse in prophage-encoded virulence gene content and integration site, supporting the key concept that prophages are critical contributors to GAS genetic diversity and population biology. Nucleotide sequence variation in the emm gene (encodes M protein) was also examined. Only three allelic variants were identified in the hypervariable portion of the emm28 gene. All but one strain had the same inferred amino acid sequence in the first 100 amino acids of the mature M28 protein. In contrast, size differences in the emm28 gene and inferred protein due to variable numbers of C-terminal repeats were common. The presence of macrolide resistance genes (mefA, ermB, and ermTR) was analyzed by PCR, and less than 2% of the strains were positive.
Antigens, Bacterial↗
The origin of Q-independent derivatives of phage lambda.
lambda qsr' (Q-independent) phages are characterised by the replacement of the region of the lambda genome that contains Q, S, R, and the late gene promoter, P'R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that lambda p4 and lambda qin A3, two such Q-independent phages, are the product of recombination between lambda and a defective lambdoid prophage (the qsr' prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr' prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K12 strain AB1157 from which lambda qsr' phages cannot be generated, the qsr' prophage has suffered an internal deletion. That the qsr' prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.
Bacteriophage lambda↗
Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112.
D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.
Amidohydrolases↗