Sequence Variability of P2-Like Prophage Genomes Carrying the Cytolethal Distending Toxin V Operon in Escherichia coli O157
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ABSTRACT Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli , in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated with Shiga-toxigenic E. coli (STEC), enterohemorrhagic E. coli (EHEC) O157 strains, and strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb-long, noninducible P2-like prophage carrying the cdt-V operon from an stx - and eae -negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF . The presence of P2-like genes ( n = 20) was investigated in a further five CDT-V-positive bovine E. coli O157 strains of various serotypes, three EHEC O157:NM strains, four strains expressing other variants of CDT, and eight CDT-negative strains. All but one CDT-V-positive atypical O157 strain uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate.Keywords:
Prophage
Cytolethal distending toxin
Temperateness
Strain (injury)
Viruses are the most abundant biological entities in the ocean and are believed to contribute to nutrient cycling, bacterial diversity, and horizontal gene exchange. However, little is known about the relationship between temperate phages and their hosts in marine environments. In this thesis, phage-host systems from the Gulf of Mexico were used to study the influence of temperate phages in bacteria. ФHAP-1 is a temperate myovirus induced with mitomycin C from Halomonas aquamarina isolate. The genome of this phage was 39,245 nucleotides long and contained 46 predicted genes. Besides genes involved in lysogeny, ФHAP-1 contained a protelomerase, which is responsible for resolution of telomeric ends in linear plasmid-like phages. Hybridization studies and PCR analysis indicated not only a lack of integration of the prophage in the host chromosome, but differences in genome arrangement between the prophage and virion forms of ФHAP-1. These results suggest that ФHAP-1 exists as a nonintegrating linear phage with telomeric ends. Eleven pigmented Bacillus spp. isolates were examined for the occurrence of lysogeny and sporulation through induction with mitomycin C and decoyinine, viii respectively. The results from these experiments suggested a variety of interactions can occur between phages and their hosts, some of which may influence sporulation. The lysogenic strain B14905 had high frequency of sporulation and was selected for further analysis. The genome of B14905 contained 4 prophage-like regions, one of which was independently sequenced from an induced lysate. PCR and TEM analysis of a mitomycin C induced lysate indicated that two of these regions were inducible prophage, one was a defective phage, and one was a non-inducible phage remnant. One of the inducible prophages contained a transcriptional regulator that is hypothesized to be involved in regulation of host sporulation. The diversity of prophage and prophage-like elements found in B14905 suggest that the genetic diversity of phages in the oceans is vast. The studies of the temperate phages from H. aquamarina and Bacillus spp. isolates illustrates that integration of molecular, genomic, and function studies can be used to provide insight into the influence of prophage on host bacteria.
Prophage
Lysogenic cycle
Temperateness
Marine bacteriophage
Halomonas
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Prophage
Lysogenic cycle
Temperateness
Lytic cycle
Siphoviridae
Lysogen
Phagemid
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Phages 933W, BAA2326, 434, and λ are evolutionarily-related temperate lambdoid phages that infect Escherichia coli. Although these are highly-similar phages, BAA2326 and 933W naturally encode Shiga toxin 2 (Stx+), but phage 434 and λ do not (Stx−). Previous reports suggest that the 933W Stx+ prophage forms less stable lysogens in E. coli than does the Stx− prophages λ, P22, and 434. The higher spontaneous induction frequency of the Stx+ prophage may be correlated with both virulence and dispersion of the Stx2-encoding phage. Here, we examined the hypothesis that lysogen instability is a common feature of Stx+ prophages. We found in both the absence and presence of prophage inducers (DNA damaging agents, salts), the Stx+ prophages induce at higher frequencies than do Stx− prophages. The observed instability of Stx+ prophages does not appear to be the result of any differences in phage development properties between Stx+ and Stx− phages. Our results indicate that differential stability of Stx+ and Stx− prophages results from both RecA-dependent and RecA-independent effects on the intracellular concentration of the respective cI repressors.
Prophage
Lysogen
Lysogenic cycle
STX2
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The defective prophage φ80 i λ c I 857 dhis has been mapped through both marker rescue and deletion analysis. Deletions have been isolated which put residual his genes close to trp genes. Analysis of these deletions shows that the histidine operon on the prophage is oriented clockwise as on the bacterial chromosome, thus opposite to the orientation of the trp operon. The presence of the his promoter-operator region is inferred by the ability of the prophage-carrying strain to derepress sequentially under conditions in which the histidine concentration is limiting. In addition to his , the gnd gene is also present on the prophage and is located between his and trp operons. The bacterial genes are inserted in the right arm of the prophage and substitute for all of the late function genes, except for the first three. These data indicate that the “sense” strand for transcription of the his operon in vivo must be the “R” strand.
Prophage
L-arabinose operon
trp operon
gal operon
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Virulence of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin‐converting phages), present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin‐converting prophages. Among other conditions, it was reported that treatment with H 2 O 2 caused induction of these prophages, though with efficiency significantly lower relative to UV‐irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin‐converting prophages as a consequence of H 2 O 2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the “bacterial altruism” and “Trojan Horse” hypotheses, which are connected to the oxidative stress, are discussed.
Prophage
Lysogenic cycle
SOS response
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ABSTRACT Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene ( aph , cat , or tet ) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The c I repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans . Although the sequences of the c I genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.
Prophage
Lytic cycle
Lysogenic cycle
Shiga-like toxin
Lysogen
SOS response
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ABSTRACT Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx 2 subtypes, and the levels of stx 2 transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx 2a by IS 1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx 2c transcription and induction of the prophage and stx 2a ::IS 1203v in Stx2a-encoding prophage generated a truncated stx 2a mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx 2a ::IS 1203v ). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.
Prophage
STX2
Shiga-like toxin
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A high-throughput 96-well plate-based method for the rapid induction of endogenous prophages from individual bacterial strains was developed. The detection of endogenous prophages was achieved by the filtration of the culture liquor following norfloxacin induction and subsequent PCRs targeting bacteriophage-carried gene markers. The induction method was tested on 188 putative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains and demonstrated the ability to detect both lambdoid and stx-carrying bacteriophages in strains for which plaques were not observed via plaque assay. Lambdoid bacteriophages were detected in 37% of the induced phage preparations via amplification of the Q gene, and Stx1- and Stx2-encoding phages were detected in 2 and 14% of the strains, respectively. The method therefore provided greater sensitivity for the detection of Stx and other lambdoid bacteriophage populations carried by STEC strains than that for the established method of plaque assay using bacterial indicator strains, enabling, for the first time, large-scale bacteriophage population and diversity studies.
Prophage
Lysogen
Lysogenic cycle
STX2
Shiga-like toxin
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Level of competence reached by Bacillus subtilis 168 lysogenic for temperate phage φ 105 was reduced compared to that reached by nonlysogenic cells. This effect was probably related to an alteration of the bacterial surface. Deoxyribonucleic acid extracted from φ 105 lysogenic bacteria was used to transform other lysogenic bacteria. About 25% linkage was found between the bacterial phe-1 marker and prophage marker ts N15. The order of a few prophage markers relative to phe-1 was established in three-factor crosses. The usefulness of this system for a study of the linkage between an integrated prophage genome and that of its host was discussed.
Lysogenic cycle
Prophage
Temperateness
Lysogen
Lytic cycle
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ABSTRACT The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H 2 O 2 ), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H 2 O 2 and also human neutrophils, an important endogenous source of H 2 O 2 , induced Stx2 expression by an EHEC clinical isolate. Both H 2 O 2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.
Prophage
STX2
Lysogen
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Citations (133)