Distinctiveness of the genomic sequence of Shiga toxin 2-converting phage isolated from Escherichia coli O157:H7 Okayama strain as compared to other Shiga toxin 2-converting phages

Shiga toxin 2-converting phage was isolated from Escherichia coli O157:H7 associated with an outbreak that occurred in Okayama, Japan in 1996 (M. Watarai, T. Sato, M. Kobayashi, T. Shimizu, S. Yamasaki, T. Tobe, C. Sasakawa and Y. Takeda, Infect. Immun. 61 (1998) 3210-3204). In this study, we analyzed the complete nucleotide sequence of Shiga toxin 2-converting phage, designated Stx2phi-I, and compared it with three recently reported Stx2-phage genomes. Stx2phi-I consisted of 61,765 bp, which included 166 open reading frames. When compared to 933W, VT2-Sakai and VT2-Sa phages, six characteristic regions (regions I-VI) were found in the Stx2 phage genomes although overall homology was more than 95% between these phages. Stx2phi-I exhibited remarkable differences in these regions as compared with VT-2 Sakai and VT2-Sa genes but not with 933W phage. Characteristic repeat sequences were found in regions I-IV where the genes responsible for the construction of head and tail are located. Regions V and VI, which are the most distinct portion in the entire phage genome were located in the upstream and downstream regions of the Stx2 operons that are responsible for the immunity and replication, and host lysis. These data indicated that Stx2phi-I is less homologous to VT2-Sakai and VT2-Sa phages, despite these three phages being found in the strains isolated at the almost same time in the same geographic region but closely related to 933W phage which was found in the E. coli O157 strain 933W isolated 14 years ago in a different geographic area.     

Cloning of enterohemorrhagic Escherichia coli phage VT-2 dam methyltransferase.

Enterobacterial GATC-specific DNA adenine methyltransferase (Dam) plays an essential role in regulation of DNA replication, methyl-directed mismatch repair, transposition and gene expression. In Salmonella typhimurium it has been shown to directly control virulence. In this paper we report cloning and expression of the dam gene from the Shiga toxin-producing VT2-Sa prophage of enterohemorrhagic Escherichia coli O157. Comparisons of the predicted amino acid sequence indicates that Dam methyltransferases of E. coli phages VT2-Sa, 933W, T1 and Haemophilus influenzae phage HP1 make up a separate subgroup of adenine-N6 methyltransferases. These proteins are similar to the gamma subfamily of amino-methyltransferases in respect to the linear order of sequence motifs and the presence of the hallmark "NPPY" tetrapeptide. However, they apparently lack an autonomous target-recognizing domain at the C-terminus of the catalytic domain and therefore we propose to dub them as a "mini-gamma" subfamily.     

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.     

Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages

The nucleotide sequence of an 11,142-bp region including the stx ₂ operon in the genome of the temperate bacteriophage 933W in the EDL933 strain of Escherichia coli O157 was determined and compared to the respective regions derived from other lambdoid bacteriophages. In phage 933W, a region of ORFs interlinked by overlapping start-stop codons (ATGA) was detected preceding the toxin gene. These ORFs show a high degree of sequence identity to genes of the nin region of phage λ. Immediately downstream of these nin genes we identified an ORF that may code for an antiterminator similar to the λ Q protein. It is concluded that toxin expression is directly associated with the initiation of cell lysis. Downstream of the stx ₂ operon we identified an ORF that is homologous to the holin gene S of bacteriophage PA-2. PCR primers were designed, which, based on a comparison of the phage sequences, appeared to be common to both stx ₁ - and stx ₂ -harbouring phages. However, only seven of the 22 STEC strains investigated from serogroups O157, O26, O103 and O111 yielded the expected PCR amplification product. The data reported here may be useful in developing new strategies for inhibiting the expression of Stx and for developing universal diagnostic primers for use in tracking the origin and evolution of Shiga toxins and the phages that carry them. Received: 24 February 1999 / Accepted: 7 September 1999     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2

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 cI 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 cI 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.     

Survival of the fittest: a role for phage-encoded eukaryotic-like kinases

Phages are often thought of as mortal enemies of bacteria. This dynamic relationship has led to the evolution of a number of processes in bacteria designed to defeat these attacks. Examples of these include blocking phage attachment, CRISPR, and restriction modification systems. Temperate phages provide another source of protection by excluding infection of heterologous phage, thwarting phage production and further infection. This strategy protects the rest of the bacterial population from attack. The lambdoid phage 933W, a source of the genes encoding Shiga toxin in the highly pathogenic O157:H7 enterohemorrhagic E. coli strain, also carries a gene encoding a eukaryotic-like tyrosine kinase, Stk. In this issue of Molecular Microbiology, Friedman et al. (2011) show that Stk, through its kinase activity, excludes infection by another lambdoid phage HK97. This exclusion is very specific as it does not affect a number of other lambdoid phages. HK97 contributes to its own demise by expressing the product of an open reading frame, orf41, which is required for Stk activation. The authors further show that autophosphorylation increases the stability of Stk and suggest that autophosphorylation contributes to Stk activity. Whether or not this exclusion activity provides a selective advantage through maintenance of Stk activity is yet to be explored.     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.     

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.     

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment

Background

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Methodology/Principal Findings

The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg²⁺. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction.

Conclusions/Significance

Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.     

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

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

Heterogeneity of Escherichia coli phages encoding Vero cytotoxins: comparison of cloned sequences determining VT1 and VT2 and development of specific gene probes

Phages coding for production of Vero cytotoxins VT1 or VT2 in strains of Escherichia coli serotype O157.H7 or O157.H- were morphologically indistinguishable. Their genome size and restriction enzyme digests of the phage DNA were similar. These phages were clearly different in these respects from a VT1-encoding phage isolated from a strain of E. coli O26.H11 (H19). However the VT1 region cloned from the phage originating in the E. coli O157.H7 strain was identical to the VT1 region previously cloned from the phage carried by H19. Sequences encoding VT2 that were cloned from the phage in E. coli O157.H- have been mapped and the VT2 region identified by transposon insertion. The cloned regions coding for VT1 or VT2 production had no similarities in the presence of restriction enzyme sites over a distance of about 2 kb, and two VT1-specific probes spanning a region of about 1.4 kb did not hybridize under stringent conditions with cloned VT2 DNA. A 2 kb HincII fragment contained the VT2 genes but hybridized to VT1-encoding phages and recombinant plasmids via flanking phage DNA. A 0.85 kb AvaI-PstI fragment was a specific probe for VT2 sequences and did not hybridize under stringent conditions to phages or plasmid recombinants encoding VT1.