Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes

Abstract

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.

Identification and Characterization of a Newly Isolated Shiga Toxin 2-Converting Phage from Shiga Toxin-ProducingEscherichia coli

“…The plaques of Stx2⌽-II developed in C600 were significantly smaller than those of Stx2⌽-I, for which the morphology of plaques resembled that of 933W. Although the full length of the Stx2⌽-I genome was similar to that of 933W (7,34,45,49), it was approximately 3 kb smaller than that of Stx2⌽-II (Fig. 2).…”

“…In this study, we sequenced the two stx2 genes in Stx2⌽-I and Stx2⌽-II and found that the two stx2 sequences were identical to each other and that they were also identical to the stx2 gene from 933W (11,21,22,54). The open reading frames containing the stx2 gene of Stx2⌽-I and Stx2⌽-II were located downstream of p gene of phage , as is the case for the stx2 gene in 933W (7,45).…”

“…Involvement of outer membrane-associated proteins of E. coli in Stx2⌽-II infection. Stx-converting phages are lambdoid phages (7,18,19,45), and therefore the involvement of the lamB gene (coding for the LamB protein) in the infection of C600 by Stx2⌽-II was checked. The lamB20::Tn5 mutation of GS20 was introduced by P1 phages into C600, and one of the lamB20::Tn5 transductants, designated MW47, was examined for phage sensitivity.…”

Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding Bacteriophages Produce Markedly Different Amounts of Shiga Toxin

“…Significant genomic divergence among Stx1-encoding phages isolated from different E. coli serotypes has been dem-onstrated by analysis of phage genome lengths and restriction fragment length polymorphisms (RFLP) (25). Recent studies have revealed some variation within O157-derived Stx2-encoding phages (6,22). In the present study, to assess the level of variation among Stx2-encoding phages derived from multiple STEC serotypes and its possible relevance to toxin regulation, we produced isogenic lysogens of seven different Stx2-encoding phages.…”

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources

“…Long-PCR restriction fragment length polymorphism (RFLP) was employed to target sequences between the p gene (analogous to the k phage replication promoter) and the VT2A subunit, because VT2 sequences can be encoded by lamboid phages. A 45°C annealing step and primers DK1 and GK7 were used (Datz et al 1996). The reaction mix was modified to contain ca 100 ng genomic DNA, 200 lM M of each deoxynucleoside triphosphate, 200 nM M of each primer, 5 ll of 10X PCR buffer, and 2AE6 U of expand high fidelity polymerase (Expand TM ; Roche Diagnostics, Lewes, UK), in a final volume of 50 ll.…”