Insertion Site Occupancy by stx ₂ Bacteriophages Depends on the Locus Availability of the Host Strain Chromosome

Abstract

Shiga toxin-producing Escherichia coli (STEC) is an emergent pathogen characterized by the expression of Shiga toxins, which are encoded in the genomes of lambdoid phages. These phages are infectious for other members of the Enterobacteriaceae and establish lysogeny when they integrate into the host chromosome. Five insertion sites, used mainly by these prophages, have been described to date. In the present study, the insertion of stx 2 prophages in these sites was analyzed in 168 STEC strains isolated from cattle. Additionally, insertion sites were determined for stx 2 phages which (i) converted diverse laboratory host strains, (ii) coexisted with another stx 2 prophage, and (iii) infected a recombinant host strain lacking the most commonly used insertion site. Results show that depending on the host strain, phages preferentially use one insertion site. For the most part, yehV is occupied in STEC strains while wrbA is preferentially selected by the same stx phages in E. coli laboratory strains. If this primary insertion site is unavailable, then a secondary insertion site is selected. It can be concluded that insertion site occupancy by stx phages depends on the host strain and on the availability of the preferred locus in the host strain.Shiga toxin-producing Escherichia coli (STEC) is an emergent pathogen causing bloody diarrhea and hemolytic uremic syndrome (7). This group comprises different serotypes; the best-known serotype, which was one of the earliest determined to belong to this group, is O157:H7 (25). Cattle are a major reservoir for STEC. Ruminant animals such as cattle, sheep, and goats are naturally colonized with STEC and release these organisms into the environment with their feces (6, 35).One of the main virulence factors in STEC is the expression of Shiga toxins (Stx). To date, two Shiga toxins, Stx 1 and Stx 2 , and several variants (15, 37) have been described. These toxins are encoded in the genomes of lambdoid phages, which establish lysogeny in STEC bacteria (13). stx bacteriophages can convert different E. coli strains, as well as some other members of the Enterobacteriaceae, into Stx-producing bacteria (1,2,13,19,29,34).A successful lysogenic event can occur only if the phage genome is integrated into the bacterial chromosome. To achieve this, phage integrase, which is regulated by gene products activated in the lysogenic pathway, and host recombinases (e.g., integration host factors), which are not regulated by any phage gene, must recognize homologous sequences present in the phage DNA and in the bacterial genome (attP and attB, respectively) (14, 26).Several studies have attempted to characterize phage integrases (3,16,23,26,27,33). Although different families of this enzyme have been established, most of them conserve certain motifs in their protein architecture that allow attachment site recognition and phage genome-bacterial genome recombination.Shiga toxin prophages can convert the host strain because they integrate their genome by using specific insertion sites. To date, five ta...

Evolution of a Self-Inducible Cytolethal Distending Toxin Type V-Encoding Bacteriophage from Escherichia coli O157:H7 to Shigella sonnei

“…The presence of cdt in phages, although not always inducible, has been reported (11) and is supported by the P2 sequences (12) or lambdoid phage sequences (15) found in the flanking regions of cdt in several bacterial isolates. In this study, a new Cdt-V phage, named ⌽AA91, was used to lysogenize S. sonnei strain 866, since this strain gave good results in studies with Cdt phages (14) and also had previously shown the capacity to generate lysogens of temperate Stx bacteriophages (21,36). No lysogens of ⌽AA91 were obtained with any of the other laboratory strains assayed.…”

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

“…They are considered members of the lambdoid family, since they share a common genome arrangement that conserves the relative positions of the genes with similar activities and associated regulatory signals (7). Although Shiga toxin-encoding bacteriophages are a heterogeneous group in terms of morphology and their genetic organization (24,33,46,54), the location of stx genes is conserved, being found next to the lytic genes and downstream of the Q antiterminator (36). Therefore, Shiga toxin production is basically linked to the induction or progression of the phage lytic cycle after activation of the SOS response in the bacterial host.…”

“…The toxin is released through cell lysis (55). Following the lytic burst, Stx phages act as vectors in the stx horizontal transmission to other members of the family Enterobacteriaceae (1,2,19,33,44,46,51).…”

BaeSR, Involved in Envelope Stress Response, Protects against Lysogenic Conversion by Shiga Toxin 2-Encoding Phages

“…The insertion sites of the phages used in this study (wrbA for all phages except A9, which inserts in yecE) (20) were confirmed to be the same in the WT and in DH5␣⌬baeSR.…”

“…DH5␣⌬baeSR with a pGEM vector without an insert was used as a negative control. The Stx2 phages used in this study differed genetically from each other, and some also had different insertion sites (14,20). Phage lysates containing 10 7 PFU/ml were used for at least three to five independent assays.…”