The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage

Abstract

Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.

FIG. 1.

Map of the chromosomal SopEΦ attachment site (attB), the circular replicative form of SopEΦ (harboring attP), and the left (attL) and right (attR) sites of the integrated SopEΦ prophage. Site-specific integration of the phage into the bacterial chromosome occurs at the core (“O”) part of attB (BOB′). The integration leads to the duplication of the core at either end of the prophage and to formation of the hybrid attL (BOP′) and attR (POB′) sites. att core sites are marked as gray boxes. Horizontal arrows indicate the positions of the primers used in this study.

FIG. 2.

Alignment of the subcloned fragment of serovar Typhimurium A36 with the attachment site from the cryptic E. coli prophage CP4-57 The proposed attB site of serovar Typhimurium A36 encodes a P4-like integrase (white box), the ssrA gene, and the 3′-terminal part of hlyD (55% identical to export protein HlyD from Xylella fastidiosa).

FIG. 3.

Plasmid preparation from serovar Typhimurium A36 (pM800) after infection with a SopEΦ^sopE::aphT lysate. (A) Plasmids of three independent clones (pM802A, -B, and -C) that were selected on LB plates supplemented with kanamycin and chloramphenicol were isolated. The larger size of pM802 plasmids suggested that SopEΦ^sopEΦ::aphT had integrated into the proposed attB site of pM800. (B) Schematic drawing of pM802. Dark gray, pM800 part; light gray, SopEΦ^sopE::aphT. att core sites are marked as gray boxes. Arrows indicate the positions of the primers used in this study.

FIG. 4.

Southern blot analysis of M106 and pM802. The chromosomal DNA of M106 (SopEΦ^sopE::aphT lysogen) and plasmid pM802 were digested with EcoRV (E) and HindIII (H) and hybridized with the entire pM802 plasmid as a probe. White dots indicate DNA fragments identical in size in M106 and pM802.

FIG. 5.

Alignment of the sequenced DNA fragments corresponding to the attR, attL, attB, and attP sites. Horizontal lines mark phage DNA, and vertical lines mark bacterial DNA. The black bar indicates the 47-bp core sequence present in all four att sites. The gray arrow marks the 3′ terminus of the ssrA gene. *, later, the attP site was confirmed by PCR and sequencing with primers 2R and 36F (see Fig. 6).

FIG. 6.

PCR amplification of the predicted att sites. Serovar Typhimurium A36 was infected with SopEΦ^sopE::aphT lysate, and lysogens were selected on kanamycin-containing LB plates. PCRs for the defined att sites were performed with primers 36R and 36F for attR, attBF and 1R for attL, and 48F and 36R for attB. attP was amplified from a plasmid preparation of mitomycin C-induced M824 with primers 36F and 2R. −, serovar Typhimurium A36; +, serovar Typhimurium A36 harboring SopEΦ^sopE::aphT (= M824).

FIG. 7.

Presence of the 47-bp attachment core site of SopEΦ in M824, serovar Typhi CT18, and serovar Typhimurium LT2. Two perfectly conserved 47-bp direct repeats flank the SopEΦ^sopE::aphT prophage in M824 and the Fels-2 prophage in serovar Typhimurium LT2. The serovar Typhi CT18 prophage at cs 93 has the 47-bp core sequence (with one mismatch) at its right end but only a remnant of 8 nt at its left end. The cryptic phage (CT18, cs 57) harbors the 47-bp sequence at its right border. The left part of this phage is absent. Whereas the right 47-bp direct repeat of SopEΦ, Fels-2, and the cryptic phage are part of the ssrA gene that is followed by hlyD, the direct repeat of the CT18 prophage at cs 93 is located in the samA gene.

FIG. 8.

Sequence comparison with attR and attL of the SopEΦ^sopE::aphT prophage in M824 and the Fels-2 prophage in the genome of serovar Typhimurium LT2. The chromosomal regions of M824 flanking the attR and attL core of the integrated SopEΦ^sopE::aphT prophage and serovar Typhimurium LT2 flanking the Fels-2 prophage are 98 and 100% identical. The right end of the SopEΦ prophage directly adjacent to the att core sequence is 98% identical between the two prophages. In contrast, the sequences at the left end of both prophages (phage part of attL) are entirely different. The values in the shaded areas indicate percent DNA sequence identity.

FIG. 9.

Alignment of genetic maps of SopEΦ with the P2 phage and prophages present in the sequenced genomes of serovar Typhi CT18 and serovar Typhimurium LT2. Boxes indicate the predicted ORFs starting with the phage integrase gene. Numbers in boxes refer to the chromosomal sequences of serovar Typhi CT18 and serovar Typhimurium LT2. (A) Alignment of SopEΦ (center), Fels-2 prophage (serovar Typhimurium LT2; bottom), and the P2 phage (accession no. NC_001895; top). For P2, gene functions are annotated. (B) Alignment of SopEΦ (center), the serovar Typhi CT18 prophage at cs 93 (top), the serovar Typhi CT18 prophage at cs 73, and the serovar Typhi CT18 incomplete prophage at cs 57 (bottom). The values in the shaded areas indicate percent amino acid sequence identity.