Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids

Abstract

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5 end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the "conserved" portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its "variable" portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this "conserved" cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry (7), yet the virulence mechanisms underlying APEC virulence are imperfectly understood. Recent studies have sought to define the APEC pathotype and have shown that several genes occur frequently among APEC, regardless of the avian host species or lesion of origin (12,36,37). Many of these genes that are characteristic of the APEC pathotype are linked to large plasmids, and many of these plasmids encode production of the bacteriocin ColV (9, 23, 36, 37), leading to their being termed ColV plasmids.ColV plasmids have long been recognized for their association with E. coli virulence (43, 51, 54). However, ColV production, the namesake trait of ColV plasmids, does not appear to contribute to the disease-causing abilities of E. coli (35), suggesting that other ColV plasmid-linked traits might be responsible for ColV plasmids' association with virulence. Indeed, we have recently completed the first sequence of a 180-kb ColV plasmid, revealing the presence of a 94-kb cluster of putative virulence genes (23). Based on studies of the prevalence of the genes of this putative virulence cluster among avian E. coli isolates, it appears that the cluster contains a "conserved" region, with its genes occurring in ...

Evolution of colicin BM plasmids: the loss of the colicin B activity gene

“…This suggests that in ColM plasmids the M operon has never been associated with colicin B genes. Among 1304 isolates examined in this study, no example of a strain with a colicin B operon in the absence of colicin M was observed, although Johnson et al (2006a) provided evidence that such strains do exist. However, without nucleotide sequence data for the B operon in colicin Balone strains, it is not possible to determine whether their colicin plasmids once encoded both colicin B and colicin M or whether they represent cases where the colicin M operon has never been present.…”

“…Many plasmids that encode multiple bacteriocins encode microcin V. Indeed, microcin V has been found together with a number of colicins known to be encoded on conjugative plasmids in E. coli: Ia, Ib, B and M . In a recent study, Johnson et al (2006a) compared the genomes of the microcin V plasmid pAPEC-O2-ColV and the colicin BM plasmid pAPEC-O1-ColBM. In fact, pAPEC-O1-ColBM actually represents a type I ColMBimm plasmid and was found to contain a putative virulence cluster (eitABCD, sitABCD, aerobactin, salmochelin, iss, tsh and etsABC) normally associated with ColV plasmids, as well as remnants of the microcin V operon.…”

“…Migration distances of DNA bands were measured directly from photographs of the gels. Colicin BM plasmids are known to be large (50-200 kb) (Johnson et al, 2006a), so when only one large plasmid was present in a wild-type strain it was assumed to be the colicin BM plasmid.…”

“…The entire colicin B and M gene region was sequenced for 60 E. coli strains selected to represent the diversity present among the strains encoding these genes. Primers for sequencing were designed as needed using published colicin B and M gene sequences (Johnson et al, 2006a;Köck et al, 1987;Ö lschläger & Braun, 1987;Schramm et al, 1987Schramm et al, , 1988 (Supplementary Table S1). DNA fragments were amplified using the PCR conditions described above, and PCR products were purified with the DNA purification reagent ExoSAP-IT (USB).…”

“…A colicin BM plasmid recently sequenced by Johnson et al (2006a) (pAPEC-O1-ColBM, accession no. DQ381420) was found to have a truncated colicin B activity gene like the colicin MBimm strains examined in this study.…”