Site‐specific integration of the conjugal Vibrio cholerae SXT element into prfC

Abstract

SummaryVibrio cholerae O139, the ®rst non-O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to the antibiotics sulphamethoxazole, trimethoprim, chloramphenicol and streptomycin. Resistances to these antibiotics are encoded by a 62 kb self-transmissible, conjugative, chromosomally integrating element designated the`SXT element'. We found that the SXT element integrates site speci®cally into both V. cholerae and Escherichia coli K-12 into the 58 end of prfC, the gene encoding peptide chain release factor 3. Integration of the SXT element interrupts the chromosomal prfC gene, but the element encodes a new 58 end of prfC that restores the reading frame of this gene. The recombinant prfC allele created upon element integration is functional. The integration and excision mechanism of the SXT element shares many features with site-speci®c recombination found in lambdoid phages. First, like l, the SXT element forms a circular extrachromosomal intermediate through speci®c recombination of the left and right ends of the integrated element. Second, chromosomal integration of the element occurs via site-speci®c recombination in a 17 bp sequence found in the circular form of the SXT element and a similar 17 bp sequence in prfC. Third, both chromosomal integration and excision of the SXT element were found to require an element-encoded int gene with strong similarities to the l integrase family. Based on the properties of the SXT element, we propose to classify this element as a CONSTIN, an acronym for a conjugative, self-transmissible, integrating element.

Multiplex PCR for detection of antibiotic resistance genes and the SXT element: application in the characterization of Vibrio cholerae

“…In contrast to El Tor strains isolated before the O139 outbreak, the re-emerged El Tor strains, like the initial O139 isolates, were resistant to Fr, Sul, Tmp, Cm and Str (Yamamoto et al, 1994). The corresponding genes were found to be located in an integrating conjugative element that was closely related but not identical to the O139 SXT element (Hochhut & Waldor, 1999;Waldor et al, 1996). Variations were noted in recent V. cholerae O139 isolates from India, i.e.…”

Subtractive hybridization reveals a high genetic diversity in the fish pathogen Photobacterium damselae subsp. piscicida: evidence of a SXT-like element

“…The ligation reactions were used as the DNA template in PCR reactions with M13fw primer (targeted to the pUC118 polylinker) and either primer P4 or P5, which are targeted to the left (attL) and right (attR) junctions of the putative SXT-like element, respectively (Hochhut & Waldor, 1999). The resulting PCR products were cloned into pGEM-T Easy (Promega) and sequenced.…”

“…Chromosomal integration of SXT-like conjugative transposons occurs via site-specific recombination between a 17 bp sequence found in the SXT element and a similar 17 bp sequence in the prfC gene that encodes the peptide chain release factor 3 (Hochhut & Waldor, 1999;McGrath & Pembroke, 2004). Recombination between the chromosomal attB and the SXT attP generates the left and right junctions attL and attR, respectively.…”

“…Recombination between the chromosomal attB and the SXT attP generates the left and right junctions attL and attR, respectively. The integration site in the chromosome of strain PC554.2 was assessed using primers targeted to the left and right junctions of this element within the bacterial chromosome (Hochhut & Waldor, 1999;Ahmed et al, 2005). PCR products were obtained that included the attL and attR junctions as well as neighbour chromosomal DNA.…”

Molecular analysis of an integrative conjugative element, ICEH, present in the chromosome of different strains of Mycoplasma hyopneumoniae

“…A number of phenotypes has been associated with this type of dissemination during conjugation of chromosomal regions: the sucrose metabolism in Salmonella enterica Seftemberg via cTnscr94 (Hochhut et al, 1997); the symbiosis traits associated with the 500 kb symbiosis island in Mesorhizobium loti (Sullivan and Ronson, 1998); the degradation of chlorocatechol mediated by gene products resident on the 105 kb clc element of Pseudomonas putida (Ravatn et al, 1998); the tetracycline resistant element Tn916 of Bacteroides (Salyers et al, 1995); the large STX unit of Vibrio cholerae (Hochhut and Waldor, 1999); and the antibiotic resistance gene cluster from Salmonella (Doublet et al, 2005). The term constin has been recently coined to describe this diverse group of conjugative, selftransmissible, integrating elements (Hochhut et al, 2001).…”