Genomic and functional analyses of SXT, an integrating antibiotic resistance gene transfer element derived from Vibrio cholerae

Abstract

SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian Vibrio cholerae. We have determined the 100-kb DNA sequence of SXT. This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources. We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision. SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid. More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility. Two SXT loci, designated setC and setD, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer. Another locus, designated setR, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.

FIG.1.

Genetic organization and ORF map of SXT. Putative ORFs are depicted by arrows showing the orientations. Colors are used to indicate similarity or function as follows: transposase (yellow), antibiotic resistance (blue), conjugative transfer (red), and regulation (purple). A hatched box indicates the position of the origin of transfer. ORFs of known or unknown function whose best hits by BLAST are found in phage genomes are colored green. The colored lines beneath the map indicate the locations of deletions discussed in Table 3: gray indicates the deletion had a wild-type phenotype, purple indicates mobilization of CloDF13 but no transfer of SXT, red indicates no or greatly reduced transfer of SXT or CloDF13, and green indicates no transfer of SXT or CloDF13 and no excision.