Genomic and functional analysis of ICEPdaSpa1, a fish-pathogen-derived SXT-related integrating conjugative element that can mobilize a virulence plasmid

Abstract

Integrating conjugative elements (ICEs) are self-transmissible mobile elements that transfer between bacteria via conjugation and integrate into the host chromosome. SXT and related ICEs became prevalent in Asian Vibrio cholerae populations in the 1990s and play an important role in the dissemination of antibiotic resistance genes in V. cholerae. Here, we carried out genomic and functional analyses of ICEPdaSpa1, an SXT-related ICE derived from a Spanish isolate of Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis. The approximately 102-kb DNA sequence of ICEPdaSpa1 shows nearly 97% DNA sequence identity to SXT in genes that encode essential ICE functions, including integration and excision, conjugal transfer, and regulation. However, approximately 25 kb of ICEPdaSpa1 DNA, including a tetracycline resistance locus, is not present in SXT. Most ICEPdaSpa1-specific DNA is inserted at loci where other SXT-related ICEs harbor element-specific DNA. ICEPdaSpa1 excises itself from the chromosome and is transmissible to other Photobacterium strains, as well as to Escherichia coli, in which it integrates into prfC. Interestingly, the P. damselae virulence plasmid pPHDP10 could be mobilized from E. coli in an ICEPdaSpa1-dependent fashion via the formation of a cointegrate between pPHDP10 and ICEPdaSpa1. pPHDP10-Cm integrated into ICEPdaSpa1 in a non-site-specific fashion independently of RecA. The ICEPdaSpa1::pPHDP10 cointegrates were stable, and markers from both elements became transmissible at frequencies similar to those observed for the transfer of ICEPdaSpa1 alone. Our findings reveal the plasticity of ICE genomes and demonstrate that ICEs can enable virulence gene transfer.

FIG. 1.

Schematic representation of portions of the ICEPdaSpa1, SXT^MO10, and R391 genomes. Conserved genes are shown as gray arrows, and DNA initially identified in ICEPdaSpa1, SXT^MO10, or R391 is shown in green, blue, or orange, respectively. The numbered yellow stars represent the sites of hot spots 1 to 4. Note that the scales used to represent the conserved and the nonconserved genes are different and that some conserved genes have been left out of the figure (as have regions without genes). The ICEPdaSpa1 and SXT^MO10 insertions in rumB are shown in more detail in Fig. 2. The left border of hot spot 3 in ICEPdaSpa1 begins in the 3′ end of s072, as s073 is absent from this ICE. In R391 and ICEPdaSpa1, s035 is split by insertion sequence elements into two ORFs, noted as s035′ and ′s035. The striped sections in s035 and ′s035 correspond to an ICE-specific variable region.

FIG. 2.

Comparison of the rumB regions in ICEPdaSpa1, SXT^MO10, and R391.

FIG. 3.

DNA sequences found at the boundaries of the four hot spots in ICEPdaSpa1, SXT_MO10, and R391. Conserved DNA is shown in gray, ICEPdaSpa1-specific DNA is shown in green, SXT-specific DNA is shown in blue, and R391-specific DNA is shown in orange. Nucleotide differences in the conserved DNA are indicated in black. The box represents the stop codon in traA.

FIG. 4.

Southern blot analysis of chromosomal DNA isolated from four independently derived Cm^r Tc^r Kn^r exconjugants from CCW088 × CAG18420 matings by using pPHDP10-Cm DNA as a probe. Lane 1, SacI-digested pPHDP10-Cm (the U refers to undigested plasmid DNA); lanes 2 to 5, SacI-digested DNA from four Cm^r Tc^r Kn^r exconjugants. The band patterns observed for the four exconjugants reveal that the pPHDP10-Cm integration site in each exconjugant differs from those in the other exconjugants; the J's mark junction fragments. Numbers at the left are molecular weight markers.