A globally distributed mobile genetic element inhibits natural transformation of Vibrio cholerae

Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA⁺ ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to ∼40% from 2006 to 2011. Thus, IdeA⁺ ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.The causative agent of the diarrheal disease cholera, Vibrio cholerae, is annually responsible for 3.5 million infections worldwide (1). This facultative pathogen naturally resides in temperate aquatic environments and causes disease when ingested in contaminated food or water. A critical nutrient for Vibrio species in the aquatic environment is the chitinous exoskeleton of crustacean zooplankton (2–4). Chitin is an insoluble polysaccharide composed of β-1,4-linked GlcNAc. In addition to serving as a carbon and nitrogen source, chitin also induces a physiological state in V. cholerae known as natural competence (5). In this state, bacteria can take up DNA from the extracellular environment and integrate this DNA into their chromosomes by homologous recombination. This cumulative process of DNA uptake and integration is known as natural transformation and is one mechanism for horizontal gene transfer (HGT) in V. cholerae. HGT by natural transformation is used by pathogenic microbes to evolve in the face of clinical intervention and immune pressure. Indeed, in V. cholerae, this mechanism of HGT is hypothesized to have generated an antigenic variant, the O139 outbreak strain, through homologous recombination and replacement of the locus responsible for O-antigen biosynthesis (6–9).Another mechanism of HGT in V. cholerae is integrating conjugative elements (ICEs) of the SXT/R391 family. These elements can range from ∼80 to 110 Kb in size and contain all of the genes required for conjugative transfer into naive hosts (10, 11); they integrate in a site-specific manner into the 5′ end of the highly conserved prfC (peptide-chain-release factor C) gene (10–12). The first natural transfer of an ICE into V. cholerae likely occurred between 1980 and 1985 (10, 13) and, by the 1990s, virtually all clinical isolates of V. cholerae contained an ICE (13). These elements confer resistance to multiple antibiotics, and it is likely that widespread use of antibiotics has rapidly selected for strains containing ICEs. There are at least 10 genetically distinct ICEs circulating in the V. cholerae population (11). These ICEs share a core set of genes, but have varied gene content at distinct sites. The most common ICE in V. cholerae is VchInd5, which is present in ∼77% of currently sequenced clinical isolates (10, 11). It is hypothesized that the current (seventh) pandemic of cholera originated in the Bay of Bengal, and strains have spread globally from this region in three overlapping waves of transmission (13, 14). Strains containing VchInd5 are globally distributed, indicating that the original transfer of VchInd5 into V. cholerae may have occurred in this region.In 2010, cholera spread to Haiti, a region that previously lacked this disease (15, 16). Phylogenetic and Bayesian analyses indicate that all strains in Haiti share a common ancestor, which was introduced into the region at the outset of the epidemic (16, 17). Consistent with this finding, strains from Haiti ubiquitously harbor a VchInd5 ICE. Throughout the epidemic, strains have acquired mutations that are likely generated intrinsically, and there is no evidence of horizontal gene transfer among these isolates (16). Consistent with this finding, strains from the Haiti outbreak were found to be poorly transformed by chitin-induced natural competence (16).In this study, we identify and characterize an ICE-encoded DNase present on VchInd5 that inhibits HGT by natural transformation in V. cholerae. We also assess the role and prevalence of this DNase in limiting transformation among clinical isolates from Haiti and Bangladesh.