Abstract
Toxigenic Vibrio cholerae strains, including strains in serogroups O1 and O139 associated with the clinical disease cholera, are ubiquitous in aquatic reservoirs, including fresh, estuarine, and marine environments. Humans acquire cholera by consuming water and/or food contaminated with the microorganism. The genome of toxigenic V. cholerae harbors a cholera-toxin producing prophage (CT-prophage) encoding genes that promote expression of cholera toxin. The CT-prophage in V. cholerae is flanked by two satellite prophages, RS1 and TLC. Using cell surface appendages (TCP and/or MSHA pili), V. cholerae can sequentially acquire TLC, RS1, and CTX phages by transduction; the genome of each of these phages ultimately integrates into V. cholerae’s genome in a site-specific manner. Here, we showed that a non-toxigenic V. cholerae O1 biotype El Tor strain, lacking the entire RS1-CTX-TLC prophage complex (designated as RCT: R for RS1, C for CTX and T for TLC prophage, respectively), was able to acquire RCT from donor genomic DNA (gDNA) of a wild-type V. cholerae strain (E7946) via chitin-induced transformation. Moreover, we demonstrated that a chitin-induced transformant (designated as AAS111) harboring RCT was capable of producing cholera toxin. We also showed that recA, rather than xerC and xerD recombinases, promoted the acquisition of RCT from donor gDNA by the recipient non-toxigenic V. cholerae strain. Our data document the existence of an alternative pathway by which a non-toxigenic V. cholerae O1 strain can transform to a toxigenic strain by using chitin induction. As chitin is an abundant natural carbon source in aquatic reservoirs where V. cholerae is present, chitin-induced transformation may be an important driver in the emergence of new toxigenic V. cholerae strains.
Highlights
Cholera, an ancient diarrheal disease characterized by profuse secretory diarrhea and caused by toxigenic strains of Vibrio cholerae, is a major public health concern in developing and poor countries lacking safe drinking water, sanitation and hygiene (Barua, 1992; Kaper et al, 1995; Faruque et al, 1998)
For chitin-induced transformation assay, we extracted and purified genomic DNA from donor AAS56 strain (Table 1), and 2 μg of the gDNA was added to the recipient strain (AAS35) grown in IOW supplemented with chitin
To determine if AAS93 (Env-9 rifR RCT orfU::specR) harboring the RCT prophages can produce cholera toxin, we first created an intact CTX prophage in that strain by replacing specR with the wild-type orfU gene using co-transformation, as described in methods section. This genetic manipulation resulted in the creation of a strain designated as AAS111 (Env-9 rifR RCT xerC::kanR) that retained the intact RCT prophages, including the wild-type orfU gene
Summary
An ancient diarrheal disease characterized by profuse secretory diarrhea and caused by toxigenic strains of Vibrio cholerae, is a major public health concern in developing and poor countries lacking safe drinking water, sanitation and hygiene (Barua, 1992; Kaper et al, 1995; Faruque et al, 1998). Cholera toxin (CT) is encoded by cholera toxin genes that are harbored by a filamentous phage (CTXφ) integrated as a CTX prophage into the genome of toxigenic strains of V. cholerae (Waldor and Mekalanos, 1996). In addition to being chromosomally integrated prophages, RS1, CTX, and TLC can form a replicative form (RF) (Waldor and Mekalanos, 1996; Faruque et al, 2002; Hassan et al, 2010). Dif site required for the successful dimer resolution of V. cholerae chromosome, is flanked by sequences that serve as binding sites for XerC and XerD recombinases (McLeod and Waldor, 2004; Faruque and Mekalanos, 2012)
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