USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus

Abstract

Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.Vibrio vulnificus is a halophilic bacterium present naturally in estuarine waters and often contaminates oysters and other shellfish (for a review, see reference 15). V. vulnificus is an opportunistic pathogen of humans, causing primary septicemia and wound infection in susceptible individuals, and is the leading cause of reported seafood-related deaths in the United States. In susceptible humans, V. vulnificus causes a rapid, fulminating disease process resulting in extensive tissue damage. Mortality rates for susceptible individuals who develop fulminating primary septicemia are greater than 50% (17). Skin infections can lead to severe cellulitis, necrotizing fasciitis, and myositis requiring surgical debridement of infected tissues or amputation of the limb (4, 29, 42). Therapeutic intervention is often difficult since death can occur in less than 24 h after contact with the bacteria. In a mouse model of infection, V. vulnificus replicates extremely rapidly in host tissues (40, 41) and kills host cells including neutrophils (41).Over 20 years of genetic analysis, only a few virulence factors have been identified and confirmed by using the molecular version of Koch's postulates (15). Among the confirmed virulence factors are capsular polysaccharide (49), acquisition of iron (34, 52), type IV p...

The Biology of Vibrio vulnificus

“…How such diversity in a single species evolved is a fascinating question but may well have involved horizontal gene transfer that is ubiquitous among vibrios, including V. vulnificus (43,44).…”

Role of GacA in virulence of Vibrio vulnificus

“…Plates were spotted with 3 ml of either exponential or stationary phase culture and incubated at 30 or 37 uC. The diameter of cell spreading was measured as an indicator of motility after 13, 21 and 24 h. Flagellar morphology was examined using NanoOrange stain (Invitrogen) on bacteria recovered from 3 % TSA plates (Grossart et al, 2000), as modified by Gulig et al (2009).…”

Genetics of Natural Competence in Vibrio cholerae and other Vibrios

“…The V. harveyi-like Vibrio QS system elucidated primarily by Bassler and colleagues [see (78) for review] was exploited in 2002 to uncover a similar system in V. cholerae (79) described in detail below, followed by the discovery in 2005 that QS is required for natural competence (6). The V. cholerae competence regulation has been studied most extensively to date; however, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio fischeri are all QS-proficient and they are capable of taking up extracellular DNA via transformation (17,18,19). Features of each of these in the Vibrio QS system will be noted if they differ from V. cholerae.…”

“…Recent studies described here suggest this may be the case in V. cholerae as well. Since Meibom et al first documented natural competence in V. cholerae in 2005 (6), several additional Vibrio species have been shown to be capable of taking up extracellular DNA (17,18,19). It is likely that conditions under which other Vibrio species are also transformable remain to be identified.…”