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

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 pilus (37), RTX toxins (21, 25, 30), and flagella (22, 26). Despite these advances, the full spectrum of virulence factors responsible for the rapid and destructive disease process has not been elucidated. A major hindrance to the molecular genetic analysis of V. vulnificus is the fact that the bacteria cannot be effectively electroporated, nor can the bacteria be chemically transformed. Therefore, the only effective means of introducing plasmid DNA is by conjugation. This limitation severely restricts the availability of plasmids for creating and complementing mutations for molecular genetic analysis.A classical method to create mutants of V. vulnificus is the use of suicide vectors containing transposons, such as TnphoA (31), mini-Tn5phoA (12), mini-Tn5Km2 (12), mini-Tn5lacZ1 (12), mini-Tn10/kan (23), and Himar (1). However, transposon mutagenesis can cause polar mutations and truncated genes. The most definitive genetic analyses can be performed by deleting the genes of interest. The standard method for deleting genes is to clone the flanking upstream and downstream sequences into an allelic exchange suicide vector and introducing the plasmid into the target strain. The suicide vector integrates into the target genome via one of the flanking DNA sequences by a single-crossover event. This process is selected using antibiotic resistance encoded on the vector. The single-crossover strain is then grown without selection to allow for a second crossover that excises the allelic exchange vector. The excision event is enriched using a counterselectable marker encoded on the allelic exchange vector. A commonly used counterselectable marker is sucrose sensitivity, encoded by the sacB gene (13). If the second crossover is in the same flanking sequence as the first, the wild-type genotype is restored; however, if the second crossover is in the opposite flanking sequence, the targeted gene is deleted.This method of mutagenesis is problematic for V. vulnificus. First, one of the easiest ways to clone PCR amplicons is by TA topoisomerase-mediated ligation (TOPO TA cloning) with commercially available vectors such as pCR2.1 TOPO (Invitrogen, Carlsbad, CA). Unfortunately, these vectors are not mobilizable; hence, they cannot be used directly with V. vulnificus. Furthermore, there are no available TA topoisomerase allelic exchange vectors. Site-specific recombination vectors such as Gateway (Invitrogen) have facilitated PCR-mediated cloning and gene expression; however, none of the relevant vectors is mobilizable. Therefore, essentially all cloning for expression in or mutagenesis of V. vulnificus requires multiple subcloning steps. Second, sucrose sensitivity counterselection is not very efficient with V. vulnificus, as it is time-consuming and involves the screening of large numbers of colonies to find truly sucrose-resistant colonies. In some cases, sacB counterselection has completely failed. Another method for introducing mutations into target strains is the lambda phage red recombinase system (11). However, this procedure involves introducing additional helper plasmids into the target strain, and mobilizable helper plasmids have not been made. Finally, no generalized transducing phages and no methods for transformation have been described for V. vulnificus. Therefore, if one desires to move mutations between strains, the mutations must be cloned into suicide vectors, and the inefficient two-step allelic exchange process must be repeated for each strain.We describe here the adaption of USER (uracil-specific excision reagent) friendly cloning (3, 14) into allelic exchange and expression vectors that are commonly used with V. vulnificus. USER friendly cloning enables the creation of 3′ overhangs in PCR amplicons by use of deoxyuridine in PCR primers and treatment with USER enzyme mix (uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII). The overhangs are complementary to overhangs created in vectors. The method is very adaptable, with immense leeway in choosing the DNA sequences of the overhangs independent of restriction enzyme cleavage sites. USER friendly cloning methods and vectors enable the rapid creation of upstream-downstream clones to delete genes or loci of interest. To alleviate inefficiencies of subcloning into V. vulnificus vectors, we created an array of USER friendly cloning allelic exchange and expression vectors that should be useful to many investigators.Meibom et al. (32) recently described the ability of Vibrio cholerae to become naturally transformable during growth in the presence of chitin, as either chitohexose or crab shell fragments. We adapted their crab shell system and determined that V. vulnificus can also become naturally transformable during growth on chitin. The bacteria take up linear DNA; hence, as long as selectable markers are used, allelic exchange mutagenesis can be accomplished without the need for inefficient counterselection involving sucrose. We used these methods to create antibiotic resistance-marked deletions in V. vulnificus of the two loci encoding flagellins, flaCDE and flaFBA, and the genes encoding the following functions: the flagellar motor motAB; a critical element of signal transduction for chemotaxis, cheY-3; an RTX toxin, rtxA1; a putative RtxA-modifying enzyme, rtxC; and a regulator of fatty acid metabolism, fadR. These genetic tools and methods will facilitate the molecular genetic analysis of V. vulnificus as well as those of other gram-negative bacteria.