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.

FIG. 1.

Motilities of Δfla, ΔmotAB, and ΔcheY-3 mutants. V. vulnificus strains were stabbed into soft LB-N agar, and the diameters of growth from motility after overnight growth were measured as described in Materials and Methods. Strains examined were wild-type strain CMCP6, ΔflaFBA::cat strain FLA680, ΔflaCDE::aph strain FLA677, ΔflaCDE::aph ΔflaFBA::cat strain FLA711, ΔmotAB::aph strain FLA674, and ΔcheY-3::aph strain FLA688. *, P ≤ 1 × 10⁻⁵ compared with CMCP6 and FLA680 and P = 0.007 compared with FLA677; **, P ≤ 0.0002 compared with CMCP6 and FLA680 (n = 3).

FIG. 2.

Flagellation of Δfla, ΔmotAB, and ΔcheY-3 mutants. Bacteria from LB-N soft-agar plates were stained with Nano Orange and examined using epifluorescence at a ×1,000 magnification as described in Materials and Methods. (A) Wild-type CMCP6; (B) ΔflaFBA::cat strain FLA680; (C) ΔflaCDE::aph strain FLA677; (D) ΔflaCDE::aph ΔflaFBA::cat strain FLA810; (E) ΔmotAB::aph strain FLA674; (F) ΔcheY-3::aph strain FLA688.

FIG. 3.

PCR confirmation of the deletion of 15.6-kb rtxA1 and creation of ΔrtxA1::aph. Genomic DNAs from V. vulnificus CMCP6 (lane A) and ΔrtxA1::aph strain FLA900 (lane B) were PCR amplified using oligonucleotide primers rtxA1-deletion-up and rtxA1-deletion-down, which amplify 1 kb upstream and downstream of the start and stop codons of rtxA1. The sizes of the products are shown to the left, based on the 1-kb ladder (lane C) and lambda phage-HindIII digestion (lane D). Southern blot analysis also confirmed the absence of rtxA1 sequences (data not shown).

FIG. 4.

Chitin transformation of wza::TnphoA from V. vulnificus CVD752 into CMCP6 yields a translucent-colony morphotype. Genomic DNA from CVD752 (M06-24/O wza::TnphoA) was chitin transformed into CMCP6, selecting for kanamycin resistance and yielding FLA1009. Kanamycin-resistant colonies acquired the translucent-colony morphotype (B and D). (A) M06-24/O; (B) CVD752; (C) CMCP6; (D) FLA1009.