Application of λ Red recombination system to Vibrio cholerae genetics: Simple methods for inactivation and modification of chromosomal genes

The λ Red-based recombination system is very useful for genetic manipulation of some Gram-negative bacteria. Here we report simple procedures for the inactivation and modification of genes of interest on Vibrio cholerae chromosome using this recombination technique. For this purpose, a polymerase chain reaction (PCR) fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus was electroporated into recipient V. cholerae strains expressing a highly proficient λ Red recombination system. Two PCR procedures were tested to generate an amplification product carrying an antibiotic resistance cassette flanked by short (50 or 100 nt) or long (1000 nt) homologous extensions, which allowed successful disruption of four chromosomal loci (ctxB, toxT, lacZ, and recA). Our results suggest that 100-nt homology between the PCR product and the target gene is sufficient to stimulate the λ Red-dependent recombination. To increase recombination efficiency, however, the PCR procedure should be used to generate a product with 1000-nt homologous extensions. Furthermore, we applied this gene replacement method to create lacZ reporter fusion to the target gene. Transcriptional fusion to the V. cholerae ctxA gene was constructed using a PCR product that contains the 100-nt homologous extension to ctxA on each side of the lacZ::cat cassette, and was shown to respond appropriately to a null mutation in the regulatory gene, toxT. Use of the techniques presented here should prompt rapid and efficient mutagenesis/modification of V. cholerae chromosomal genes.