Elsevier

Gene

Volume 438, Issues 1–2, 1 June 2009, Pages 57-64
Gene

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

https://doi.org/10.1016/j.gene.2009.02.015Get rights and content

Abstract

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.

Introduction

The accumulation of an increasing number of sequenced genomes has opened a new field of biology, comparative genomics, while the development of efficient methods to analyze the functions of newly identified genes is also required. In Vibrio cholerae, the method traditionally used to inactivate chromosomal genes requires several DNA cloning steps into a suicide vector in Escherichia coli and subsequent conjugational transfer into V. cholerae (Miller and Mekalanos, 1988). Thus, this methodology limits large-scale mutagenesis in post-genomic studies. Recently, a simple mutagenesis method without DNA cloning was developed in E. coli (Datsenko and Wanner, 2000, Yu et al., 2000). The procedure involves the deletion of chromosomal genes via homologous recombination between the chromosomal region of interest and a polymerase chain reaction (PCR) product that contains an antibiotic resistance cassette flanked by sequences homologous to the target DNA. Linear DNA can be obtained in one-step PCR using primers that contain 36 to 50-nucleotide (nt) homologous extensions of the target gene. This method also utilizes the λ Red gam, bet, and exo gene products, which encode an efficient homologous recombination system. Gam protein is capable of inhibiting the Exonuclease V activity of RecBCD, allowing for the transformation of linear DNA (Unger et al., 1972). The bet and exo gene products are capable of promoting homologous recombination in short homologous regions between the PCR product and the chromosome (Unger and Clark, 1972). Furthermore, Datsenko and Wanner (2000) created antibiotic resistance cassettes flanked by FLP recognition target (FRT) sites for use in constructing λ Red-mediated insertions. These constructs allow subsequent removal of the antibiotic resistance cassette by FLP recombinase produced from a conditionally replicating plasmid.

The λ Red-based technology developed in E. coli has been successfully used for other Gram-negative bacteria, including Salmonella (Husseiny and Hensel, 2005), Yersinia (Derbise et al., 2003), Shigella (Beloin and Dorman, 2003), Serratia (Rossi et al., 2003), Klebsiella (Janes et al., 2001) and Pseudomonas (Lesic and Rahme, 2008), but alterations are occasionally required (Derbise et al., 2003, Lesic and Rahme, 2008). For example, in Y. pseudotuberculosis, 55-nt homology extensions were generally not sufficient to allow recombination, while PCR products with extensions of approximately 500-nt, generated using a three-step PCR procedure, could promote gene disruption (Derbise et al., 2003).

Here we describe the application of the λ Red-based method to inactivate V. cholerae genes, in which chromosomal single gene deletions were generated using a one-step PCR product containing 100-nt flanking regions homologous to the target sequence. We further examined the effectiveness of using a two-step PCR product with 1000-nt homology extensions to obtain mutants. Finally, we applied this method to create a lacZ reporter fusion gene on the V. cholerae chromosome.

Section snippets

Bacterial strains, plasmids, media, and DNA manipulation

V. cholerae strains N16961, V080085, V080080, their constructed mutants, and the plasmids used in this study are listed in Table 1. The bacteria were routinely grown in Luria-Bertarni (LB) broth and agar, and SOC as described previously (Sambrook et al., 1989), and for studies of the expression of cholera toxin in AKI medium without NaHCO3, as reported previously (Iwanaga et al., 1986). The antibiotic concentrations were 100 μg/ml ampicillin (Ap) and 1 μg/ml chloramphenicol (Cm). l-arabinose

λ Red recombination using PCR product with short homologous extensions to the target gene

In E. coli, λ Red-mediated recombination occurs efficiently when a PCR product with a short homologous extension (approximately 50 nt) to a target gene (Datsenko and Wanner, 2000, Yu et al., 2000) is used as the donor DNA; however, this is not always the case in other bacteria (Derbise et al., 2003, Lesic and Rahme, 2008). For example, in Y. pseudotuberculosis, PCR products with relatively long extensions (approximately 500-nt) were required for gene disruption by λ Red-mediated recombination (

Acknowledgments

We thank Sunao Iyoda, Jiro Mitobe, and Victor J. DiRita for their helpful suggestions. This work was partly supported by grants-in-aid from the Ministry of Health, Labour, and Welfare of Japan (H18-Shokuhin-Ippan-003, H20-Shinko-Ippan-013, H20-Shinko-Ippan-015, and International Health Cooperation Research 18C-5).

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