Blue-white selection of regulatory genes that affect the expression of dehalogenase IVa of Burkholderia cepacia MBA4

Abstract

We have developed a method for rapid screening of genes that affected the expression of dehalogenase IVa of Burkholderia cepacia MBA4. The promoter region of the dehalogenase gene was used to drive the expression of a beta-galactosidase gene. A plasmid containing this reporter was first electroporated into MBA4, and a Tn5 containing suicidal plasmid was introduced subsequently. The use of electroporation was necessary because Escherichia coli mediated transconjugation was ineffective in plasmid-carrying MBA4. The number of integrants generated was directly proportional to the amount of plasmid DNA used. Integrants with an elevated beta-galactosidase activity were isolated. Mutants with a disruption in a putative iron-transporter gene and in a putative response regulator receiver gene were identified. The basal dehalogenase transcript levels of these mutants were higher than the wild type. These mutants also grow faster than the wild type in chloroacetate-containing medium. This methodology of isolating regulatory mutants is theoretically feasible and convenient for any kinds of bacteria. © 2007 Springer-Verlag.