Construction of chromosomal recA mutants of Pseudomonas putida PpG2

Abstract

The recA gene of Pseudomonas putida PpG2 was cloned by complementation of the recA mutations of Escherichia coli strains DH5 alpha and HB101. The nucleotide sequence of the DNA fragment was determined and shown to contain recA and a downstream partial open reading frame. Two mutants of P. putida PpG2, strains JS387 and JS388, were constructed by insertional inactivation of recA with a tetracycline-resistance gene in both orientations. Both mutants acquired sensitivity to methyl methanesulfonate (MMS) and both failed to undergo homologous recombination. While the recA mutation of P. putida JS388 was complemented in trans by recA of P. putida, the JS387 mutant was difficult to transform and transformants exhibited varying degrees of sensitivity to MMS. Therefore, P. putida JS388 can be used as a carrier of recombinant plasmids, but JS387 is not a suitable host for this purpose.