Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotactic um, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino a cid residues essential for function of the recombinase, protease, and ATPas e in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid res idues believed to be the ATP binding domain of E. coli RecA, 17 are found t o be identical in G. oxydans RecA. Interestingly, nucleotide sequence align ment between the SOS box of G. orphans recA gene and those from different m icroorganisms revealed that all the DNA sequences examined have dyad symmet ry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such re placement of the wild-type recA with a kanamycin resistance gene in the chr omosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation th an the wild-type strain, suggesting that the recA gene of G. oxydans ATCC93 24 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strai n, illustrating that G. oxydans LCC96 could be used as a host to take up th e recombinant plasmid for gene manipulation.