Molecular characterization ofGluconobacter oxydans recAgene and its inhibitory effect on the function of the host wild-typerecAgene

Abstract

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.

Sequence analysis of theGluconobacter oxydansRecA protein and construction of arecA-deficient mutant

“…Its coding region has 1032 base pairs, specifying 344 amino acids. The deduced protein has a molecular mass of 37.84 kDa, which is consistent with the experiment of in vitro protein synthesis previously documented (Liu et al 1998). Interestingly, in the 5′ non-coding region, an E. coli-like lexA repressor-binding site (so-called SOS box) was found in the promoter region of the G. oxydans recA gene (Liu et al 1998).…”

“…The deduced protein has a molecular mass of 37.84 kDa, which is consistent with the experiment of in vitro protein synthesis previously documented (Liu et al 1998). Interestingly, in the 5′ non-coding region, an E. coli-like lexA repressor-binding site (so-called SOS box) was found in the promoter region of the G. oxydans recA gene (Liu et al 1998). Since the SOS box was reported to be a LexA binding site (Ball et al 1990), the presence of the SOS box in the regulatory region of G. oxydans recA gene strongly suggests that a similar LexAmediated regulation system for recA gene expression may exist in G. oxydans.…”

“…Previously, we have isolated and characterized the recA gene of G. oxydans ATCC 9324 (Liu et al 1998). We report here the full-length homology of amino acid sequences between the G. oxydans RecA and those from other bacteria and show that the residues essential for the function of recombinase, protease, and ATPase of the E. coli RecA are conserved in G. oxydans.…”

“…The nucleotide sequence of the G. oxydans recA gene has been determined in our previous works and deposited in the GenBank Nucleotide Sequence Data Library under accession number U21001 (Liu et al 1998). Its coding region has 1032 base pairs, specifying 344 amino acids.…”

“…Conserved amino acids are shaded. Sequences are from the following sources: Ec, E. coli (Sancar et al 1980), Am, A. magnetotacticum (Berson et al 1990), Pa, P. aeruginosa (Sano et al 1987), and Go, G. oxydans (Liu et al 1998). The number at the end of each line indicates the number of amino acid residues in that portion of the protein.…”