Cloning and Characterization of the recA Gene of Alkaliphilic Bacillus sp. C- 125
Hideto TAKAMI*, and Koki HORIKOSHI
The DEEPSTAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima, Yokosuka 237, Japan
Among alkaliphiles isolated so far, Bacillus sp. C-125 is best characterized strain physiologically and biochemically. The study on adaptation mechanism to alkaline environment was initiated in the early 1980s and molecular biological approaches have also started since 1988. Some genes which can recover alkaliphily in the alkali- sensitive mutants was isolated from the strain C-125 in 1990 (1). Although alkali- sensitive mutants recovered alkaliphily by introducing pALK 1 or pALK2 plasmid, it was found that this occurred by homologous recombination to the chromosome of the mutant. It seemed that the gene product encoded on the plasmid did not contribute directly for recovery of alkaliphily. In most prokaryotes, the recA gene encodes the major protein involved in homologous recombination whereas there is no information of the recA gene from alkaliphilic Bacillus strain. Actually, various recA-defective mutants of E. coli have been used for the genetic manipulation. Thus, it is clear that the recA-defective mutants of the strain C-125 is necessary for further molecular biological study on alkaliphily. In this study, we attempted to isolate and characterize the recA gene from alkaliphilic Bacillus sp. C-125. The recA gene from alkaliphilic Bacillus sp. C-125 has been isolated from a genomic library by hybridization with the Bacillus subtilis recA gene. The nucleotide sequence of the isolated gene consists of 1041 bp encoding a polypeptide of 349 amino acids. The putative RecA protein showed 81.3% and 64.8% amino acid sequence similarities to the RecA proteins from Bacillus subtilis and E. coli, respectively.
The construction of recA-defective mutant of alkaliphilic Bacillus sp. C-125 is being attempted by replacement of the active recA gene by an in vitro inactivated gene copy for further investigations.
1. Kudo, T., Hino, M., Kitada, M., and Horikoshi, K. (1990) J. Bacteriol., 172, 7282-7283.
