Structural Analysis of the Gene Region Encoding the Replication Origin of an Alkaliphilic Bacillus sp. Strain C-125
Noriaki MASUI*, Fumie FUJI, Chie HIRAMA, Kaoru NAKASONE, Yuka NAKAMURA, Yoshihiro TAKAKI, Hideto TAKAMI, Akira INOUE, and Koki HORIKOSHI
a The DEEPSTAR group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho,Yokosuka 237, Japan
The alkaliphilic Bacillus sp. strain C-125 can grow in an alkaline environment. To understand how this alkaliphile is adapted to grow at high pH, analysis using both physiological and molecular biological approaches have been performed (1).
Recently we started another approach, genome analysis, which can obtain whole genetic information to aid understanding of alkaliphily. Furthermore, as a part of genome analysis, we are interested in the replication origin of alkaliphiles. The replication origin region of the bacterial chromosome is essential for DNA replication. For cell division to occur in an alkaline environment, the alkaliphile may not only need the conserved mechanism of DNA replication in bacteria, but also a specific mechanism only present in alkaliphiles. Therefore, analysis of this region in alkaliphiles is of considerabe importance. In this conference, we report molecular cloning and mapping of the replication origin from an alkaliphilic Bacillus sp. strain C-125 and its comparative analysis.
From the gyrB sequence of this strain, a 9.8 kb DNA fragment encoding the gyrB gene was cloned by inverse PCR and sequenced. This 9.8 kb DNA fragment contained seven open reading frames (ORF's) which were identified as dnaN, recF, gyrB, gyrA and three functionally unknown ORFs (ORF 1, 2 and 3), by database using the BLAST program. Comparison of amino acid sequences of dnaN, recE gyrB, gyrA and the unknown ORF3 ( situated upstream of the recF gene ) showed the highest homology to those of Bacillus subtilis. Especially, recE gyrB and gyrA genes showed 65%, 78% and 76% homologies, respectively. These results suggest that C-125 is closely related to B. subtilis. The genetic organization of B. subtilis replication origin region is ordered: (including functionally unknown ORFs (ORF1 and ORF2)) dnaA-dnaN-ORFl-recF-ORF2-gyrB-gyrA-rrnO (2). The cloned fragment of C-125 was ordered: (including functionally unknown ORFs (ORF1, 2 and 3)) dnaN-ORFl-recF-ORF2-gyrB-gyrA-ORF3. In B. subtilis, rrnO which is one of the rRNA operons was encoded downstream of the gyrA gene. In C-125, a functionally unknown ORF5 is encoded and the rrnO gene was not identified downstream of ORF5. This result shows that genetic organization of the alkaliphilic Bacillus sp. strain C-125 is at least different from that of B. subtilis downstream of the gyrA gene. This difference is possibly related to alkaliphily. dnaA, essential for initiation of chromosomal replication was not contained in the DNA fragment we cloned in this study. Further, cloning of DNA fragments containing the dnaA gene and the downstream region of gyrA are now in progress.
1. Horikoshi, K., Microorganisms in Alkaline Environments, VCH Verlagsgenellschaft, Weinheim, Germany.
2. Ogasawara, N., Nakai, S., and Yoshikawa, H. (1994) DNA Research, 1, 1-14.
