Physical Map of the Genome of the Alkaliphilic Bacillus sp. Strain C-125
Kaoru NAKASONE*, Fumie FUJI, Chie HIRAMA, Noriaki MASUI, Yuka NAKAMURA, Yoshihiro TAKAKI, Hideto TAKAMI, Akira INOUE, and Koki HORIKOSHI
The DEEP STAR group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka 237, Japan
The facultative alkaliphilic Bacillus sp. strain C-125 grows between pH 6.8 and 10.8. To understand how the organism is adapted to live or survive at high pH, analyses by physiological and molecular biological approaches have been performed. Recently we have started another approach, genome analysis of this strain that can obtain whole genetic information which not only enable us to understand the alkaliphily, but also to obtain new useful genes. Therefore, as a first step, construction of a physical map of the alkaliphile is essential. In this conference, we report the construction of a physical map of the alkaliphilic Bacillus sp. strain C-125 and demonstrate that it will help fine-mapping strategies for this organism.
The bacterium was grown at 37℃ in Horikoshi-II medium and intact chromosomal DNA was prepared in agarose plugs after harvesting the cells. Analysis of the genome using several restriction endonucleases showed that rare-cut restriction endonucleases, AscI (5'GG/CGCGCC) and Sse83871 (5'CCTGCA/GG), digest the chromosomal DNA into a relatively small number of large fragments. They could be resolved by PFGE and these enzymes are, therefore, the most appropriate for the construction of a physical map. The pulsed-field electrophoretic analysis shows that both AscI and Sse83871 digestions yielded 20 resolvable restriction fragments ranging from 600 kb to 20 kb. In order to isolate linking clones, AscI- and Sse83871-linking clone libraries using HindIII or EcoRI were constructed. These libraries were screened to identify genuine linker clones that linked separate AscI and Sse83871, and were used as probes in hybridization experiments with PFGE-separated chromosomal restriction fragments produced by these enzymes to determine the linkages between the different fragments. The genome size of the strain was estimated at 4.2 Mb by adding together the sizes of the fragments from AscI and Sse83871 digests, taking mean values from at least three independent PFGE runs utilizing different electrophoretic conditions. Using several genetic markers such as rpoA, rpoB, rpoC, sigA, gyrA and gyrB, the position of several genetic loci were localized on the physical map.
