Structural Analysis of Alpha Subunits from an Alkaliphilic Bacterium, Bacillus Sp. C- 125
Yoshihiro TAKAKI*, Noriaki MASUI, Yuka NAKAMURA, Chie HIRAMA, Fumie FUJI, Kaoru NAKASONE, 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 alkaliphilic Bacillus Sp. C-125 is able to grow well at alkaline pH. The mechanism which prescribe the range has not been perfectly clarified, although both its physiology and biochemistry have been studied (1). In order to adapt to this environment, this bacterium might have evolved sophisticated systems which can sense changes in external pH, and then respond by turning on and off specific sets of genes. In controlling gene expression, the RNA polymerase is considered to play a key role by rapidly modulating its promoter selectivity. Here we turn our attention to structural analysis and characterization of the genes encoding components of the RNA polymerase of this alkaliphilie. In this conference, we report cloning, characterization, and organization of the rpoA gene encoding the RNA polymeraseα subunit in C-125.
Utilizing synthetic degenerate oligonucleotide primers to a conserved region of both B. subtilis (2) and Streptomyces coelicolor (3) RNA polymeraseα subunits, a partial DNA fragment encoding the gene was cloned and sequenced. Southern hybridization of the chromosomal DNA digested by HindIII was carried out by using this PCR product as a probe, showing one hybridized band of 3 kb. This DNA fragment was cloned by inverse PCR using oligonucleotide primers synthesized from the partial rpoA DNA fragment and then sequenced. Identification of an open reading frame predicted by the sequence data was done with the database using the BLAST program.
Six open reading frames were identified in this fragment, their translated amino acid sequences showed the highest homologies to their counterparts in B. subtilis. Genetic organization was the same as that of B. subtilis, which was infA-rpmJ-rpsM- rpsK-rpoA-rplQ. In addition, this gene cluster was used as a gentic marker for location on a physical map of the C-125 chromosome by southern hybridization using AscI-liking clones. Theα subunit gene encoded a protein of 314 amino acids with a deduced molecular mass of 34,805 Da. Compared with amino acid sequences of other known bacterial tz subunits, the gene has homology of 84% to that of the B. subtilisα subunit, whereas S. coelicolor and Escherichia coli (4) showed 47% and 47% homologies, respectively. These results suggest that C-125 is closely related to B. subtilis. Further, several conserved domains in the C-125 α subunit were observed in
