Molecular Biology of Triangular Disc-shaped Extremely Halophilic Archaeon Haloarcula japonica
Satoshi Nakamura and Rie Yatsunami
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
A predominantly triangular disc-shaped extremely halophilic microorganism, strain TR-1, has been isolated from a saltern soil located at Noto Peninsula in Japan (see review 1 )). Extremely halophilic microorganisms usually belong to the domain "archaea". The taxonomical characteristics of the strain led us to propose a new species Haloarcula japonica. The Ha. japonica cells have been shown to divide asymmetrically. Here, we present systematically the current results of our molecular biological studies on Ha. japonica.
The shape-maintaining mechanism and the cell division process of Ha. japonica attracted our attention because of its characteristic morphology and asymmetric cell division. We have demonstrated the occurrence of a glycoprotein on the cell surface of Ha. japonica. The cell surface glycoprotein (CSG) seemed to be important in maintaining the characteristic shape of Ha. japonica2). The gene encoding the Ha. japonica CSG has been cloned and the primary structure of the CSG has been deduced, as well as potential N-glycosylation sites3). The amino acid sequence of the Ha. japonica CSG showed 52 and 43% identities with those from rod-shaped Halobacterium salinarum and pleomorphic disc-shaped Haloferax volcanii, respectively. FtsZ is a tubulin-like protein that is essential for cell division in bacteria. Recently, an ftsZ homologue was cloned from Ha. japonica and the gene was successfully expressed in Escherichia coli. Preparation of antibodies against the recombinant Ha. japonica FtsZ is now underway to visualize FtsZ rings of Ha. japonica cells by immunostaining technique.
Bacteriorhodopsin (bR, a proton pump) and halorhodopsin (hR, an anion pump) are light-driven ion pumps and required for photoenergy conversion coupled with ATP synthase in Hb. salinarum. Several new members of ion pumps have been reported in the last decade. We have cloned the genes encoding bR-like4) and hR-like proteins from Ha. japonica. Sequence analyses revealed that the bR-like protein belonged to the cruxrhodopsin (cR) family and that the hR-like protein did to the cruxhalorhodopsin (chR) family. Northern analyses indicated that transcription of the cR gene was induced by high light and low oxygen tension5), while the chR gene was inducible by high light. Furthermore, the possible A1-ATP synthase gene was found just upstream of the cR gene6).
Ferredoxins (Fds) are iron-sulfur proteins and are supposed to act as electron carrier. A Fd was purified from Ha. japonica and characterized to contain a [2Fe-2S] cluster7). The Ha. japonica Fd gene has been cloned and sequenced8). The deduced amino acid sequence of the Ha. japonica Fd was 43, 89 and 98% identical with those of spinach, Hb. salinarum and Ha. marismortui Fds, respectively. The Ha. japonica Fd, as well as the other Fds from halophilic archaea, is longer in polypeptide length than the spinach Fd and has an extra N-terminal region. This region contains large amount of acidic amino acids. In order to elucidate the role of the extra N-terminal region, protein engineering technique is a powerful tool. As a first step for the protein engineering study, we have succeeded in expression of the Ha. japonica Fd gene in E. coli and Ha. Japonica.
References
1) K. Horikoshi et al. Experientia, 49, 497-502 (1993).
2) S. Nakamura et al. Biosci. Biotechnol. Biochem., 56, 996-998 (1992).
3) H. Wakai et al. Extremophiles, 1, 29-35 (1997).
4) R. Yatsunami et al. Extremophiles, 4, 109-114 (2000).
5) R. Yatsunami et al. Nucleic Acids Symp. Ser., 42, 73-74 (1999).
6) R. Yatsunami et al. Nucleic Acids Symp. Ser., 42, 75-76 (1999).
7) D. Sugimori et al. BioMetals, 13, 23-28 (2000).
8) A. Ikeda et al. Nucleic Acids Symp. Ser., 37, 109-110 (1997).
