|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1995 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1994 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We succeeded to clone and sequence the nqr operon that encodes Na^+-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus. The nqr operon was composed of six structural genes, and nqr1, nqr3 and nqr6 were found to encode alpha, gamma and beta-subunits, respectively, of the purified enzyme complex. From these informations, we prepared probes for each genes by a PCR method and the probes were labeled with digoxigenin-dUTP.We selected 5 different species of moderately halophilic bacteria, which have been demonstrated to have an Na^+ pump by the biochemical tethniques, and their chromosomal DNAs were isolated. Each DNA was digested by an appropriate restriction enzyme and the products were applied to agarose gel electrophoresis. The gel was blotted to a membrane, and the reactivities of each band for the probes were performed by a method of Southern hybridization. A DNA fragment from V.costicola reacted with four different probes (nqr1,3,5, and6) from V.alginolyticus, suggesting a close correlation between the two species. DNA fragments from Halovibrio reacted with labeled probes from nqr5 and nqr6. On the contrary, DNA fragments from Pseudomonas beijelinckii, Ps. halosaccharolytica and Deleya halophila reacted only faintly with these probes. These results suggested the presence of a different kind of Na^+ pump in these bacterial species.