Nitrite reductase gene cloning of Amycolatopsis mediterranei U-32

Southern blot analysis showed great homology existed between niaD (NR gene) of Aspergillus nidulans and A. mediterranei U-32 chromosome DNA. A 5.0kb PstI fragment from A. mediterranei U-32 complementary to A. nidulans niaD gene was cloned in E. coli NM522 using niaD as a probe. An identical DNA band was observed through back-hybridization of the cloned DNA fragment to PstI digest of A. mediterranei U-32 chromosome DNA. Its 2.1 kb SmaI-EcoRV fragment can only hybridize with total RNA from nitrate-cultured mycelium but not with that from ammonia-cultured mycelium. These data suggested that the cloned DNA fragment contains NR gene of A. mediterranei U-32. This is the first report on NR gene cloning from a aerobic bacterium. It was deduced from the molecular weight of the nitrate reductase that the coding sequence of NR gene is almost 1.5 kb in size. Further hybridization analysis indicated that the cloned DNA fragment covers the full-length NR gene of A. mediterranei U-32. We also constructed the physical map of the recombinant plasmid pJL1 with various restriction endonucleases, among them ten with no restriction site, six with unique site and two with double sites on the insert.