Butirosin is an interesting 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic produced by non-actinomycete Bacilli. Recently we were successful in purification of 2-deoxy-,scy//0-inosose synthase from butirosin-producer Bacillus circulans as the key enzyme for the biosynthesis of DOS, in cloning of the responsible gene (btrC), and in its overexpression in Escherichia coli. The present study involved gene-walking approach, which allowed us to find a gene cluster around btrC. The function of each gene was further investigated by gene disruption, and the disruptants of btrB, btrC, btrD and btrM showed no antibiotic producing activity. Therefore, the gene cluster found so far was determined to be a part of the butirosin biosynthetic gene cluster. Functions of some ORFsare also discussed in terms ofbutirosin biosynthesis on the basis of database search.

The 2-deoxystreptamine aglycon is a commonstructural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-1sic>'//<9-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scy//<9-inosose. The recent success of isolating the 2-deoxy-,s'cy//6>-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach. With the aid of DNAprobes constructed on the basis of the amino-terminal sequence of the purified 42kDa subunit of the enzyme, the responsible gene btrC was successfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coll, and the 2-deoxy-scy//oinosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Our previous proposal for the similarity of 2-deoxy-scylloinosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those ofbiosynthetic enzymes for other related microbial products is also discussed.

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Using inverse PCR, two new genes (btrN and btrS) were identified upstream of the putative glycosyltransferase gene btrM in the butirosin-biosynthetic btr gene cluster of Bacillus circulans. The upstream gene btrS showed significant homology with stsC of Streptomyces griseus, which encodes L-glutamine: scyllo-inosose aminotransferase in the biosynthesis of streptomycin. The function of BtrS was further confirmed by heterologous expression in Escherichia coli and chemical identification of the conversion of 2-deoxy-scyllo-inosose into 2-deoxy-scyllo-inosamine.The identification of BtrS as L-glutamine: 2-deoxy-scyllo-inosose aminotransferase is the first report of the aminotransferase gene responsible for 2-deoxystreptamine biosynthesis.

The denitrifying bacterium strain MT-1 was isolated from the mud of the Mariana Trench. The optimal temperature and pressure for growth of this bacterium were found to be 30 degrees C and 0.1 MPa, respectively. However, it showed greater tolerance to low temperature (4 degrees C) and high hydrostatic pressure (50 MPa) as compared with denitrifiers obtained from land. From the results, it can be said that this organism is adapted to the environment of the deep sea. Strain MT-1 was shown to belong to the genus Pseudomonas by analysis of its 16S rDNA. The cytochrome contents of the bacterium were similar to those of Ps. stutzeri in spectrophotometric studies.