IDENTIFICATION OF CELLULAR PROTEINS INVOLVED IN NIKKOMYCIN PRODUCTIONIN STREPTOMYCES-TENDAE TU901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cell ular proteins. Ten gene products (P1-P10) were identified that were sy nthesized when nikkomycin was produced; these proteins were not detect ed in non-producing mutants, N-terminal sequences of six of the 10 pro teins were obtained by microsequencing of protein spots excised from p reparative two-dimensional gels. Protein P8 was identified as L-histid ine aminotransferase (HisAT), which has been previously correlated wit h nikkomycin production. By using oligonucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb PvuII fragment, respectively, from the genome of Streptomyces tendae Tu901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligon ucleotide probe hybridizing sites indicated that the genes encoding pr otein P2 and P6 are closely spaced on the 8 kb BamHI fragment, and the latter is located on the overlapping region. DNA sequence analysis re vealed that proteins P1 and P2 are encoded by a single gene, orfP1, th at is translated at two initiation codons, The orfP1 gene was interrup ted by homologous recombination using the integrating vector pWHM3. Th e gene-disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The d ata presented show that reverse genetics was successfully used to isol ate genes involved in nikkomycin production.