V. Mohrle et al., IDENTIFICATION OF CELLULAR PROTEINS INVOLVED IN NIKKOMYCIN PRODUCTIONIN STREPTOMYCES-TENDAE TU901, Molecular microbiology, 15(3), 1995, pp. 561-571
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.