Christiane Bormann

The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins

Streptomycetes form hydrophobic aerial hyphae that eventually septate into hydrophobic spores. Both aerial hyphae and spores possess a typical surface layer called the rodlet layer. We present here evidence that rodlet formation is conserved in the streptomycetes. The formation of the rodlet layer is the result of the interplay between rodlins and chaplins. A strain of Streptomyces coelicolor in which the rodlin genes rdlA and/or rdlB were deleted no longer formed the rodlet layer. Instead, these surfaces were decorated with fine fibrils. Deletion of all eight chaplin genes (strain ΔchpABCDEFGH) resulted in the absence of the rodlet layer as well as the fibrils at surfaces of aerial hyphae and spores. Apart from coating these surfaces, chaplins are involved in the escape of hyphae into the air, as was shown by the strong reduction in the number of aerial hyphae in the ΔchpABCDEFGH strain. The decrease in the number of aerial hyphae correlated with a lower expression of the rdl genes in the colony. Yet, expression per aerial hypha was similar to that in the wild‐type strain, indicating that expression of the rdl genes is initiated after the hypha has sensed that it has grown into the air.

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two‐dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non‐producing mutants. N‐terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two‐dimensional gels. Protein P8 was identified as l‐histidine amino‐transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo‐nucleotide 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 Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene‐disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.

Genetic complementation of Streptomyces tendae deficient in nikkomycin production

SummaryStreptomyces tendae Tü 901 produces the nucleoside peptide antibiotic nikkomycin. In shot-gun cloning experiments using pIJ699 as vector we isolated a 9.4-kb DNA fragment fromS. tendae which complemented the nikkomycin nonproducing mutant NP9 to the formation of nikkomycin C/Cx and Kx. Nikkomycins were identified by HPLC analyses and their characteristic UV spectra. In Southern hybridization experiments the cloned DNA exclusively reacted withS. tendae DNA sequences. As shown by Northern dot blotting, transcripts of the isolated DNA fragment were only detected during stationary growth and correlated with the extent of nikkomycin production. When the recombinant plasmid pNP113 containing the 9.4-kb DNA fragment was transferred into the over-producing mutant Tü901/ S2566, transformants exhibited a significantly decreased capacity for forming nikkomycin. Southern analysis of genomic DNA of these transformants revealed that severe rearrangements occurred in DNA sequences homologous to the 9.4-kb insert of pNP113.

Histidine aminotransferase activity in Streptomyces tendae and its correlation with nikkomycin production

Streptomyces tendae Tü901 produces nikkomycins belonging to the nucleoside peptide antibiotics. Mutants defective in histidine catabolism were isolated and characterized with regard to their histidine ammonium-lyase activity and antibiotic synthesis. In the histidine ammonialyase-negative mutant hut-11 which was unimpaired in nikkomycin production histidine aminotransferase activity was detected as an additional histidine metabolizing enzyme. A protein exhibiting histidine aminotransferase activity could be demonstrated on non-denaturing gels of hut-11 crude extracts. Using optimized assay conditions, histidine aminotransferase activity was investigated in the strain hut-11 during growth in nikkomycin production medium. Maximal activity was reached at the end of exponential growth prior to nikkomycin production. In the presence of bromopyruvate, an effective inhibitor of histidine aminotransferase activity in vitro, production of nikkomycin Z and X was markedly reduced in hut-11.

Purification and characterization of L-histidine aminotransferase from nikkomycin-producing Streptomyces tendae Tu901

Cell extracts of Streptomyces tendae grown in nikkomycin production media contained an enzyme (HisAT) that transaminated L-histidine as the sole amino substrate with pyruvate as the amino group acceptor. HisAT was purified about 190-fold from the crude extract of S. tendae. The enzyme was determined by gel filtration and SDS-PAGE to be a homodimer with a subunit molecular mass of approximately 45 kDa. The aminotransferase had maximum activity at pH 7.0 and 37 °dGC. The enzyme was highly specific for L-histidine; pyruvate, 2-oxobutyrate, 2-oxovalerate and 2-oxocaproate were used as keto acceptors to about the same extent. The reaction mechanism was ping-pong. The K m values for L-histidine and pyruvate, determined from Lineweaver-Burk plots, were 25 mM and 10 mM, respectively. Neither cell extracts of non-producing S. tendae mutants nor extracts of Streptomyces lividans, a species that does not synthesize nikkomycins, showed transaminating activity with a narrow substrate specificity for L-histidine as the amino donor. This strongly suggests that the formation of HisAT is essential for nikkomycin production.