The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74,683 D and that the immunity protein had a molecular weight of 10,057 D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.
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 oligonucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb BamHI fragment and a 6.5 kb PvuII 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 BamHI 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.
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 amino‐transferase 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.
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 OC. The enzyme was highly specific for L-histidine; pyruvate, 2-oxobutyrate, 2-oxovalerate and Zoxocaproate were used as keto acceptors to about the same extent. The reaction mechanism was ping-pong. The K, 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 liuidans, 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.
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.
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