The galP1 promoter is responsible for galactose-dependent, glucose-sensitive transcription of the galactose utilization operon of Streptomyces coelicolor and Streptomyces lividans. We describe the characterization of mutations that were positioned directly upstream of the apparent transcription start site of galP1 and that resulted in deregulated expression. Certain combinations of base changes within a series of hexamers that lie within two pairs of direct repeat sequences resulted in significant expression from galP1 in the absence of inducer. These motifs are further implicated in regulation by the observation that DNA fragments containing the hexamers and direct repeat sequences resulted in increased transcription from the chromosomal copy of galP1 on multicopy plasmids in the absence of galactose. We suggest that these hexamers and direct repeat sequences constitute an operator for the negative regulation of the Streptomyces gal operon.
To isolate Streptomyces tendae mutants blocked in the biosynthesis of the nikkomycin nucleoside base 4-formyl-4-imidazoline-2-one, an assay was developed to detect the formation of nikkomycins containing this base during growth on solid medium. The assay is based on the reaction of the 4-formylimidazolonestructure of nikkomycinswith the aldehyde reagent barbituric acid leading to red-colored products. Among18,000 AT-methyl-JV'-nitro-TV-nitrosoguanidine treated clones tested in the barbituric acid assay, weisolated one mutant which was incapable of forming any nikkomycins containing the 4-formylimidazolone base (nikkomycins Cx, X and I) but instead produced nikkomycins containing uracil (nikkomycins C, Z and J). led to the isolation of Tu901/AECl and AEC2which produced exclusively nikkomycins Kz and Kx. According to their nikkomycin spectrum, these strains were blocked at the hydroxylation step occuring at the pyridyl residue during biosynthesis of the nikkomycin amino acid. 913Nikkomycins belong to the nucleoside peptide antibiotics and act as potent inhibitors of chitin synthetases from fungi and insects1~4). Streptomyces tendae Tu901 produces a spectrum of various nikkomycins with 4-formyl-4-imidazoline-2-one and uracil as variable bases5>6). The nucleoside moiety of biologically active nikkomycins produced by the wild type as major components is peptidically linked to the unusual amino acid 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-3-methylbutyric acid (nikkomycin D, Fig. 1). Studies on the biosynthesis of nikkomycins revealed that the imidazolone base is derived from L-histidine7) and uracil from pyrimidine metabolism8). Furthermore, L-lysine was shown to be the precursor of the pyridyl residue and the attached hydroxymethylene carbon in nikkomycin D9). There is little knownabout enzymatic reactions and intermediate structures of nikkomycin biosynthesis. Therefore we decided to screen for mutants blocked in nikkomycin biosynthesis which could be employed as hosts for cloning experiments to isolate nikkomycin biosynthetic genes. We describe the isolation of strains of S. tendae with mutations affecting the biosynthesis of the imidazolone base and nikkomycin D.
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.
We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions ؊34, ؊36, and ؊11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The ؊35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.Sequences that signal transcription initiation in bacteria play an important role in gene regulation. Among the best studied of these sequences are those involved in RNA polymerase recognition. The observation that sequences centered around Ϫ10 and Ϫ35 bp upstream of the transcription start site are highly conserved in many promoters (35), the fact that these sequences are required for promoter activity (46), and the discovery of RNA polymerase heterogeneity in Bacillus subtilis (26) supported the model that in most cases the sigma subunit of RNA polymerase makes sequence-specific contacts in these two regions of bacterial promoters. The strongest evidence comes from experiments that show allele specificity of suppression between mutations in the sigma subunit of RNA polymerase and compensating base changes in the DNA sequence of promoters. Amino acid changes in various sigma proteins, H (8, 47), A (22), and E (43) of B. subtilis and 70 of Escherichia coli (13,38), show that these proteins contact the Ϫ10 and Ϫ35 regions of their cognate promoters.While the RNA polymerase holoenzymes that catalyze transcription in Streptomyces spp. are presumed to be functionally similar to those of other bacteria, relatively little is known about sequences that are required for RNA polymerase recognition in this complex bacterium. At least eight RNA polymerase holoenzymes have been identified in Streptomyces, and promoters with putative RNA polymerase binding sites similar to known consensus sequences have been described (reviewed in references 3 and 41). A consensus sequence prototypical of eubacteria (TTGACA centered around Ϫ35 and TATAAT centered around Ϫ10) appears in several Streptomyces promoters, and at least four genes whose predicted products resemble E. coli 70 have been cloned and characterized (2, 4, 42). One of these genes is apparently essential (4). The existence of an additional sigma factor that may be functio...
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.