Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.
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An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria. (+info)
Phosphatoquinones A and B, novel tyrosine phosphatase inhibitors produced by Streptomyces sp.
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Phosphatoquinones A and B were isolated from the cultured broth of Streptomyces sp. TA-0363 and their structures were elucidated by spectroscopic analyses. Phosphatoquinones A and B inhibited the protein tyrosine phosphatase activity prepared from human Ball-1 cells with IC50 of 28 microM and 2.9 microM, respectively. (+info)
Biosynthesis of polyketomycin produced by Streptomyces diastatochromogenes Tu 6028.
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The biosynthesis of polyketomycin was investigated by feeding 13C-labeled acetate and propionate to the growing cultures of Streptomyces diastatochromogenes Tu 6028. 13C NMR spectral analysis demonstrated the polyketide origin of the aglycone and the dimethylsalicyloyl moieties. The O-methyl group and 6-CH3 of the aglycone as well as 3B-CH3 of L-axenose and 3C-CH3 of the salicyloyl residue were labeled by feeding L-[methyl-13C]methionine. Both deoxysugars emerged from D-glucose. The biosynthesis of the aglycone and the assembly of the glycoside are discussed. The polyketomycin producing strain may be a candidate for further exploration in combinatorial biosynthesis. (+info)
Production of nikkomycins Bx and Bz by mutasynthesis with genetically engineered Streptomyces tendae Tu901.
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The previously described Streptomyces tendae nikC::aph mutant was used to mutasynthesize nikkomycins Bx and Bz. The mutant is deficient in L-lysine 2-aminotransferase, which transaminates lysine to form piperideine 2-carboxylate, the precursor of the peptidyl side chain of the biologically active nikkomycins I, J, X, and Z, and is therefore unable to produce these nikkomycins. The mutant accumulates the biologically inactive biosynthetic nucleoside precursors nikkomycins Cx and Cz. Resting cell cultures of the mutant fed with benzoic acid produced the biologically active nikkomycins Bx and Bz, which contain 2-amino-4-hydroxy-3-methyl-4-(4'-hydroxyphenyl)butanoic acid as the peptidyl side chain. The structures of nikkomycins Bx and Bz were confirmed by mass spectrometry and NMR. Nikkomycins Bx and Bz exhibit significantly higher pH stability than their analogues nikkomycins X and Z. (+info)
Bioconversion of milbemycin-related compounds: biosynthetic pathway of milbemycins.
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Streptomyces hygroscopicus subsp. aureolacrimosus SANK 60286 and SANK 60576 produce many kinds of milbemycins. Among them, milbemycin alpha11, alpha14, A3, and A4 have the most effective acaricidal activity. In this study, we investigated the terminal biosynthetic pathway to milbemycin alpha14 and A4 which accumulated as the final products in these strains. Using cerulenin, a specific inhibitor of fatty acid and polyketide biosynthesis, we conducted bioconversion experiments with cultures of several mutants, including milbemycin A4- and alpha14-producing strains. The bioconversions of milbemycin beta6 to milbemycin A4 and milbemycin A4 to milbemycin alpha14 could be identified. For the biosynthesis of milbemycin A4 from milbemycin beta6 in the milbemycin A4-high producing strain, there appeared to be two separate pathways exhibiting different sequences of furan ring formation and C-5 keto reduction steps. (+info)
Biosynthesis of the pyrroindomycins by Streptomyces rugosporus LL-42D005; characterization of nutrient requirements.
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Streptomyces rugosporus LL-42D005 was shown to produce the novel pyrroindomycin antibiotics. Production of pyrroindomycin (alpha) and chloro-pyrroindomycin (beta) was characterized in a semi-defined fermentation medium containing glucose, casein, phosphate, vitamins and minerals. Accumulation of pyrroindomycin beta increased with increasing concentrations of glucose, reaching maximum titers at approximately 5g/L glucose. Glucose concentrations greater than 7.5 g/L decreased pyrroindomycin beta yields. Inhibition of pyrroindomycin accumulation at higher glucose concentrations could be reversed by increasing the casein concentration. Ammonium chloride, arginine or glutamine could replace casein as the sole nitrogen source for growth and pyrroindomycin production. Glucose, fructose or mannitol were utilized as the sole carbon source, while sucrose, maltose, glycerol, corn oil and starch were poorly metabolized. Incubation of this isolate in a vitamin-deficient medium resulted in a delay in growth and pyrroindomycin production; this delay was eliminated by the addition of biotin. Addition of L-tryptophan to the medium resulted in the production of pyrroindomycin alpha as the major species. (+info)
Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.
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Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K. (+info)
Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli.
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Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebE gene prevents high-affinity uptake of cellobiose. (+info)