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At least 19 recordsLinked to original sources

PubMed1996

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.

Aminoglycosides↗

PubMed2004

Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae.

An isolate of Streptomyces tendae produced a extracellular protease which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 21 kDa. Optimum activity was at 70 degrees C and pH 6. It was stable at 55 degrees C for 30 min and between pH 4 and 9. It was resistant to neutral detergents and organic solvents such as Triton X-100, Tween 80, methanol, ethanol, acetone, and 2-propanol at 5% (v/v). The enzyme was completely inhibited by 5 mM PMSF, indicating it to be a serine protease. N-terminal amino acid sequence did not show any homology with other known proteolytic enzymes. The protease may therefore be a novel neutral serine protease, which is stable at high temperature and over a broad range of pH.

Amino Acid Sequence↗

PubMed1990

Genetic complementation of Streptomyces tendae deficient in nikkomycin production.

Streptomyces 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 from S. 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 with S. 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.

Aminoglycosides↗

PubMed1999

Production of nikkomycins Bx and Bz by mutasynthesis with genetically engineered Streptomyces tendae Tü901.

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.

Aminoglycosides↗

PubMed1987

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction.

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.

Aminoglycosides↗

PubMed1995

Identification of cellular proteins involved in nikkomycin production in Streptomyces 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 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.

Amino Acid Sequence↗

PubMed1992

Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture.

Continuous cultures with Streptomyces tendae revealed some interesting facts. In a continuous culture running for more than 2500 h the production of either nikkomycines or juglomycins could be selected by varying the feed composition. Decreasing the phosphate supply in the feed broth from the initial concentration of 2.5 mM to 1.0 mM enhanced the productivity of nikkomycins and decreased the productivity of juglomycins. When switching back to the initial conditions of the experiment after 2000 h nearly the same production behaviour as at the beginning of the fermentation could be observed. This indicated a stable behaviour of the population with regard to nikkomycin productivity. The long continuous fermentation showed the ability of S. tendae Tü 901/8c to produce nikkomycin at a high level for at least 1500 h. In a second continuous culture it was shown that the productivity of the nikkomycins and juglomycins decreased and increased, respectively, with increasing dilution rate. Comparing batch cultures with continuous fermentations, higher juglomycin productivity was found in the latter. These facts indicate that the strain responds to complex interacting physiological controls, by producing either nikkomycins or juglomycins in a higher amount.

Aminoglycosides↗

PubMed1992

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.

Aminoglycosides↗

PubMed1984

Tendamistat (HOE 467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158. Isolation, biochemical properties.

Culture fluids of Streptomyces tendae 4158 (ATCC 31210) contain a new kind of polypeptide alpha-amylase inhibitor, tendamistat (HOE 467). Several methods of isolating this inhibitor are described, including two rapid crystallisation methods, which produce homogeneous material. A characteristic of tendamistat is its tight-binding, pH-independent inhibition kinetics and the specific inhibition of the mammalian alpha-amylase form a stoichiometric 1:1 complex, which cannot be separated into its individual components by sodium dodecyl sulphate or molecular sieve chromatography. Studies of the mode of action reveal that the alpha-amylase-inhibiting activity is linked to the intact disulphide bridges of the inhibitor. It is assumed that the multipoint protein-protein bond exists between the enzyme and tendamistat. It is shown that extracellular tendamistat inhibits amylase formed by streptomyces. We therefore assume a regulatory function in the microorganism. By-products of tendamistat, which possess similar enzyme-inhibiting properties, are also described.

Binding Sites↗

PubMed1992

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.

Aminoglycosides↗

PubMed1989

Isolation of Streptomyces tendae mutants with an altered nikkomycin spectrum.

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-formylimidazolone structure of nikkomycins with the aldehyde reagent barbituric acid leading to red-colored products. Among 18,000 N-methyl-N'-nitro-N-nitrosoguanidine treated clones tested in the barbituric acid assay, we isolated 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). In addition, we isolated strains with mutations affecting the biosynthesis of 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-3-methylbutyric acid, the unusual amino acid of nikkomycins Z, X, J and I. By analyzing colonies derived from single spores or protoplasts of S. tendae Tü901/395, a mutant producing besides nikkomycins Z, X, J and I, also nikkomycins Kz/Kx and Oz/Ox, we obtained strains which only formed nikkomycins Kz/Kx and Oz/Ox with 2-amino-4-hydroxy-4-(2-pyridyl)butyric acid and 2-amino-4-hydroxy-4-(5-hydroxy-2-pyridyl)-butyric acid as amino acids. Mutation of such a strain (Tü901/395-11) by UV365nm in the presence of 8-methoxypsoralen and selection of S-2-aminoethyl-L-cysteine-resistant clones led to the isolation of Tü901/AEC1 and AEC2 which produced exclusively nikkomycins Kz and Kx. According to their nikkomycin spectrum, these strains were blocked at the hydroxylation step occurring at the pyridyl residue during biosynthesis of the nikkomycin amino acid.

Aminoglycosides↗

PubMed1981

[The sequence of the alpha-amylase inhibitor Hoe-467 A (alpha-amylase inactivator Hoe-467 A) from Streptomyces tendae 4158].

An alpha-amylase inhibitor isolated from Streptomyces tendae, strain 4158 was re-purified by chromatography on CM- and DEAE-cellulose column. Two inhibitors could be characterized: alpha-amylase inhibitor Hoe-467 A (with aspartic acid as N-terminal residue), and alpha-amylase inhibitor Hoe-467 S (with serine as N-terminal residue). The primary structure was determined by automatic Edman-degradation procedures of the aziranized inhibitor and tryptic peptides, derived from digestions of the performic oxidized, aziranized and maleylated inhibitor, respectively. The alpha-amylase inhibitor Hoe-467 A consists of 74 residues and has a calculated molecular weight of 7958. It is composed of all common amino acids except methionine and phenylalanine. Digestion with pepsin was carried out to determine the disulfide bonds. Two fractions could be isolated, containing one cystine each giving information about the positions of the disulfide bridges. The possible clinical application of the inactivator (diabetes mellitus) is pointed out.

Amino Acid Sequence↗

PubMedDate not supplied

Carbazole antibiotics synthesis in a Streptomyces tendae bald mutant, created by acriflavine treatment.

Acriflavine treatment on Streptomyces tendae generated a bald mutant (bld-1) with an altered antibiotic pattern. The parental strain produced nikkomycins and juglomycins, whereas the mutant bld-1 was only capable of juglomycin synthesis. The existence of a mutant defective in morphogenesis and in nikkomycin biosynthesis suggests a common regulation of these processes. An interesting finding of this study is that mutant bld-1 produced two carbazole derivatives, hitherto never seen in cultures of the parental strain. It seems likely that the DNA intercalating dye acriflavine, by mutagenesis, had activated cryptic genes which are involved in carbazole synthesis. The two carbazole derivatives were identified as the neuronal cell protecting compounds CS-79B and carquinostatin A, recently isolated from a wild-type of S. exfoliatus. We found that both substances showed antibacterial activity.

Acriflavine↗

PubMed1989

Metabolic products of microorganisms. 252. Isolation of new nikkomycins from Streptomyces tendae.

Two new nikkomycins were isolated from the culture broth of Streptomyces tendae Tü 901/PF 53+-3. The new compounds are the dipeptide nikkomycin pseudo-Z (psi Z) and tripeptide nikkomycin pseudo-J (psi J), which are analogues to nikkomycins Z and J. Nikkomycins pseudo-Z and pseudo-J have a C-glycosidic linkage between uracil and 5-amino-5-deoxy-D-allo-furanuronic acid, which is comparable to the C-glycosidic bond in pseudouridine. The new Cc-nucleoside nikkomycins exhibit a lower biological activity than the CN-nucleoside nikkomycins.

Aminoglycosides↗

PubMed1993

Use of Tn4560 to generate nikkomycin non-producing mutants of Streptomyces tendae.

Transposon Tn4560 was used to generate three nikkomycin non-producing mutants in Streptomyces tendae ATCC 31160. Southern hybridization confirmed that Tn4560 was present in 10-12-kb BamHI fragments of the chromosomes of the mutants. Biologically active nikkomycins were not detected in culture broths of the mutants as determined by bioassays and HPLC. Differences in the HPLC profiles of culture broths suggest that Tn4560 inserted into different genes in the mutants.

Aminoglycosides↗

PubMed2001

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.

Amino Acid Sequence↗

PubMed1999

Characterization of a novel, antifungal, chitin-binding protein from Streptomyces tendae Tü901 that interferes with growth polarity.

The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity against Paecilomyces variotii in agar diffusion assays. The 384-bp afp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes for Streptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls of P. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth of Aspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.

Amino Acid Sequence↗

PubMed1990

Metabolic products of microorganisms. 255. Nikkomycins Wz and Wx, new chitin synthetase inhibitors from Streptomyces tendae.

Two new dipeptidyl nikkomycins of the Z and X type were isolated from the culture broth of Streptomyces tendae TU 901/395-11/32 and characterized. They show a variation in the amino acid moiety of the molecule. Nikkomycin Wz is composed of L-tyrosine and 5-amino-5-deoxy-D-allo-furanuronic acid N-glycosidally bound to uracil, whereas nikkomycin Wx is composed of L-tyrosine and 5-amino-5-deoxy-D-allo-furanuronic acid N-glycosidally bound to 4-formyl-4-imidazolin-2-one. The new nikkomycins are good inhibitors of chitin synthetase from Coprinus cinereus but they did not inhibit growth of fungi and yeasts.

Aminoglycosides↗