Classification of acidophilic, neutrotolerant and neutrophilic streptomycetes by nucleotide sequencing of 5S ribosomal RNA.
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Complete 5S ribosomal RNA sequences were obtained for four acidophilic actinomycetes, seven neutrophilic streptomycetes and a strain of Streptoverticillium baldaccii. All of the organisms contained RNAs belonging to the 120 nucleotide type. An evolutionary tree was generated after combining the test data with results from similar studies on representative Gram-positive bacteria. The acidophilic, neutrotolerant and neutrophilic actinomycetes were recovered in a distinct cluster that was equated with the genus Streptomyces. The sequence data support the view that the genera Chainia, Elytrosporangium, Kitasatoa and Microellobosporia should be considered as synonyms of the genus Streptomyces. The recovery of the Streptoverticillium baldaccii strain on the fringe of the Streptomyces cluster is also consistent with current trends in the taxonomy of these organisms. Further work is needed to determine the taxonomic status of the two streptomycete subgroups that comprised the streptomycete cluster. (+info)
Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes.
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Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function. (+info)
Antagonistic enzymes may generate alternate phase transitions leading to ephemeral gels.
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In some biological processes, two enzymes with antagonistic activities--the one creating a bond, the other destroying it--are involved in a reaction cycle. Several catalysts have the ability to modify the rheological properties of biological media participating in the production of a solid gel phase which later dissolves. Transglutaminase, catalyzing intermolecular protein cross-linking, is considered here as a reverse protease as far as the physical state of a proteic gel is concerned. A kinetic model including diffusion constraints and based on a protease/transglutaminase cycle interconverting insoluble gel and soluble proteolysis fragments showed that alternate sol/gel and gel/sol transitions could occur within such a system, generating transient gel phases. Then, ephemeral gels were obtained in vitro using an experimental system consisting of gelatin, transglutaminase, and thermolysin. Modulating the enzyme activity ratio allows us to "program" the global behavior: polymerization/solubilization cycle of a mixture containing at least one protein and two enzymes without any change in temperature or medium composition. (+info)
Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif.
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The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine-glycine (GG) motif in Region I and the glycine-serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP-PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S-S489G) contained a serine residue in the GG motif, and exhibited approximately a 9-27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP-PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S-S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability. (+info)
Medium optimization for enhanced production of cytosine-substituted mildiomycin analogue (MIL-C) by Streptoverticillium rimofaciens ZJU 5119.
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Kitasatospora saccharophila sp. nov. and Kitasatospora kazusanensis sp. nov., isolated from soil and transfer of Streptomyces atroaurantiacus to the genus Kitasatospora as Kitasatospora atroaurantiaca comb. nov.
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A polyphasic study was undertaken to establish the taxonomic positions of two isolates, SK15(T) and SK60(T), from soil samples that were found to have morphological and chemical properties consistent with Kitasatospora strains. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strains SK15(T) and SK60(T) form novel evolutionary lineages within the radiation of the genus Kitasatospora and share the highest 16S rRNA gene sequences similarities with their closest relatives, Kitasatospora setae IAM 15325(T) (97.8%) and Kitasatospora mediocidica IAM 15162(T )(97.5%), respectively. However, the results of DNA-DNA hybridization experiment and phenotypic data demonstrated that strains SK15(T) and SK60(T) are distinct from their closest phylogenetic neighbors and other Kitasatospora species. For chemotaxonomic characteristics, the cell-wall peptidoglycan of strains contained both meso- and LL-diaminopimelic acids as the diamino acids, the predominant quinone system was MK-9(H(6)) and MK-9(H(8)), whole-cell hydrolysates were rich in galactose, mannose and ribose, and the major fatty acids were C(16:0), anteiso-C(15:0), iso-C(15:0) and iso-C(16:0). On the basis of both phenotypic and phylogenetic evidence, strains SK15(T) and SK60(T) were assigned to represent two novel species of the genus Kitasatospora, for which the names Kitasatospora saccharophila sp. nov. (type strain SK15(T)=JCM 14559(T)=KCTC 19566(T)) and Kitasatospora kazusanensis sp. nov. (type strain SK60(T)=JCM 14560(T)=KCTC 19565(T)) are proposed. It is also proposed that Streptomyces atroaurantiacus should be transferred to the genus Kitasatospora as Kitasatospora atroaurantiaca comb. nov. (type strain NBRC 14327(T)=DSM 41649(T)). (+info)
YS-822A, a new polyene macrolide antibiotic. II. Planar structure of YS-822A.
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The planar structure of a new polyene macrolide antibiotic, YS-822A, which was isolated from the culture filtrate of a mutant strain H-8 of Streptoverticillium eurocidicum var. asterocidicus S-822, was established as I on the basis of spectroscopic evidences and by comparison spectroscopic data with nystatin A1 (II) and amphotericin A (III), representative polyene macrolide antibiotics. Molecular formula of YS-822A was established as C37H59NO14 (MW 741) by elemental analysis, NMR, and FAB mass spectra. The UV spectrum of YS-822A was very similar to that of nystatin A1, suggesting that YS-822A also has a conjugated all-trans-tetraene moiety. 1H and 13C NMR spectra of YS-822A showed a number of broad and overlapped signals, but the 1H-1H and 13C-1H COSY spectra implied the existence of a mycosamine moiety and several other partial structures. The connectivity of these partial structures was established by extensive 2D NMR experiments, including homonuclear Hartmann-Hahn and heteronuclear multiple-bond connectivity measurements, which led to the determination of the gross planar structure of YS-822A as I. (+info)