High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

Streptomyces tunisiensis sp. nov., a novel Streptomyces species with antibacterial activity

A novel actinomycete strain designated CN-207^T was isolated from northern Tunisian soil. This strain exhibited potent broad spectrum antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several other Gram-positive and Gram-negative bacteria. Strain CN-207^T developed greyish aerial mycelium and pale grey substrate mycelium on yeast extract/malt agar. The isolate produced branching vegetative mycelia with sporangiophores bearing sporangia developing at a late stage of growth. The sporangia contained smooth, non-motile spores. Chemotaxonomic characteristics of strain CN-207^T were typical of the Streptomyces genus. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CN-207^T belonged to the genus Streptomyces, and was most closely related to Streptomyces griseoincarnatus DSM 40274^T, Streptomyces variabilis DSM 40179^T, Streptomyces labedae DSM 41446^T and Streptomyces erythrogriseus DSM 40116^T. Low DNA–DNA relatedness values were recorded between strain CN-207^T and its closest phylogenetic neighbours. Strain CN-207^T was also distinguished from the nearest phylogenetic neighbours using a combination of morphological and phenotypic characteristics. On the basis of its phenotypic and molecular properties, strain CN-207^T is considered as a novel species of the Streptomyces genus, for which the name Streptomyces tunisiensis sp. nov. is proposed. The type strain is CN-207^T (=JCM 17589^T = DSM 42037^T).     

Streptomyces lividans possesses a GroEL-like chaperonin

Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin. This protein is specifically synthesized in bacterial cell cultures upon heat shock induction. It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E. coli. The S. lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E. coli and B. subtilis.     

Streptomyces genetics: a genomic perspective

Streptomycetes are gram-positive, soil-inhabiting bacteria of the order Actinomycetales. These organisms exhibit an unusual, developmentally complex life cycle and produce many economically important secondary metabolites, such as antibiotics, immunosuppressants, insecticides, and anti-tumor agents. Streptomyces species have been the subject of genetic investigation for over 50 years, with many studies focusing on the developmental cycle and the production of secondary metabolites. This information provides a solid foundation for the application of structural and functional genomics to the actinomycetes. The complete DNA sequence of the model organism, Streptomyces coelicolor M145, has been published recently, with others expected to follow soon. As more genomic sequences become available, the rational genetic manipulation of these organisms to elucidate metabolic and regulatory networks, to increase the production of commercially important compounds, and to create novel secondary metabolites will be greatly facilitated. This review presents the current state of the field of genomics as it is being applied to the actinomycetes.     

Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans.

A genomic library of Streptomyces sp. KB210-8SY, prepared in the plasmid vector pACYC184, was screened to obtain the gene encoding sarcosine oxidase with probes based on the amino acid sequence of the protein. A plasmid pSOXS13, which was isolated from a clone identified by hybridization with the probes, contained a 8.4-kb insert of Streptomyces DNA. When the 2.0-kb MIuI/EcoRV DNA fragment of pSOXS13 was inserted into the Streptomyces vector pIJ680 and introduced into S. lividans, the transformants produced 100-fold more sarcosine oxidase intracellularly than KB210-8SY. The nucleotides of the 1.7-kb fragment containing the sarcosine oxidase gene were sequenced. An open reading frame encoded a mature sarcosine oxidase consisting of 388 amino acids, with a calculated molecular mass of 42,107 daltons.     

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Streptomyces plicatus as a model biocontrol agent

Three hundred and seventy two isolates belonging to the genusStreptomyces were isolated and screened for chitinase production.Streptomyces plicatus was found to be the best producer. The highest chitinase production were incubated for 3 d at 30 °C on buffered culture medium (pH 8.0) containing chitin plus sucrose and calcium nitrate as carbon and nitrogen sources.S. plicatus chitinase had a highly significant inhibitory effect on spore germination, germ tube elongation and radial growth ofFusarium oxysporum f.sp.lycopersict., Altrernaria alternata andVerticillium albo-atrum, the causal organisms ofFusarium wilt, stem canker andVerticillium wilt diseases of tomato. Application ofS. plicatus to the root system of tomato plants before transplantation markedly protected tomato plants against the tested phytopathogenic fungiin vivo.     

New Olimycins from a Cold-Seep-Derived Streptomyces olivaceus

Cold-seeps are areas of the ocean floor in which hydrogen sulfide and methane are released into the open water. The cold-seep microbes are an emerging source of novel bioactive natural products. Four new ansa-ring opened linear ansamycin analogues, named olimycins E−H ( 1 – 4 ) were isolated from the cold-seep-derived Streptomyces olivaceus OUCLQ19-3. The planar and stereochemical structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations.     

Cloning and expression of a lignin peroxidase gene from Streptomyces viridosporus in Streptomyces lividans.

A lignin peroxidase gene was cloned from Streptomyces viridosporus T7A into Streptomyces lividans TK64 in plasmid pIJ702. BglII-digested genomic DNA (4-10 kb) of S. viridosporus was shotgun-cloned into S. lividans after insertion into the melanin (mel+) gene of pIJ702. Transformants expressing pIJ702 with insert DNA were selected based upon the appearance of thiostrepton resistant (tsrr)/mel-colonies on regeneration medium. Lignin peroxidase-expressing clones were isolated from this population by screening of transformants on a tsr-poly B-411 dye agar medium. In the presence of H2O2 excreted by S. lividans, colonies of lignin peroxidase-expressing clones decolorized the dye. Among 1000 transformants screened, 2 dye-decolorizing clones were found. One, pIJ702/TK64.1 (TK64.1), was further characterized. TK64.1 expressed significant extracellular 2,4-dichlorophenol (2.4-DCP) peroxidase activity (= assay for S. viridosporus lignin peroxidase). Under the cultural conditions employed, plasmidless S. lividans TK64 had a low background level of 2.4-DCP oxidizing activity. TK64.1 excreted an extracellular peroxidase not observed in S. lividans TK64, but similar to S. viridosporus lignin peroxidase ALip-P3, as shown by activity stain assays on nondenaturing polyacrylamide gels. The gene was located on a 4 kb fragment of S. viridosporus genomic DNA. When peroxidase-encoding plasmid, pIJ702.LP, was purified and used to transform three different S. lividans strains (TK64, TK23, TK24), all transformants tested decolorized poly B-411. When grown on lignocellulose in solid state processes, genetically engineered S. lividans TK64.1 degraded the lignocellulose slightly better than did S. lividans TK64. This is the first report of the cloning of a bacterial gene coding for a lignin-degrading enzyme.     

Collagen hydrolysis by a new Streptomyces species

A soil streptomycete hydrolysed collagen extracted from bovine Achilles tendon, calf skin, carp swim bladder and rat tail tendon. Enzyme activity was highest with rat tail tendon collagen as substrate. The taxonomy of the streptomycete was studied according to internationally approved methods. Comparative studies revealed that it resembled Streptomyces humidus , S. rochei and S. distatochromogenes in some respects, but differed in many cultural and physiological characteristics. Based on these differences the organism was given a new identity as Streptomyces species A.