Streptomyces antimicrobicus sp. nov., a novel clay soil-derived actinobacterium producing antimicrobials against drug-resistant bacteria.

A novel actinobacterium, designated strain SMC 277T, was isolated from the clay soil in paddy field of Chonburi Province, Thailand, and characterized using polyphasic taxonomy. Strain SMC 277T formed straight chains of nonmotile cylindrical spores with smooth surface developed on aerial mycelia. The typical chemotaxonomic properties of members of the genus Streptomyces were observed in strain SMC 277T, e.g., cell wall peptidoglycan, whole cell sugars, major menaquinones, cellular fatty acids, and polar lipids. Chemotaxonomic data combined with mycelium and spore morphologies supported the assignment of strain SMC 277T to the genus Streptomyces. The results of comparative analysis of the 16S rRNA gene sequences confirmed that strain SMC 277T represented a member of the genus Streptomyces. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMC 277T shared the highest sequence similarity with Streptomyces bambusae NBRC 110903T (98.8%). Genome sequencing revealed a genome size of 6.55 Mbp and a digital G+C content of 73.4 mol%. In addition to the differences in phenotypic characteristics (morphology and physiology), values of ANI (ANIb and ANIm), AAI and dDDH between strain SMC 277T and its closest relative S. bambusae NBRC 110903T were 81.84, 86.77, 76.91 and 26.1%, respectively. Genome annotation and secondary metabolite gene cluster analysis predicted that SMC 277T contained 35 biosynthetic gene clusters encoding diverse bioactive secondary metabolites. It is in agreement with observed antimicrobial activity against drug-resistant bacteria associated with nosocomial infections (methicillin-resistant Staphylococcus aureus, extended-spectrum ß-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Acinetobacter baumannii). On the basis of these genotypic and phenotypic characteristics, strain SMC 277T can be characterized to represent a novel species of the genus Streptomyces, for which the name Streptomyces antimicrobicus is proposed. The type strain is SMC 277T (= TBRC 15568T = NBRC 115422T).

Comparative genome features and secondary metabolite biosynthetic potential of Kutzneria chonburiensis and other species of the genus Kutzneria.

Actinobacteria are well known as a rich source of diversity of bioactive secondary metabolites. Kutzneria, a rare actinobacteria belonging to the family Pseudonocardiaceae has abundance of secondary metabolite biosynthetic gene clusters (BGCs) and is one of important source of natural products and worthy of priority investigation. Currently, Kutzneria chonburiensis SMC256T has been the latest type-strain of the genus and its genome sequence has not been reported yet. Therefore, we present the first report of new complete genome sequence of SMC256T (genome size of 10.4 Mbp) with genome annotation and feature comparison between SMC256T and other publicly available Kutzneria species. The results from comparative and functional genomic analyses regarding the phylogenomic and the clusters of orthologous groups of proteins (COGs) analyses indicated that SMC256T is most closely related to Kutzneria sp. 744, Kutzneria kofuensis, Kutzneria sp. CA-103260 and Kutzneria buriramensis. Furthermore, a total of 322 BGCs were also detected and showed diversity among the Kutzneria genomes. Out of which, 38 clusters showing the best hit to the most known BGCs were predicted in the SMC256Tgenome. We observed that six clusters responsible for biosynthesis of antimicrobials/antitumor metabolites were strain-specific in Kutzneria chonburiensis. These putative metabolites include virginiamycin S1, lysolipin I, esmeraldin, rakicidin, aclacinomycin and streptoseomycin. Based on these findings, the genome of Kutzneria chonburiensis contains distinct and unidentified BGCs different from other members of the genus, and the use of integrative genomic-based approach would be a useful alternative effort to target, isolate and identify putative and undiscovered secondary metabolites suspected to have new and/or specific bioactivity in the Kutzneria.

Nonomuraea montanisoli sp. nov., isolated from mountain forest soil.

A novel actinomycete, strain SMC 257T, was isolated from a soil sample collected from mountain forest, Nan Province, Thailand. Strain SMC 257T formed tightly closed spiral spore chains on aerial mycelia. A polyphasic approach was used for the taxonomic study of this strain. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMC 257T belonged to the genus Nonomuraea, and the closest phylogenetically related species were Nonomuraea roseoviolacea subsp. carminata JCM 9946T (98.9 % 16S rRNA gene sequence similarity), Nonomuraea rhodomycinica TBRC 6557T (98.4 %), and Nonomuraea roseoviolacea subsp. roseoviolacea JCM 3145T (98.3 %). Genome sequencing revealed a genome size of 9.76 Mbp and a G+C content of 72.3 mol%. The genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values that distinguished this novel strain from its closest related species were species boundary of 95-96 % and 70 %, respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars were glucose, ribose, madurose and mannose. The major menaquinone was MK-9(H4). The polar lipid profile consisted of phosphatidylethanolamine, hydroxyphosphatidylethanolamine, lysophosphatidylethanolamine, diphosphatidylglycerol, N-phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The predominant cellular fatty acids were C17 : 0 10-methyl and iso-C16 : 0. Based on comparative analysis of phenotypic, chemotaxonomic and genotypic data, strain SMC 257T is considered to represent a novel species of the genus Nonomuraea, for which the name Nonomuraea montanisoli is proposed. The type strain is SMC 257T (=TBRC 13065T=NBRC 114772T).

Pseudonocardia mangrovi sp. nov., isolated from soil.

A novel Gram-stain-positive, aerobic actinomycete, designated strain SMC 195T, was isolated from soil collected from a mangrove forest in Thailand. The strain produced extensively branched substrate and aerial mycelia. The substrate mycelium was fragmented into rod-shaped elements, and spore chains consisting of smooth and rod-shaped spores were formed on the aerial mycelium. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that SMC 195T represented a member of the genus Pseudonocardia, and the most closely phylogenetically related species were Pseudonocardia yuanmonensisJCM 18055T (99.2 % 16S rRNA gene sequence similarity), Pseudonocardia halophobicaNRRL B-16514T (98.9 %) and Pseudonocardia kujensisNRRL B-24890T (98.7 %). However, the DNA-DNA relatedness values between SMC 195Tand the closest phylogenetically related species were significantly below 70 %. The G+C content of the genomic DNA was 74±0.8 mol%. The cell wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars consisted of arabinose, galactose, glucose, rhamnose and ribose. The menaquinone was MK-8(H4) only. The major cellular fatty acid was the branched fatty acid iso-C16 : 0 (33.6 %). The polar lipids detected were phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and unidentified glycolipids. On the basis of the results from phenotypic, chemotaxonomic and genotypic studies, it is concluded that SMC 195T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia mangrovi sp. nov. is proposed. The type strain is SMC 195T (=TBRC 7778T=NBRC 113150T).

Kutzneria chonburiensis sp. nov., isolated from soil.

A novel actinomycete strain, SMC 256T, which developed small, globose sporangia at the ends of long sporangiophores on aerial mycelium, was isolated from soil collected in a mountain forest of Thailand. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SMC 256T belonged to the genus Kutzneria, and the closest phylogenetically related species were Kutzneria buriramensis BCC 29373T (98.9 % 16S rRNA gene sequence similarity), Kutzneria kofuensis ATCC 27102T (98.2 %), Kutzneria albida ATCC 25243T (97.9 %) and Kutzneria viridogrisea ATCC 25242T (97.4 %). The DNA-DNA relatedness values that distinguished strain SMC 256T from previously described members of the genus Kutzneria were significantly below 70 %. The G+C content of the genomic DNA was 71.8 mol%. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars consisted of rhamnose, ribose, mannose, glucose and galactose. The predominant menaquinone was MK-9(H4). Mycolic acids were not detected. The diagnostic phospholipids were hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, unidentified phosphoglycolipids, unidentified phospholipids and an unidentified lipid. The predominant cellular fatty acids were iso-C16 : 0, C17 : 1 and C17 : 0 10-methyl. Following the evidence of phenotypic, chemotaxonomic and genotypic studies, it is proposed that strain SMC 256T represents a novel species in the genus Kutzneria, namely Kutzneria chonburiensis sp. nov. The type strain is SMC 256T ( = BCC 72675T = NBRC 110610T).

Formation of catalytically active cross-species heterodimers of thymidylate synthase from Plasmodium falciparum and Plasmodium vivax.

Thymidylate synthase (TS) of Plasmodium dihydrofolate reductase-thymidylate synthase (DHFR-TS) functions as a homodimeric enzyme with two active sites located near the subunit interface. The dimerization is essential for catalysis, since the active site of each subunit contains amino acid residues contributed from the other TS domain. In P. falciparum DHFR-TS, it has been shown that the active sites require Cys-490 from one domain and Arg-470 donated from the other domain. Mutants of these two series can complement one another giving rise to active enzyme. Here, the potential to form cross-species heterodimers between P. falciparum and P. vivax TS has been explored. Formation of cross-species heterodimer was tested by co-transformation of TS-inactive Cys-490 mutants of P. falciparum or P. vivax with corresponding TS-inactive Arg-486 mutants of P. vivax or P. falciparum into thymidine-requiring Escherichia coli. Active heterodimers were detected by subunit complementation and 6-[(3)H]-FdUMP binding assays. All combinations of the mutants tested, except for (Pf)R470A+(Pv)C506Y, were able to form catalytically active cross-species heterodimers. The single active site formed by (Pf)R470D+(Pv)C506Y and (Pv)R486D+(Pf)C490A pairs of cross-species heterodimers has k(cat) and K(m) values similar to those of intra-species heterodimers of P. falciparum and P. vivax. This is the first report to demonstrate that the TS subunit interface between Plasmodium species is sufficiently conserved to allow formation of fully active cross-species heterodimer.

Subunit complementation of thymidylate synthase in Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase.

Thymidylate synthase of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) functions as a dimeric enzyme with extensive contact between the two TS domains. Structural data of PfDHFR-TS shows that the formation of the two TS active sites involves contribution of the amino acid residues from both TS domains. Arg-470 donated from the adjoining domain is shown to hydrogen-bond to dUMP, while Cys-490 is a key nucleophile for TS catalysis by attacking C-6 of dUMP. However, mutants of the two series could complement one another, giving rise to active enzyme. By means of subunit complementation assay using Arg-470 and Cys-490 mutants, it is shown that co-transformants of both TS-inactive Arg-470 and Cys-490 mutants can complement the growth of thymidine auxotroph chi2913RecA(DE3) by formation of a functional TS heterodimer contributing from both Arg-470 and Cys-490 mutant subunits. 6-[3H]-FdUMP thymidylate synthase activity assay further elaborate the essence of restoration of TS activity. The TS k(cat) value of the R470D+C490A heterodimer is decreased by half from that of the wild-type PfDHFR-TS. However, the Km values for dUMP and CH2H4folate of the R470D+C490A heterodimer are similar to those of wild-type enzyme, indicating that the catalytic efficiency of the functional TS from the R470D+C490A heterodimer is similar to the wild-type TS enzyme in P. falciparum DHFR-TS.