Misa Otoguro

Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter

The present paper describes a simple enrichment technique which enables rapid and selective isolation of diverse zoosporic actinomycete genera directly from soil and plant litter. This technique, designated the rehydration and centrifugation (RC) method, consists of immersing the air-dried source material in 10 mM phosphate buffer containing 10% soil extract, letting the preparation stand at 30 °C for 90 min, followed by centrifugation of the fluid at 1,500×g for 20 min. Portions of the supernatant containing actinomycete zoospores are plated on the humic acid-vitamin agar which is supplemented with nalidixic acid and trimethoprim as the selective inhibitors for Gram-negative bacteria and bacilli. The phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material. The centrifugation stage greatly eliminated streptomycetes and other non-motile actinomycetes from the liquid phase, thereby facilitating selective growth of rare, motile actinomycetes on the isolation plates subsequent to inoculation. Ten different soil and leaf-litter samples, taken from fields, forests, and stream banks, were examined. The RC method consistently achieved preferential isolation of motile actinomycetes in all samples, which accounted for 37–86% of the total microbial population recovered. The most frequently isolated motile actinomycetes were Actinoplanes and Dactylosporangium. Strains of Actinokineospora, Catenuloplanes and Kineosporia were also recovered, depending on the nature of the samples examined. Other motile actinomycetes that were occasionally isolated in small numbers included Actinosynnema, Geodermatophilus and Sporichthya.

Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary Snapshot of the Family Streptomycetaceae

Kitasatospora setae NBRC 14216T (=KM-6054T) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8 783 278 bp with terminal inverted repeats of 127 148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.

Transfer of Actinomadura spadix Nonomura and Ohara 1971 to Actinoallomurus spadix gen. nov., comb. nov., and description of Actinoallomurus amamiensis sp. nov., Actinoallomurus caesius sp. nov., Actinoallomurus coprocola sp. nov., Actinoallomurus fulvus sp. nov., Actinoallomurus iriomotensis sp. nov., Actinoallomurus luridus sp. nov., Actinoallomurus purpureus sp. nov. and Actinoallomurus yoronensis sp. nov.

Ten actinomycete strains that form chains of spiral or looped spores were isolated from soil and dung samples in Japan. They contained D- and L-lysine, meso-diaminopimelic acid (A2pm), D-glutamic acid and D- and L-alanine in the cell-wall peptidoglycan, madurose as a characteristic whole-cell sugar, MK-9(H6) and MK-9(H8) as the major isoprenoid quinones and iso-C16:0 as the major cellular fatty acid and showed genomic DNA G+C contents of 69-74 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolated actinomycete strains consistently formed a monophyletic cluster with Actinomadura spadix NBRC 14099T and a separate line of descent in the phylogenetic cluster of the family Thermomonosporaceae. Actinomadura spadix NBRC 14099T also contained D- and L-lysine in addition to meso-A2pm. This genetic and phenotypic evidence revealed that the actinomycete strains could be clearly differentiated from the other members of the family Thermomonosporaceae and that they warranted separate genus status. We conclude that Actinomadura spadix should be assigned the status of the type species of a new genus as Actinoallomurus spadix gen. nov., comb. nov. (type strain NBRC 14099T=ATCC 27298T=BCRC 13386T=CBS 261.72T=CIP 105479T=DSM 43459T=JCM 3146T=KCTC 9252T=NCIMB 11118T=NRRL B-16128T). Further, we conclude that the ten new isolates should be assigned to the novel species Actinoallomurus amamiensis sp. nov. (type strain TT00-28T=NBRC 103682T=KCTC 19537T), Actinoallomurus caesius sp. nov. (type strain A3015T=NBRC 103678T=KCTC 19535T), Actinoallomurus coprocola sp. nov. (type strain TT04-09T=NBRC 103688T=KCTC 19542T), Actinoallomurus fulvus sp. nov. (type strain TT99-66T=NBRC 103680T=KCTC 19536T), Actinoallomurus iriomotensis sp. nov. (type strain TT02-47T=NBRC 103685T=KCTC 19539T), Actinoallomurus luridus sp. nov. (type strain TT02-15T=NBRC 103683T=KCTC 19538T), Actinoallomurus purpureus sp. nov. (type strain TTN02-30T=NBRC 103687T=KCTC 19541T) and Actinoallomurus yoronensis sp. nov. (type strain TTN02-22T=NBRC 103686T=KCTC 19540T).

Taxonomic and ecological studies of actinomycetes from Vietnam: isolation and genus-level diversity

Actinomycetes were isolated from 109 soil and 93 leaf-litter samples collected at five sites in Vietnam between 2005 and 2008 using the rehydration-centrifugation (RC) method, sodium dodecyl sulfate-yeast extract dilution method, dry-heating method and oil-separation method in conjunction with humic acid-vitamin agar as an isolation medium. A total of 1882 strains were identified as Vietnamese (VN)-actinomycetes including 1080 (57%) streptomycetes (the genus Streptomyces isolates) and 802 (43%) non-streptomycetes. The 16S ribosomal RNA gene sequences of the VN-actinomycetes were analyzed using BLAST searches. The results showed that these isolates belonged to 53 genera distributed among 21 families. Approximately 90% of these strains were members of three families: Streptomycetaceae (1087 strains, 58%); Micromonosporaceae (516 strains, 27%); and Streptosporangiaceae (89 strains, 5%). Motile actinomycetes of the genera Actinoplanes, Kineosporia and Cryptosporangium, which have quite common morphological characteristics, were frequently isolated from leaf-litter samples using the RC method. It is possible that these three genera acquired common properties during a process of convergent evolution. By contrast, strains belonging to the suborder Streptosporangineae were exclusively isolated from soils. A comparison of the sampling sites revealed no significant difference in taxonomic diversity between these sites. Among the non-streptomycetes, 156 strains (19%) were considered as new taxa distributed into 21 genera belonging to 12 families. Interestingly, the isolation of actinomycetes from leaf-litter samples using the RC method proved to be the most efficient way to isolate new actinomycetes in Vietnam, especially the Micromonosporaceae species.

Actinomycetospora iriomotensis sp. nov., a novel actinomycete isolated from a lichen sample

An actinomycete strain, IR73-Li102T, was isolated from a lichen sample obtained from Iriomote Island, Japan, and subsequently characterized using a polyphasic approach. Comparative 16S rRNA gene sequence analysis revealed that strain IR73-Li102T had the highest sequence similarities with Actinomycetospora chiangmaiensis YIM 0006T (98.3%), A. corticola 014-5T (98.1%) and A. rishiriensis RI109-Li102T (98.0%). However, DNA–DNA hybridization assays, as well as physiological and biochemical analyzes, showed that strain IR73-Li102T could be clearly differentiated from its closest phylogenetic relatives. The strain contained meso-diaminopimelic acid, and arabinose and galactose were present in whole-cell hydrolysates. The predominant menaquinone was MK-8(H4), and the diagnostic phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acid was iso-C16:0 (58%). The chemotaxonomic properties of strain IR73-Li102T were consistent with those shared by members of the genus Actinomycetospora. On the basis of the phenotypic features and DNA–DNA hybridization data, strain IR73-Li102T (= NBRC 106365T = KCTC 19783T) represents a novel species of the genus Actinomycetospora, for which the name Actinomycetospora iriomotensis sp. nov. is proposed.

Actinokineospora baliensis sp. nov., Actinokineospora cibodasensis sp. nov. and Actinokineospora cianjurensis sp. nov., isolated from soil and plant litter.

Six actinomycete strains isolated from soil and plant-litter samples in Indonesia were studied for their taxonomic position by using a polyphasic approach. Phylogenetically, all the strains were located in the broad cluster of the genus Actinokineospora. Chemotaxonomic data [cell-wall diamino acid, meso-diaminopimelic acid; cell-wall peptidoglycan, type III (A1γ); major sugars, galactose and arabinose; major menaquinone, MK-9(H₄); major fatty acid, iso-C₁₆:₀; major phospholipid, phosphatidylethanolamine] supported the affiliation of all six strains to the genus Actinokineospora. The results of DNA-DNA hybridization with DNA from type strains of Actinokineospora species with validly published names revealed three DNA-DNA relatedness groups. Group I (ID03-0561(T)) showed low relatedness to the other strains studied. The three strains in group II (ID03-0784(T), ID03-0808 and ID03-0809) formed a group with high relatedness (98-100 %) and showed low relatedness to the other strains studied. The two strains in group III (ID03-0810(T) and ID03-0813) showed 58-68 % relatedness to Actinokineospora terrae NBRC 15668(T) and showed low relatedness (2-24 %) to the other strains studied. The description of three novel species is proposed: Actinokineospora baliensis sp. nov., for the single strain in group I (type strain ID03-0561(T) =BTCC B-554(T) =NBRC 104211(T)), Actinokineospora cibodasensis sp. nov., for the strains in group II (type strain ID03-0784(T) =BTCC B-555(T) =NBRC 104212(T)), and Actinokineospora cianjurensis sp. nov., for the strains in group III (type strain ID03-0810(T) =BTCC B-558(T) =NBRC 105526(T)).

JBIR-94 and JBIR-125, Antioxidative Phenolic Compounds from Streptomyces sp. R56-07

Two phenolic compounds, JBIR-94 (1) and JBIR-125 (2), were isolated from the fermentation broth of strain R56-07, which was identified by phylogenetic methods as a novel species of Streptomyces. The structures of 1 and 2 were assigned on the basis of 1D and 2D NMR spectroscopy and MS analyses. Compounds 1 and 2 exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with an IC(50) value of 11.4 and 35.1 μM, respectively. These compounds are the first examples of hydroxycinnamic acid amides containing putrescine or spermidine produced by actinomycetes.

Complete genome sequence of the motile actinomycete Actinoplanes missouriensis 431T (= NBRC 102363T)

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds. Here, we describe the features of A. missouriensis 431T, together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

Flexivirga alba gen. nov., sp. nov., an actinobacterial taxon in the family Dermacoccaceae

A novel actinobacterial strain ST13T isolated from soil near wastewater treatment facilities of an electroplating plant was subjected to a polyphasic taxonomic study. Cells of this organism were non-sporulating, and were irregular coccoid to comma shaped. The peptidoglycan of strain ST13T contained glutamic acid, serine, alanine, glycine and lysine, and represented the peptidoglycan type A4α. The whole-cell sugars contained ribose, glucose, galactose, rhamnose and mannose. The predominant menaquinone was MK-8(H4). The major fatty acid was iso-C16:0. The polar lipid contained phosphatidylglycerol. The DNA G+C content was 67.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ST13T fell within the radius of the family Dermacoccaceae, and its closest neighbor was Luteipulveratus mongoliensis MN07-A0370T (95.1%). However, strain ST13T did not make a coherent clade with members of the recognized organisms. On the basis of the phylogenetic and phenotypic characteristics of this actinobacterium, a novel genus and species, Flexivirga alba gen. nov., sp. nov., is proposed. The type strain of F. alba is ST13T (=NBRC 107580T=DSM 24460T).

Actinophytocola timorensis sp. nov. and Actinophytocola corallina sp. nov., isolated from soil

Two actinomycete strains, ID05-A0653(T) and ID06-A0464(T), were isolated from soils of West Timor and Lombok island, respectively, in Indonesia. 16S rRNA gene sequence analysis clearly demonstrated that the isolates belonged to the family Pseudonocardiaceae and were closely related to the genus Actinophytocola. Strains ID05-A0653(T) and ID06-A0464(T) exhibited 98.1 and 98.2 % 16S rRNA gene sequence similarity, respectively, with Actinophytocola oryzae GMKU 367(T). The isolates grew well on ISP media and produced white aerial mycelium. Short spore chains were formed directly on the substrate mycelium. The isolates contained meso-diaminopimelic acid, arabinose and galactose as cell-wall components, MK-9(H(4)) as the sole isoprenoid quinone, iso-C(16 : 0) as the major cellular fatty acid and phosphatidylethanolamine as the diagnostic polar lipid. The DNA G+C contents of strains ID05-A0653(T) and ID06-A0464(T) were 69.7 and 71.2 mol%, respectively. On the basis of phenotypic characteristics, DNA-DNA relatedness and 16S rRNA gene sequence comparisons, strains ID05-A0653(T) and ID06-A0464(T) each represent a novel species of the genus Actinophytocola, for which the names Actinophytocola timorensis sp. nov. (type strain ID05-A0653(T)  = BTCC B-673(T)  = NBRC 105524(T)) and Actinophytocola corallina sp. nov. (type strain ID06-A0464(T)  = BTCC B-674(T)  = NBRC 105525(T)) are proposed.