The taxonomic diversity of actinomycetes from soils in a cool-temperate area (74 samples from Rishiri Island) and a subtropical area (94 samples from Iriomote Island) of Japan was compared. Soil samples were treated with five different pre-treatment methods and subsequently cultured on humic acid-vitamin agar, resulting in the selective isolation of 1,234 actinomycete strains. These isolates were classified using 16S rDNA sequence analysis. 16S rDNA sequence similarity values of (cid:1) 97 % and (cid:1) 98 : 7 % were tentatively used to identify isolates at the genus and species ranks, respectively. The actinomycete assemblages isolated comprised diverse species. Specifically, 668 of the Rishiri isolates fell into 17 families, 40 genera, and 178 species, and 566 of the Iriomote isolates belonged to 21 families, 59 genera, and 194 species. Diversity analyses based on the Shannon index revealed that there was no significant difference between the species diversity of the Rishiri isolates and that of the Iriomote isolates. However, there was little taxonomic overlap between the Rishiri isolates and the Iriomote isolates, as only 66 of the species occurred in both groups. Several species that were reportedly isolated from other tropical areas were found among the Iriomote isolates but not among the Rishiri isolates. This result suggests that climate may influence the species types of soil-inhabiting actinomycetes.
Sinefungin is a nucleoside antibiotic in which L-ornithine is covalently bound to adenosine. 14C-Incorporation studies indicate that L-Arg and ATP are precursor substrates. To identify the biosynthetic intermediates in the sinefungin biosynthetic pathway, a shunt metabolite accumulated in a resting cell system was isolated from the cell suspension broth, and identified as ornithine-lactam (OrnLcm). The enzyme that catalyzes the formation of OrnLcm was purified from the lysed cell extract of Streptomyces incarnatus NRRL8089 by four-steps of column chromatography using DEAE-Toyopearl, Sephacryl S-200 HR gel filtration, Butyl-Toyopearl and hydroxy-apatite. The molecular mass of the purified protein was determined to be 43 kDa by SDS-PAGE. The N-terminal sequence MKVGIRP was identical to those of L-alanine dehydrogenases (40 kDa) encoded in the genomes of Streptomyces griseus, Streptomyces avermitilis, Streptomyces coelicolor, and other Gram-positive bacteria. The purified enzyme catalyzed the NAD-dependent oxidation of L-Ala, as well as the formation of OrnLcm from L-Arg. It did not show any activity towards L-Orn or D-Arg. Although it has not yet been clarified whether OrnLcm serves as a direct substrate for enzymatic C-C bond formation intracellularly, the lactam intermediate has the advantage of facile transport across the cell membrane as well as the enhanced nucleophilicity at the C5 carbon.
Terrestrial Actinobacteria have served as a primary source of bioactive compounds. However, a rapid reduction in the discovery of new compounds strongly necessitates new investigational approaches, including screening Actinobacteria from marine habitats. We therefore studied and compared the diversity of culturable Actinobacteria associated with 18 marine sponge samples. A total of 462 strains of Actinobacteria belonging to 19 genera were isolated; 24 of these strains represented new species in six genera. Only 28 strains showed seawater-dependent growth. Eleven strains belonging to the genera Streptomyces (8 strains), Rhodococcus (2), and Nocardiopsis (1) required ≥50% (v/v) seawater for their growth, whereas an additional 17 strains showed better growth in the presence of 50% seawater (v/v). Screening of strains for antimicrobial activity showed that 144, 24, and 77 strains exhibited activity against Micrococcus luteus, Escherichia coli, and Candida albicans, respectively.
Actinomycetes isolated from marine sediments in neritic zone in the sea around Japan were compared with terrestrial isolates. Among actinomycetes that are macroscopically Streptomyces and Micromonospora, marine isolates of both genera were found to have higher NaCl tolerance than terrestrial ones. Around 37% of Streptomyces and 26% of Micromonospora that were isolated from the marine environment could tolerate up to 12% and 5% NaCl, respectively. However, no terrestrial isolates could tolerate NaCl at these concentrations. The results of the 16S rRNA gene sequence analysis of 10 strains with high NaCl tolerance among these marine isolates showed that they had a close phylogenetic relationship with terrestrial strains isolated previously. The isolation of actinomycetes from marine sediments and terrestrial soils in the presence or absence of 6% NaCl in media revealed that the selective pressure of NaCl demonstrated no clear difference between both isolates. Terrestrial strains isolated by using a medium supplemented with 6% NaCl exhibited a high frequency (95%) of NaCl tolerance up to 9%. The highly NaCl tolerant strains isolated from terrestrial soils were frequently found to produce antimicrobial substances in the presence of seawater. Almost 75% of strains tested showed antimicrobial activities against Bacillus subtilis PCI 219 and 40% against Candida albicans 3147. It was suggested that the frequency of microorganisms with antimicrobial properties was increased in the medium supplemented with seawater.
The eshA gene was originally found to encode a protein required for the extension of sporogenic hyphae during submerged spore formation in Streptomyces griseus NRRL B-2682. An eshA-disrupted strain of S. griseus IFO13189 was reported to be conditionally deficient in streptomycin production and aerial mycelium formation. Our previous transcriptomic analyses indicated that AdpA, a global transcriptional regulator of morphological and physiological differentiation, induced eshA (SGR1270) transcription in S. griseus IFO13350. Here, we examined the transcriptional regulation of eshA by AdpA and the involvement of eshA in the morphological and physiological differentiation of S. griseus IFO13350. Transcriptional analysis by S1 nuclease mapping showed that eshA was transcribed throughout growth on solid medium. In contrast, no eshA transcription was detected in an adpA deletion mutant. Recombinant His-tagged AdpA bound to a region upstream from the eshA promoter in vitro. However, mutation of the AdpA-binding sequence did not affect the transcription of eshA in vivo, indicating that AdpA indirectly activates eshA transcription. Streptomycin production by an eshA deletion mutant grown on TSB plates was lower than that of the wild-type strain. However, the eshA deletion mutant grew and formed aerial mycelia and spores following the same time course as the wild-type strain on various media.
During a study of the distribution of actinomycetes in a subtropical zone in Japan, two strains forming short sporangiophores on the substrate mycelium were isolated from soil. The 16S rRNA gene sequences of the isolates indicated that these organisms formed a monophyletic cluster with members of the genus Virgisporangium in the family Micromonosporaceae. These strains formed narrow sporangia on short sporangiophores directly above the substrate mycelium. The sporangia contained motile spores. The strains contained 3-OH-diaminopimelic acid in the cell wall and glucose, ribose, mannose, galactose, xylose and 3-O-methylmannose as whole-cell sugars. The predominant menaquinones were MK-10(H4) and MK-10(H6). The diagnostic phospholipid was phosphatidylethanolamine. Cis 9 C17:1 and C17:0 were detected as the major cellular fatty acids. The G+C content of the DNA was 71.7 mol%. DNA–DNA relatedness analysis showed that the two isolates represented the same genomic species. The results of morphological, chemotaxonomic and 16S rRNA gene sequence analyses, as well as DNA–DNA hybridization studies, confirmed that these isolates certainly belonged to a new species of the genus Virgisporangium. We propose a novel taxon of the genus Virgisporangium as Virgisporangium aliadipatigenens sp. nov., with the type strain IR20-55T (= NBRC 105644T).
In this study, we investigated the taxonomic diversity of actinomycetes isolated from swine manure compost that had undergone self-heating. Pretreatment with sodium dodecyl sulfate and subsequent cultivation of the compost sample on humic acid-vitamin agar at 30 and 50°C resulted in the selective isolation of 125 actinomycete strains. These isolates were classified using a combination of morphological characterization and 16S rDNA sequence analysis, with sequence similarities of ≥97% and ≥99% used to identify isolates at genus and species level, respectively. The isolated actinomycete assemblage consisted of a diverse array of species, with the 125 isolates falling into 10 families, 15 genera, and 22 species. The most dominant thermophilic actinomycetes were Saccharomonospora (Sam.) viridis and Thermobifida fusca, followed by Actinomadura spp. Other species that were frequently isolated at 30°C included Glycomyces spp., Nocardiopsis (Nop.) alba, Nop. composta, Rhodococcus rhodochrous, and Sam. azurea.
Virginiamycin M1 (VM1), produced by Streptomyces virginiae, is a polyunsaturated macrolactone antibiotic belonging to the streptogramin family. The 10-kb regulatory gene cluster of VM1 production contains the varM gene, which encodes the type II ATP-binding cassette (ABC) transporter. This transporter is presumably involved in self-resistance to avoid suicide of the producer strain. Northern blot analyses revealed that varM expression is transcriptionally controlled by the VM1 molecule, but not by virginiamycin S, which is simultaneously produced to form a synergistic pair of the streptogramin family. Sequential addition of VM1 at increasing concentrations secured constitutive activation of varM expression, which suggested that the varM expression reflected an intracellular concentration of VM1. Heterologous expression of varM in Streptomyces lividans generated strains with no significant VM1 resistance. The mechanism underlying varM function in VM1 production is discussed.
The dereplication of isolates is an important step in the search for microorganisms that are producers of novel bioactive compounds. We have tested the RiboPrinter system, an automated ribotyping instrument, for the detection of polyketide synthase (PKS) genes and checking of strain similarity. Using a 1.1 kb probe derived from the ketosynthase (KS) domain of eryAIII instead of the standard ribosomal probe, the system was able to detect PKS genes in Streptomyces strains. Band patterns were similar among four Streptomyces violaceoruber strains harboring similar PKS genes. Conversely, some strains taxonomically identified as the same species showed different band patterns. Two unknown isolates were appropriately discriminated from their closest strains by band patterns. These results demonstrate that an automated Southern hybridization method employing a KS-probe will enable easy and rapid dereplication based on an index of PKS genes.
In the present study, Streptomyces sp. CW1 was isolated from a constructed wetland system mesocosm and identified as such based on 16S rDNA analysis, and additional biochemical properties were evaluated. This isolate was found to be halotolerant (up to 11% NaCl) and able to grow and utilize biopolymers such as: agarose, agar, gellan gum (a bacterial polysaccharide), polypectate and chitin as the sole carbon and energy source. Since the primary isolation was performed based on its capability to degrade phenol, the high cell yield coefficient showed rapid growth on phenol (0.82–0.98 mg dry biomass/mg phenol). Its halotolerance and the capability to biodegrade biopolymers found mainly in the marine environments, suggest on its primordial oceanic origin. In the present study some of its characteristics are described and discussed along with its beneficial use for wastewater purification processes in constructed wetland systems.
A total of 377 actinomycetes were isolated from eight soil samples collected from the Phatup Cave Forest Park and Phanangkhoi cave in northern Thailand. One hundred and sixty eight isolates (44.56%) were non-streptomycete based on their spore formations and isomeric forms of diaminopimelic acid. Among these, 50 isolates belong to the genus Micromonospora due to their characteristic single spore formation. Eleven randomly selected isolates of these rare actinomycetes were identified by using phenotypic data combined with 16S rDNA sequence-based phylogenetic analysis. Very rare genera such as Spirillospora, Catellatospora and Nonomuraea were also found. This is the first recorded isolation of Spirillospora and Nonomuraea from a cave soil. Phylogenetic analysis of 16S rRNA gene sequence data revealed that 5 of 11 randomly selected isolates might represent a new species. Two of them showed anti Gram-positive bacteria and anti-cancer activity. Caves are attractive places to look for new actinomycete species that might be a source of novel bioactive compounds.
To collect new kinds of microorganisms for screening of biologically active substances, we focused on spider materials (webs, cuticle, egg sac), previously uninvestigated sources of such organisms. Using a new method of pre-treatment with 70% ethanol, 1,159 strains of actinomycetes were isolated from 196 spider materials, based on their morphological features. Of these, 293 strains were identified as non-filamentous actinomycetes from their 16S rRNA gene sequences. More detailed examination indicated that 139 strains belonged to the suborders Micrococcineae, Frankineae and Propionibacterineae, and they included some novel strains of non-filamentous actinomycetes. Thus, spider materials provide a more useful source of non-filamentous actinomycetes than do soil samples.
A Gram-positive bacterium, designated strain Kis4-19T, was isolated from the intestinal tract of a fish, and its taxonomic position was investigated by a polyphasic approach. The cells of strain Kis4-19T were coccus-shaped, non-motile and non-sporulating. The peptidoglycan type of this organism was A3γ; LL-diaminopimeric acid (LL-A2pm) was the diagnostic diamino acid of the peptidoglycan. The predominant menaquinone was MK-8(H4), and the major fatty acids were iso-C15:0, iso-C14:0 and iso-C16:0. Galactose was detected as a cell-wall sugar. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and lyso-phosphatidylethanolamine. The DNA G+C content was 71.1 mol%. Sequencing analysis of the 16S rRNA gene indicated that strain Kis4-19T was closely related to Arsenicicoccus bolidensis CCUG 47306T (97.5%). However, DNA–DNA hybridization results and phenotypic characteristics revealed that the strain Kis4-19T differed from A. bolidensis. Therefore, strain Kis4-19T represents a novel species of the genus Arsenicicoccus, for which the name Arsenicicoccus piscis sp. nov. is proposed. The type strain is Kis4-19T (=NBRC 105830T =DSM 22760T).
The nucleoside antibiotic A-500359s are produced by Streptomyces griseus SANK 60196. During a screening program of A-500359s high-producing strains, several interesting mutants were isolated and classified into three groups according to two characteristics, spore-forming ability and the retention of a giant linear plasmid SGF180, as follows: [spore+ SGF180-], [bald SGF180+] and [bald SGF180-]. A-500359s production was markedly decreased in all the mutants [spore+ SGF180-] and completely lacking in all the bald-type mutants. To understand the regulatory mechanisms of A-500359s production in these mutants, co-cultivation analyses were conducted in several mutant combinations, and the effect of an addition of an EtOAc extract, which was prepared from a culture broth of an A-500359s producer, was tested. A-500359s production was clearly restored when mutant [spore+ SGF180-] was co-cultivated with mutant [bald SGF180+] and the A-500359s production of the mutant [bald SGF180+] was activated by the addition of an EtOAc extract. The results suggested that SGF180 plays an important role in A-500359s production and that A-500359s biosynthesis might be controlled by a low molecular weight compound, such as an A-factor-like compound, synthesized by the product of afsA gene that was lost in all the tested bald-type mutants.
Nucleocidin, a fairly broad antibacterial and trypanocidal agent produced by Streptomyces calvus, has the unique structure of 4′-α-fluoro-5′-O-sulfamoyl adenosine. This nucleoside antibiotic has been a target for organic synthesis in past decades; however, microbial large-scale production has not been established due to low yield and poor reproducibility. To activate the dormant secondary metabolism of S. calvus, we examined the effect of an rpoB mutation that was induced by ultraviolet light irradiation. The resulting rifampicin-resistant strains showed remarkably improved antibiotic activity, which was extracted by n-butanol and identified by Electron-Spray-Ionization Mass Spectrometry. DNA sequencing identified double mutations, C1309A and C1318A, in the rpoB gene (according to the numbering of Streptomyces coelicolor rpoB). The resulting amino acid substitutions, H437N and R440S, corresponded to two of the previously reported amino acid substitutions that allowed the activation of dormant actinorhodin production in S. lividans 66.
Semi-quantitative data and the identity or mass spectroscopic data are reported for 120 volatile metabolites from 26 selected Streptomyces isolates grown on suitable media. Approximately half of the metabolites are terpene derivatives.
An actinomycete, designated as CMU-PNK470, was isolated from the soil in Phanangkoi cave in northern Thailand and subjected to phenotypic and genotypic characterization. Morphological and chemical properties indicated that this isolate belonged to the genus Spirillospora. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed its placement in the genus Spirillospora and it was most closely related to Spirillospora albida (98.86%). This is the first reported isolation of Spirillospora albida from a cave habitat. The crude extract of this strain showed antimicrobial activity against three Gram-positive bacteria, Bacillus cereus and Methicillin-resistant Staphylococcus aureus with a MIC value of 23.1 μg/ml and Paenibacillus larvae with a MIC value of 185 μg/ml. It also reduced the number of viable Human Small lung cancer cells (NCI-H187) to less than 50% at a concentration of 10.18 μg/ml.
