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Streptacidiphilus gen. nov., acidophilic actinomycetes with wall chemotype I and emendation of the family Streptomycetaceae (Waksman and Henrici (1943)AL) emend. Rainey et al. 1997.

The taxonomic position of acidophilic actinomycetes selectively isolated from acidic soils and litter was examined using a polyphasic approach. The distinct 16S rDNA phyletic branch formed by representative strains was equated with related monophyletic clades that corresponded to the genera Kitasatospora and Streptomyces. The acidophilic isolates also exhibited a distinctive pH profile, a unique 16S rDNA signature, and contained major amounts of LL-diaminopimelic acid, galactose and rhamnose in whole-organism hydrolysates. It is proposed that these acidophilic actinomycetes be assigned to a new genus, Streptacidiphilus gen. nov., on the basis of genotypic and phenotypic differences. Three species were defined on the basis of DNA:DNA pairing and phenotypic data, namely, Streptacidiphilus albus sp. nov., the type species, Streptacidiphilus neutrinimicus sp. nov. and Streptacidiphilus carbonis sp. nov. Members of the genera Kitasatospora, Streptacidiphilus and Streptomyces share a number of key characteristics and form a stable monophyletic branch in the 16S rDNA tree. It is, therefore, proposed that the description of the family Streptomycetaceae be emended to account for properties shown by Kitasatospora and Streptacidiphilus species.

Bacterial Typing Techniques↗

Inhibition of epsilon-poly-L-lysine biosynthesis in Streptomycetaceae bacteria by short-chain polyols.

Antimicrobial epsilon-poly-L-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). In this study, by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.

Culture Media↗

Phylogenetic diversity of acidophilic sporoactinobacteria isolated from various soils.

Spore forming actinobacteria (sporoactinobacteria) isolated from soils with an acidic pH in Pinus thunbergii forests and coal mine waste were subjected to taxonomic characterization. For the isolation of acidophilic actinobacteria, acidified starch casein agar (pH adjusted to 4-5) was used. The numbers of actinobacteria growing in acidic media were between 3.2 x 10(4) and 8.0 x 10(6) CFU/g soil. Forty three acidophilic actinobacterial strains were isolated and their 16S rDNA sequences were determined. The isolates were divided into eight distinctive phylogenetic clusters within the variation encompassed by the family Streptomycetaceae. Four clusters among them were assigned to the genus Streptacidiphilus, whereas the remaining four were assigned to Streptomyces. The clusters belonging to either Streptomyces or Streptacidiphilus did not form monophyletic clade. The growth pH profiles indicated that the representative isolates grew best between pH 5 and 6. It is evident from this study that acidity has played a critical role in the differentiation of the family Streptomycetaceae, and also that different mechanisms might have resulted in the evolution of two groups, Streptacidiphilus (strict acidophiles) and neutrotolerant acidophilic Streptomyces. The effect of geographic separation was clearly seen among the Streptacidiphilus isolates, which may be a key factor in speciation of the genus.

Coal↗

Production of epsilon-poly-L-lysine by newly isolated Kitasatospora sp. PL6-3.

A novel epsilon-poly-L-lysine (epsilon-PL)-producing strain PL6-3 was isolated from soil, and was identified as a strain of Kitasatospora sp. This is the first detailed report of production of epsilon-PL by a strain in the genera of Kitasatospora. By controlling the culture pH at 4.0, the yield of epsilon-PL from PL6-3 reached 13.9 g/L after 120 h of cultivation in fed-batch fermentation. The morphological characteristics of Kitasatospora sp. PL6-3 in culture broth were different from those reported from strains of Streptomycetaceae, as no mycelium pellets were observed during the course of fermentation of PL6-3, which was beneficial to the assimilation of nutrition and secretion of the products. Furthermore, the molecular mass of the purified epsilon-PL from PL6-3 was determined to be 5.01 kDa by SDS-PAGE and 5.05 kDa by gel permeation chromatography, indicating that the epsilon-PL produced by this strain might be composed of 40 lysine residues. Usually, epsilon-PL with more lysine residues showed higher antimicrobial activity; however, it was difficult to obtain epsilon-PL with more than 36 lysine residues in this study. As a result, epsilon-PL from Kitasatospora sp. PL6-3, which contains more lysine residues than that from other strains, is more promising in the field of food preservatives.

Polylysine↗

Culturable marine actinomycete diversity from tropical Pacific Ocean sediments.

Actinomycetes were cultivated using a variety of media and selective isolation techniques from 275 marine samples collected around the island of Guam. In total, 6425 actinomycete colonies were observed and 983 (15%) of these, representing the range of morphological diversity observed from each sample, were obtained in pure culture. The majority of the strains isolated (58%) required seawater for growth indicating a high degree of marine adaptation. The dominant actinomycete recovered (568 strains) belonged to the seawater-requiring marine taxon 'Salinospora', a new genus within the family Micromonosporaceae. A formal description of this taxon has been accepted for publication (Maldonado et al., 2005) and includes a revision of the generic epithet to Salinispora gen. nov. Members of two major new clades related to Streptomyces spp., tentatively called MAR2 and MAR3, were cultivated and appear to represent new genera within the Streptomycetaceae. In total, five new marine phylotypes, including two within the Thermomonosporaceae that appear to represent new taxa, were obtained in culture. These results support the existence of taxonomically diverse populations of phylogenetically distinct actinomycetes residing in the marine environment. These bacteria can be readily cultured using low nutrient media and represent an unexplored resource for pharmaceutical drug discovery.

Actinomycetales↗

Studies on the aerobic non-symbiotic nitrogen-fixing bacteria, other than Azotobacter, in Egyptian soils.

Twenty isolates of micro-organisms capable of growing on nitrogen-deficient medium and found as contaminants in Azotobacter cultures were isolated from Egyptian soils and studied for their morphological, cultural, and physiological properties. These micro-organisms s are members of Rhizobiaceae, Pseudomonadaceae, Achromobacteriaceae, Enterobacteriaceae, Micrococcaceae, Bacillaceae, and Streptomycetaceae as well as some yeasts. In nitrogen-free medium the micro-organism fixed only small amounts of atmospheric nitrogen, hardly exceeding 3 ppm and because of their low sugar consumption rates, efficiences of N2-fixation sometimes reaching 18 mg nitrogen fixed/g carbon oxidized were recorded. Addition of 15 ppm combined nitrogen to the medium increased the amounts of nitrogen fixed to 3--9 ppm.

Aerobiosis↗

Antibiotic terpenoid chloro-dihydroquinones from a new marine actinomycete.

As part of our continuing interest in exploring the chemistry of actinomycete bacteria uniquely adapted for survival in ocean sediments, we encountered several new strains, which by 16S rDNA sequence-based phylogenetic analysis were recognized as members of a new genus (tentatively called MAR4) within the family Streptomycetaceae. We report here the isolation and structure elucidation of three new chlorinated dihydroquinones (1-3) and one previously reported analogue, 4, from our strain CNQ-525, isolated from ocean sediments collected at a depth of 152 m near La Jolla, California. The compounds formally possess new carbon skeletons, but are related to several previously reported metabolites of the napyradiomycin class. The structures of the new molecules, which possess significant antibiotic properties and cancer cell cytotoxicities, were assigned by comprehensive spectral measurements and by comparison with NMR and other spectral data from the antibiotic A80915C (5), the full stereostructure of which was recently assigned by X-ray diffraction methods.

Actinobacteria↗

Numerical classification of Streptomyces and related genera.

Four hundred and seventy-five strains, which included 394 type cultures of Streptomyces and representatives of 14 other actinomycete genera, were studied. Overall similarities of these strains for 139 unit characters were determined by the SSM and SJ coefficients and clustering by the UPGMA algorithm. Test error and overlap between the phena defined were within acceptable limits. Cluster-groups were defined by the SSM coefficient at the 70.1% similarity (S) level and by the SJ coefficient at the 50% S-level. Clusters were distinguished at the 77.5% SSM and 63% SJ S-levels. Groupings obtained with the two coefficients were generally similar, but there were some changes in the definition and membership of cluster-groups and clusters. The phenetic data obtained, together with those from previous diverse studies, indicated that the genera Actinopycnidium, Actinosporangium, Chainia, Elytrosporangium, Kitasatoa and Microellobosporia should be reduced to synonyms of Streptomyces, while Intrasporangium, Nocardioides and Streptoverticillium remained as distinct genera in the family Streptomycetaceae. Nocardiopsis dassonvillei also showed strong phenetic affinity to Streptomyces, despite its chemotaxonomic differences. Actinomadura sensu stricto was phenetically distinguishable from Streptomyces and 'Nocardia' mediterranea was recognized as a taxon distinct from both these genera and from Nocardia sensu stricto. Most of the Streptomyces type cultures fell into one large cluster-group. At the 77.5% SSM S-level, they were recovered in 19 major and 40 minor clusters, with 18 strains recovered as single member clusters. The status of the latter as species was therefore confirmed. Most of the minor clusters, consisting of two to five strains, can also be regarded as species. The major clusters varied in size (from 6 to 71 strains) and in there homogeneity. Therefore, it is suggested that they be regarded as species-groups until further information is available. The results provide a basis for the reduction of the large number of Streptomyces species which have been described. They also demonstrate that the previous use of a limited number of subjectively chosen characters to define species-groups or species has resulted in artificial classifications.

Culture Media↗

Neutrotolerant acidophilic Streptomyces species isolated from acidic soils in China: Streptomyces guanduensis sp. nov., Streptomyces paucisporeus sp. nov., Streptomyces rubidus sp. nov. and Streptomyces yanglinensis sp. nov.

The taxonomic status of six neutrotolerant acidophilic streptomycetes isolated from acidic soils in Yunnan Province, China, was established using a polyphasic approach. The morphological and chemotaxonomic characteristics revealed that the isolates belong to the genus Streptomyces. Almost complete 16S rRNA gene sequences of the isolates were determined and aligned with available corresponding sequences of representatives of the family Streptomycetaceae; phylogenetic trees were inferred using four tree-making algorithms. The isolates formed a distinct, albeit heterogeneous, subclade in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces yeochonensis, but were readily distinguishable from the latter using DNA-DNA hybridization and phenotypic data. It was evident from the genotypic and phenotypic data that the isolates belonged to four novel Streptomyces species, for which the following names are proposed: Streptomyces guanduensis sp. nov. (type strain 701T=CGMCC 4.2022T=JCM 13274T), Streptomyces paucisporeus sp. nov. (type strain 1413T=CGMCC 4.2025T=JCM 13276T), Streptomyces rubidus sp. nov. (type strain 13c15T=CGMCC 4.2026T=JCM 13277T) and Streptomyces yanglinensis sp. nov. (type strain 1307T=CGMCC 4.2023T=JCM 13275T); isolates 317 and 913 belong to this latter species.

Bacterial Typing Techniques↗

Distribution of microbes producing antimicrobial epsilon-poly-L-lysine polymers in soil microflora determined by a novel method.

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), epsilon-poly-L-lysine (epsilon-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected epsilon-PL producers by electrostatic interaction with the secreted basic polymers. All epsilon-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family Streptomycetaceae and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of epsilon-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an epsilon-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.

Bacteria↗

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.

Amino Acid Sequence↗

Studies on antibiotic 230 as a molluscicide.

In the course of studying molluscicidal effects of microorganisms, a microorganism belonging to Streptomycetaceae, Streptomyces griseolus (Strain 230) was isolated from soil and a certain microbial ingredient proved to have a molluscicidal activity. The effective substance was named antibiotic 230. As a result of several investigations on the antibiotic, the following chemical features were made clear. The chemical substance was an indol-carbazole-heterocyclic compound, the molecular weight 446 and the molecular formula C28H26N4O3. The antibiotic demonstrated a molluscicidal effect against Oncomelania snails and their eggs both in the laboratory and in the field. The compound had a wide bacteriostasis spectrum and a slight toxicity to fish in addition to the above, but it was non-toxic to plants.

Animals↗

[Study on biodiversity of type I & II polyketide synthesis genes positive microorganisms].

Some soil samples were collected from different places in Yunnan Provinces, China. 876 bacteria or actinomycete strains were isolated using Glucose-Peptone-Yeast extract agar, Starch-Casein agar and Glycerol-Asparagine agar with these soil samples, of which about 100 strains belong to genus Streptomyces and the others belong to rare actinomycetes or bacteria. With polyketide synthesis gene screening, 75 strains were picked up as type I & II polyketide synthesis gene positive strains. Then 10 strains were chosen for 16S rDNA amplification and systematic analysis based on comparing results with their anti-bacteria activity, morphology, and physiological characteristics analysis. They were classified to be at least 7 families and 8 genera, such as genus Streptomyces of the family Streptomycetaceae, two genera Streptosporangium and Nonomuraea of the family Streptosporangiaceae, genus Mycobacterium of the family Mycobacteriaceae, genus Nocardia of the family Nocardiaceae, genus Achromobacter of the family, another two strains belong to the suborder Micrococcineae and the family Oxalobacteraceae', respectively. Eight of them were classified into six potential novel species and two new genera with polyphasic taxonomic methods. The results showed that designing new strategies for isolation and identification of microorganisms from natural environments was the key step to exploit microbial resources.

Actinobacteria↗