Actinomadura mexicana sp. nov. and Actinomadura meyerii sp. nov., two novel soil sporoactinomycetes

The taxonomic position of two soil isolates, strains A288(T) and A290(T) [provisionally assigned to the genus Actinomadura] was clarified in a polyphasic study. The organisms showed a combination of chemotaxonomic and morphological properties typical of actinomadurae. They also formed distinct phyletic lines in the 16S rRNA Actinomadura gene tree; strain A288(T) was associated with A. nitritigenes whereas strain A290(T) was closely related to a group that consisted of A. citrea, A. coerulea, A. glauciflava, A. luteofluorescens and A. verrucosospora. Strains A288(T) and A290(T) showed key phenotypic features which readily distinguish them from one another and from representatives of related validly described species of Actinomadura. It is proposed that the two organisms be classified as new species of the genus Actinomadura. The names proposed for the new taxa are Actinomadura mexicana (A290(T) = DSM 44485(T) = NRRL B-24203(T)), and Actinomadura meyerii (A288(T) = DSM 44485(T) = NRRL B-24203(T)).     

A taxonomic review of the genus Microbispora and a proposal to transfer two species to the genus Actinomadura and to combine ten species into Microbispora rosea

We conducted a taxonomic review of the genus Microbispora using chemotaxonomic and DNA-DNA hybridization techniques, and reached the following conclusions: Microbispora viridis should be transferred to the genus Actinomadura as Actinomadura rugatobispora comb. nov., nom. nov. (type strain SF2240 = IFO 14382 = JCM 3366) and Microbispora echinospora should be transferred to the genus Actinomadura as Actinomadura echinospora comb. nov. (type strain JCM 3148 = ATCC 27300). We also propose that Microbispora rosea, Microbispora amethystogenes, Microbispora chromogenes, Microbispora diastatica, Microbispora indica, Microbispora karnatakensis and Microbispora parva should be combined into the species Microbispora rosea subsp. rosea (type strain JCM 3006 = ATCC 12950), and that Microbispora aerata, Microbispora thermodiastatica and Microbispora thermorosea should be combined and transferred to the new subspecies Microbispora rosea subsp. aerata comb. nov. (type strain IFO 12581 = ATCC 15448). Microbispora bispora clearly differs from these ten strains at the species level.     

Majority of Actinomadura clinical isolates from sputa or bronchoalveolar lavage fluid in Japan belongs to the cluster of Actinomadura cremea and Actinomadura nitritigenes, and the description of Actinomadura chibensis sp. nov

In Japan during 1996-2004, 21 actinomycete strains that have madurose as the diagnostic cell-wall sugar and show true branching in their substrate and aerial mycelia were isolated from sputa or bronchoalveolar lavage (BAL) fluid of patients with pulmonary infections or who were suspected of having related infections. Chemotaxonomic studies showed that all the isolates belong to the genus Actinomadura. Among them, six and seven strains were classified respectively into clusters of Actinomadura nitritigenes and Actinomadura cremea based on 16S rDNA analyses because their 16S rDNA similarities to those respective species were greater than 99.5%. To our knowledge, this is first report that strains of above two species were isolated from clinical specimens. Neither Actinomadura madurae nor Actinomadura pelletieri strain was isolated, and one new species, Actinomadura chibensis, was proposed; the remaining seven strains were not assigned into any known species, suggesting the presence of another new Actinomadura species.     

Actinomadura of the sierozem soils of Turkmenia and their antagonistic properties

Occurrence of actinomycetes of the genus Actinomadura in the grey earth soils of Turkmenistan was studied. The Gause organic medium No. 2 with rubomycin proved to be most favourable for isolation of Actinomadura from the soil samples. The number of Actinomadura in 1 g of the soil varied from 3 to 686 000 depending on the sample which constituted 0.2-11 per cent of the total number of the actinomycetes. It was shown that Actinomadura are rather widely distributed in the grey earth soils of Turkmenistan. They were detected practically in all the soil samples tested. The number of Actinomadura significantly depended on the level of the soil cultivation. The number of Actinomadura in the samples of cultivated soils was higher than that in the virgin land samples. The isolates were classified as belonging to 16 species of actinomadura, 5 of which proved to be new Actinomadura species. It was shown with the streak plate method that Actinomadura had moderate antagonistic properties. The majority of the isolates were active against gram-positive organisms.     

Echinomycin production by Actinomadura sp. INA 654]

An actinomycete strain designated as Actinomadura sp. INA 654 was isolated from a chernozem soil sample in the Voronezh Region by the soil sample treatment with millimetric waves (EHF band). The strain produced an antibiotic complex of 2 components, named A-654-I and A-654-II. Investigation of their physico-chemical properties showed that A-654-I was identical to echinomycin, a heteropeptide lactone of the quinoxaline group with antitumor activity, while A-654-II proved to be likely a new natural compound. Production of echinomycin by a representative of the Actinomadura genus was detected for the first time. Up to now, only representatives of the Streptomyces genus were known to produce echinomycin.     

Actinomadura fibrosa sp. nov. isolated from soil

A new species of the genus Actinomadura which belongs to the Actinomadura madurae group of Goodfellow et al. was isolated from soil collected in Togo, West Africa. Traditional taxonomic methods plus contemporary fatty acid analysis techniques were used to establish the position of this species. Both physiological characteristics and fatty acid composition differentiate this strain from previously described species. This culture produces a new polyether antibiotic. It is characterized by the production of white to pink aerial hyphae on a limited number of media. The aerial hyphae appear asporogenous, forming thick fibers and projections instead of true spores. The reverse side is a distinctive reddish orange. This organism is resistant to 5% NaCl and grows at temperatures between 20 and 45 degrees C. Whole cells contain meso-diaminopimelic acid, galactose, glucose, mannose, madurose, phosphatidylinositol, and diphosphatidylglycerol. The menaquinones detected were MK-9(H6) and minor amounts of MK-9(H8). The name proposed for this new species is Actinomadura fibrosa; the type strain is strain NRRL 18348.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 as a novel approach to actinomycete taxonomy: application to the genera Actinomadura and Microtetraspora.

Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura, and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30 preparations from several strains proved that we were indeed dealing with the specified protein homologous to ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.     

Molecular-genetic and chemotaxonomic studies on Actinomadura and Nocardiopsis

The relationships of 24 strains of 13 species of Actinomadura and 4 strains of Nocardiopsis dassonvillei were determined by nucleic acid hybridization studies. DNA-rRNA cistron similarity and DNA homology values reveal that Actinomadura is genetically heterogeneous. One cluster contained the type species Actinomadura madurae, Actinomadura pelletieri, Actinomadura verrucosospora, Actinomadura malachitica, Actinomadura citrea and 'Actinomadura kijaniata'. A second cluster embraced Actinomadura pusilla, Actinomadura roseoviolacea, Actinomadura libanotica, Actinomadura roseola and Actinomadura ferruginea. The internal homogeneity of the two Actinomadura clusters was demonstrated by a high similarity in the menaquinone and fatty acid composition of the strains enclosed. Actinomadura spadix, Actinomadura spiralis, and two strains of Actinomadura madurae were found to be unrelated to each other and could not be allocated to one of the two major Actinomadura clusters. Nocardiopsis dassonvillei was genetically and phenotypically clearly separated from all Actinomadura species investigated.     

Distribution of teichoic acids in cultures of the genus Actinomadura

The occurrence of teichoic acids in cultures of Actinomadura genus was studied. All 30 strains examined in this survey contained alditol phosphate polymers. Most of the cultures had poly(glycerol phosphate) teichoic acids. Some of the poly(glycerol phosphate) chains bear madurose as a glycosyl substituent. In seven cultures glycerol teichoic acids with an unusual localization of the phosphodiester linkage were detected. Ribitol teichoic acids were found in six organisms.     

Heterogeneity of the genus Actinomadura Lechevalier a. Lechevalier]

Studies of the morphology of Actinomadura spp. have shown that the genus comprises several morphological types of actinomycetes which differ by the presence and structure of the sporophores on the aerial and substrate mycelium. The actinomycetes differ also by the fatty acid composition of lipids in the mycelium, and can be subdivided into three groups: (1) fatty acids with a straight chain prevail; (2) fatty acids with branched chains of the iso- and anteiso structure predominate; and (3) fatty acids with straight and branched chains are contained in almost equal amounts. Therefore the genus separated on the basis of differences in the composition of the cell wall was heterogeneous according to the properties used in the taxonomy of the actinomycetes at the level of genus. These data prove that morphological properties have to be taken into account in the taxonomy of the actinomycetes.     

Antimicrobial compounds produced by Actinomadura sp. AC104 isolated from an Algerian Saharan soil

During a search for nonpolyenic antifungal antibiotics, an actinomycete designated AC104 was isolated from a Saharan soil sample by a dilution agar plating method using a chitin - vitamins B medium supplemented with rifampicin. Isolate AC104 presented the morphological and the chemical characteristics of the genus Actinomadura. On the basis of 76 physiological tests and 16S rDNA analysis, this isolate was determined to be quite different from the known species of Actinomadura. It is active against filamentous fungi and both Gram-positive and Gram-negative bacteria. The production of antibiotic substances was investigated using several culture media. The highest antimicrobial activities were obtained on ISP2 medium. The benzenic extract contained five bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 104A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase high-pressure liquid chromatography. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass, and 1H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species.     

Ultrastructure of the mycelium and spores of Actinomadura fastidiosa sp. nov]

A new species of the Actinomadura genus, A. fastidiosa sp. nov., is described. The ultrastructure of the vegetative mycelium and spores of this organism was studied. The vegetative cells have a multilayered cell wall, often consisting of five layers with different thickness and electron density. The spores are similar to the vegetative cells by their inner structure but have a thicker wall.     

Selective isolation of actinomycetes of the genus Actinomadura from soil

Some approaches to the selective isolation of actinomycetes of the genus Actinomadura from soil are described. The approach that involves thermal treatment of soil samples and their plating onto Gauze 1 medium with the antibiotics nystatin, nalidixic acid, and rubomycin provides for an increased amount of actinomaduras isolated from the soil actinomycete complex and for a decreased amount of streptomycetes.     

Isolation of Actinomycetales from the soil of Kazakhstan on selective media with antibiotics

About 3000 actinomycetes were isolated from various soil samples collected in 11 regions of Kazakhstan. 62.7 per cent of them proved to be antagonists. For isolation of the strains, selective media supplemented with antibiotics were used. Kanamycin promoted growth of Actinomadura and Nocardia. Rubomycin promoted growth of Actinomadura. Tavromycetin and roseofungin were used as selective agents for the first time. Tavromycetin favoured isolation of Actinomadura and Nocardia. Roseofungin favoured isolation of Actinomadura. Light chestnut and serozemic soils were the most rich in antagonists (67.1 and 61.3 per cent, respectively) while saline and chestnut soils were the poorest in antagonists (32.2 and 30.6 per cent, respectively). Actinomadura were more frequent in light-chestnut light-loamy and serozemic soils. Half of the antibiotics isolated in the form of concentrates were identified with the known antibiotics or classified as belonging to various groups. A culture producing a novel antibiotic was isolated.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.     

New species, Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. and their antagonistic properties

Two new species of Actinomadura isolated from soil samples of the Turkmen SSR, i.e. Actinomadura fulvescens sp. nov. and Actinomadura turkmeniaca sp. nov. are described. The first species is characterized by formation of short (1-2 turns) spiral spore chains, smooth spores, scanty white aerial mycelium, colourless or yellowish substrate mycelium on synthetic media and brownish-yellow substrate mycelium and soluble pigment of the same colour on organic media. No melanoid pigment is secreted. The type culture is designated as INA 3321. The cultures of A. fulvescens show antibiotic activity. A. turkmeniaca is characterized by formation of short straight or spiral spore chains, smooth spores, scanty white aerial mycelium, substrate mycelium and soluble pigment of pinkish-violet colour, absence of melanoid pigment. The type culture is designated as INA 3344. The strains of this species have low antibiotic activity. The study on the use of carbon sources by the representatives of 7 species (9 strains) of Actinomadura showed that the majority of the cultures (5 species, 7 strains) produced no growth on the Pridham and Gottlieb medium (ISP-9) with various carbon sources, including glucose. Possibly this medium cannot be used as the main medium for investigation of the spectrum of carbohydrate consumption in Actinomadura.     

Development of a self-cloning system for Actinomadura verrucosospora and identification of polyketide synthase genes essential for production of the angucyclic antibiotic pradimicin.

A self-cloning system for Actinomadura verrucosospora, a producer of the angucyclic antibiotic pradimicin A (PRM A), has been developed. The system is based on reproducible and reliable protoplasting and regeneration conditions for A. verrucosospora and a novel plasmid vector that consists of a replicon from a newly found Actinomadura plasmid and a selectable marker cloned from the Actinomadura strain. The system has an efficiency of more than 10(5) CFU/microgram of DNA. Using this system, we have cloned and identified the polyketide synthase (PKS) genes essential for PRM A biosynthesis from A. verrucosospora. Nucleotide sequence analysis of the 3.5-kb SalI-SphI fragment showed that ketosynthase subunits (open reading frame 1 [ORF1] and ORF2) of the essential PKS genes have strong similarities (59 to 89%) to those for angucyclic antibiotic biosynthesis.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein: a new approach for actinomycete taxonomy.

The ribosomal (r)-proteins from eleven Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). The protein patterns were specific for each species. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and 2D-PAGE analysis of the r-protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that S. griseolavendus was a variant of S. lavendulae. Actinomycete r-protein AT-L30 exhibited electrophoretic mobility that is specific for each genus. On the basis of this observation, we analyzed AT-L30 r-proteins from numerous strains of species belonging to the genera Actinomadura, Microtetraspora, Streptosporangium, Nocardia, Rhodococcus and mycolate-less wall chemotype-IV actinomycetes. The results strongly supported the conclusions of previous work and thus proved the efficacy of r-protein analysis as a novel approach for taxonomy of actinomycetes.     

Diversity of antifungal actinomycetes in various vegetative soils of Korea

Diversity of actinomycetes and their antifungal activities against some plant pathogenic fungi were examined in various vegetative soils from 14 different sites in the western part of Korea. Actinomycete counts ranged from 1.17 x 10(6) to 4.20 x 10(6) cfu x g(-1) dried soil. A total of 1510 actinomycetes were isolated from the soil samples. Streptomyces was predominant in soils with a pH range of 5.1-6.5, 9.1-13.0% moisture, and 9.1-11.0% organic matter. Most Micromonospora, Dactylosporangium, and Streptosporangium were distributed in soils with pH 4.0-5.0, 2.0-9.0% moisture, and 4.0-7.0% organic matter. Actinomadura and nocardioform actinomycetes were abundant in soils with pH 4.0-5.0 and 13.1-20.0% moisture and with 9.1-11.0 and 4.0-7.0% organic matter, respectively. Populations of Streptomyces were predominant in all the soils, but were highest in grassland and lowest in mountain-forest soils. Micromonospora was most abundant in pepper-field soil and nocardioform actinomycetes were highest in rice paddy field soil. Dactylosporangium was predominant in lake-mud sediments and pepper-field soil, Streptosporangium in lake-mud sediments, and Actinomadura in mountain-forest soil. Antifungal actinomycetes were abundant in orchard soil and lake mud. More than 50% of antifungal isolates from most soils were classified as genus Streptomyces. Actinomycete isolates that showed strong antifungal activity against Alternaria mali, Colletotrichum gloeosporioides, Fusarium oxysporum f.sp. cucumerinum, and Rhizoctonia solani were predominant in pepper-field soils, whereas those against Magnaporthe grisea and Phytophthora capsici were abundant in radish-field soils.