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Identification of coryneform bacteria and related taxa by Fourier-transform infrared (FT-IR) spectroscopy.

An extensive Fourier-transform infrared (FT-IR) spectroscopy database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae (Actinomycetales, Actinobacteria) as well as other morphologically similar genera was established. The database consists of averaged IR spectra from 730 reference strains, covering 220 different species out of 46 genera. A total of 192 species are represented by type strains. The identity of 352 reference strains was determined by comparative 16S rDNA sequence analysis and, if necessary, strains were reclassified accordingly. FT-IR frequency ranges, weights and reproducibility levels were optimized for this section of high-G+C gram-positive bacteria. In an internal validation, 98.1% of 208 strains were correctly identified at the species level. A simulated external validation which was carried out using 544 strains from 54 species out of 16 genera resulted in a correct identification of 87.3% at the species level and 95.4% at the genus level. The performance of this identification system is well within the range of those having been reported in the literature for the identification of coryneform bacteria by phenotypical methods. Coryneform and related taxa display a certain degree of overlapping distribution of different taxonomical markers, leading to a limited differentiation capacity of non-genotypical identification methods in general. However, easy handling, rapid identification within 25 h starting from a single colony, a satisfactory differentiation capacity and low cost, render FT-IR technology clearly superior over other routine methods for the identification of coryneform bacteria and related taxa.

Actinomycetales↗

New genus-specific primers for the PCR identification of novel isolates of the genera Nocardiopsis and Saccharothrix.

The taxonomic position of the genera Saccharothrix and Nocardiopsis has evolved in recent years to accommodate an increasing number of actinomycete strains that cannot be clearly distinguished by morphological characters. More recently, the taxonomic reorganization of the genus Saccharothrix has determined the creation of new, related genera, increasing the complexity of the identification of this taxon. Nevertheless, today these genera can still only be identified by applying chemotaxonomic and molecular criteria, and no other tools are available for the rapid distinction of members of the two genera. Phylogenetic analysis based on 16S rDNA nucleotide sequences of reference strains has shown that both genera represent complete distinct lineages within the order Actinomycetales. Differences in the nucleotide sequences of the 16S rDNAs of reference strains were used to design two pairs of genus-specific primers to identify novel members of the genera Nocardiopsis and Saccharothrix by PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates that exhibited morphological characteristics common to both genera. The diversity and taxonomic position of the isolates identified with these tools is also discussed.

Actinomycetales↗

Glaucescin, a bacteriocin-like substance from Streptomyces glaucescens.

Streptomyces glaucescens ETH 22794 produced a variety of antibiotic substances. Besides low molecular weight antibiotics like hydroxystreptomycin and the tetracenomycins, this strain excreted glaucescin, a high molecular weight product with bacteriocin-like properties. In plate tests the antagonism of glaucescin against Streptomyces canadiensis was masked by the large inhibition zone caused by the tetracenomycins. Glaucescin activity was revealed when a tetracenomycin-resistant mutant of S. canadiensis NRRL 3155 was used as an indicator. Glaucesin was produced on complex and minimal solid and liquid media. It was not inducible by mitomycin C. The killing activity of glaucescin was thermolabile and resistant to DNAase, RNAase, various proteinases, and lipase. Its apparent molecular weight was estimated as 196000 by gel filtration and glycerol gradient centrifugation. Glaucescin preferentially killed outgrowing spores of S. canadiensis. Resting spores and mycelium were considerably less sensitive to the inhibitor, and adsorption of glaucescin by S. canadiensis paralleled sensitivity. The activity spectrum of the bacteriocin was restricted to spore-forming Actinomycetales. Non spore-forming nocardiae and a variety of Gram-positive and Gram-negative bacteria were resistant to glaucescin.

Actinomycetales↗

A phylogenetic analysis of the family Dermatophilaceae.

The comparative analysis of the 16S ribosomal ribonucleic acid (rRNA) of Geodermatophilus obscurus DSM 43060 and Dermatophilus congolensis DSM 43037 revealed that these members of the family Dermatophilaceae were only remotely related. While G. obscurus represented an individual and separate line of descent within the phylogenetically defined order Actinomycetales, D. congolensis was closely related to representatives of Arthrobacter, Micrococcus, Cellulomonas, Brevibacterium, Promicromonospora and Microbacterium.

Actinomycetales↗

Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes.

The whiB sporulation gene of Streptomyces coelicolor was shown [Davis, N. K. & Chater, K. F. (1992). Mol Gen Genet 232, 351-358] to encode a small, cysteine-rich putative transcription factor unlike any that had been described previously. The large database of DNA sequences of mycobacteria (like Streptomyces, members of the Actinomycetales) has revealed a family of genes encoding proteins related to WhiB. Mycobacterium tuberculosis contains at least six such genes (whiB homologues in mycobacteria: whmA-F) and a likely seventh, whmG. Using conserved features of Whm proteins, a PCR-based approach led to the discovery that S. coelicolor A3(2) contains several similar genes. Cloning and sequencing of these whiB-like (wbI) genes revealed likely orthologues of four of the whm genes of M. tuberculosis. In all, S. coelicolor contains at least five wbI genes in addition to whiB itself. All five were shown by RT-PCR to be transcribed. A Southern blotting survey using each wbI gene as a probe showed that nearly all of a series of representatives of ten actinomycete genera (including morphologically simple organisms) contain close homologues of several wbI genes, suggesting that the ancient progenitor of all these organisms already contained a family of such genes, which have not been found in any other organisms.

Actinomycetales↗

Comparison of the 23S ribosomal RNA genes and the spacer region between the 16S and 23S rRNA genes of the closely related Mycobacterium avium and Mycobacterium paratuberculosis and the fast-growing Mycobacterium phlei.

The 23S rDNA sequences of Mycobacterium paratuberculosis, M. avium and M. phlei and the sequences of the spacer regions between the 16S and 23S rRNA genes were determined. The overall 23S rDNA sequence identity between M. paratuberculosis and M. avium was 99.7% (nine mismatches), showing the very close relatedness of these mycobacteria. Evolutionary distances between the five known mycobacterial 23S rRNA/rDNA sequences and those of other Gram-positive G + C-rich bacteria were determined. The 23S rDNA sequences of mycobacteria showed two inserted regions compared to the other bacteria. A mycobacterial unique region contained one mismatch between M. paratuberculosis and M. avium. An Actinomycetales-specific insertion, consisting of 111 nucleotides, was completely identical for M. paratuberculosis and M. avium. The sequence of the intergenic spacer region between 16S and 23S rDNA had a length of 278 bp for M. paratuberculosis and M. avium with only two mismatches. The spacer region of the fast-growing M. phlei was 85 bp longer. No tRNA-encoding region was found in the spacer region.

Actinomycetales↗

Serinicoccus marinus gen. nov., sp. nov., a novel actinomycete with L-ornithine and L-serine in the peptidoglycan.

A Gram-positive bacterial strain containing L-ornithine as the diagnostic diamino acid was isolated from a sea-water-sample from the East Sea, Korea. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain JC1078T represents a phyletic line within the suborder Micrococcineae of the order Actinomycetales, adjacent to the genus Ornithinimicrobium. The highest sequence similarity values to the isolate were observed against Ornithinimicrobium humiphilum (94.3 %) and Kytococcus sedentarius (94.1 %). The strain was strictly aerobic and moderately halophilic with optimal growth at 2-3 % (w/v) NaCl. Cells were non-motile, non-sporulating and coccoid-shaped. The cell wall contains L-ornithine, glutamic acid, alanine, glycine and serine. The major menaquinone was MK-8(H4). The predominant cellular fatty acids were of the iso- and anteiso-methyl-branched types. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unknown glycolipid. The acyl type of the glycan chain of peptidoglycan is acetyl. The DNA G + C content was 72 mol%. The combination of physiological, biochemical and chemotaxonomical data clearly separated the marine isolate from other members of the suborder Micrococcineae. On the basis of polyphasic evidence, it is proposed to classify strain JC1078T in a novel genus and species, for which the name Serinicoccus marinus gen. nov., sp. nov. is proposed. The type strain is JC1078T (= IMSNU 14026T = KCTC 9980T = DSM 15273T).

Actinomycetales↗

Oryzihumus leptocrescens gen. nov., sp. nov.

Three novel strains were isolated from a soil sample collected in Japan using GPM agar plates supplemented with superoxide dismutase and/or catalase. The strains were Gram-positive, catalase-positive, irregular rod-shaped bacteria with meso-diaminopimelic acid as a peptidoglycan diagnostic diamino acid, and the acyl type of the peptidoglycan was acetyl. The major menaquinone was MK-8(H4). Mycolic acids were not detected. The G+C content of the DNA was 72-73 mol%. On the basis of morphological and chemotaxonomic properties and a phylogenetic analysis using 16S rRNA gene sequences, these strains were classified as a novel genus and species, Oryzihumus leptocrescens gen. nov., sp. nov., in the family Intrasporangiaceae of the order Actinomycetales. The type strain is KV-628T (=NRRL B-24347T=JCM 12835T=NBRC 100762T).

Actinomycetales↗

Demequina aestuarii gen. nov., sp. nov., a novel actinomycete of the suborder Micrococcineae, and reclassification of Cellulomonas fermentans Bagnara et al. 1985 as Actinotalea fermentans gen. nov., comb. nov.

An actinobacterial strain containing demethylmenaquinone DMK-9(H(4)) as the diagnostic isoprenoid quinone was isolated from a tidal flat sediment sample, from South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain JC2054(T) represents a distinct phyletic line within the suborder Micrococcineae of the order Actinomycetales. The closest phylogenetic neighbour was Cellulomonas fermentans, with 94.7 % 16S rRNA gene sequence similarity. The novel isolate was strictly aerobic and slightly halophilic, with optimum growth occurring in 2-4 % (w/v) NaCl. Cells were non-motile, non-sporulating and rod-shaped. The peptidoglycan type was of the A-type of cross-linkage. l-ornithine was the diamino acid and d-glutamate represented the N-terminus of the interpeptide bridge. The predominant fatty acids were anteiso-branched and straight-chain fatty acids. The major polar lipids were phosphatidylinositol, diphosphatidylglycerol and an unknown phospholipid. The menaquinone composition of C. fermentans was determined to be MK-10(H(4)), MK-9(H(4)) and MK-8(H(4)) in the ratio 56 : 2 : 1. On the basis of the polyphasic evidence presented in this study, it is proposed that strain JC2054(T) should be classified as representing a novel genus and species of the suborder Micrococcineae, with the name Demequina aestuarii gen. nov., sp. nov. The type strain is JC2054(T) (=IMSNU 14027(T)=KCTC 9919(T)=JCM 12123(T)). In addition, it was clear from the phylogenetic analysis and chemotaxonomic data that C. fermentans does not belong to the genus Cellulomonas or any other recognized genera. Therefore, C. fermentans should be reclassified as representing a novel genus, for which the name Actinotalea fermentans gen. nov., comb. nov. is proposed, with strain DSM 3133(T) (=ATCC 43279(T)=CFBP 4259(T)=CIP 103003(T)=NBRC 15517(T)=JCM 9966(T)=LMG 16154(T)) as the type strain.

Actinomycetales↗

Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes.

The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil (Jospeh et al., Appl Environ Microbiol 69, 7210-7215, 2003). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics.

Actinomycetales↗

Influence of maize mucilage on the diversity and activity of the denitrifying community.

In order to understand the effect of the maize rhizosphere on denitrification, the diversity and the activity of the denitrifying community were studied in soil amended with maize mucilage. Diversity of the denitrifying community was investigated by polymerase chain reaction (PCR) amplification of total community DNA extracted from soils using gene fragments, encoding the nitrate reductase (narG) and the nitrous oxide reductase (nosZ), as molecular markers. To assess the underlying diversity, PCR products were cloned and 10 gene libraries were obtained for each targeted gene. Libraries containing 738 and 713 narG and nosZ clones, respectively, were screened by restriction fragment analysis, and grouped based on their RFLP (restriction fragment length polymorphism) patterns. In all, 117 and 171 different clone families have been identified for narG and nosZ and representatives of RFLP families containing at least two clones were sequenced. Rarefaction curves of both genes did not reach a clear saturation, indicating that analysis of an increasing number of clones would have revealed further diversity. Recovered NarG sequences were related to NarG from Actinomycetales and from Proteobacteria but most of them are not related to NarG from known bacteria. In contrast, most of the NosZ sequences were related to NosZ from alpha, beta, and gammaProteobacteria. Denitrifying activity was monitored by incubating the control and amended soils anaerobically in presence of acetylene. The N2O production rates revealed denitrifying activity to be greater in amended soil than in control soil. Altogether, our results revealed that mucilage addition to the soil results in a strong impact on the activity of the denitrifying community and minor changes on its diversity.

Actinomycetales↗

Taxonomic characterization of two rubber degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hyper variable regions of 16S rDNA sequences.

Two cis-1,4-polyisoprene (isoprene rubber) degrading bacteria, strains VH2 and Y2K, were identified as strains of the species Gordonia polyisoprenivorans belonging to the Corynebacterineae, a suborder of the order Actinomycetales. Both showed characteristic growth and degradation of isoprene rubber as described previously for the type strain of G. polyisoprenivorans Kd2 (DSM 44302(T)). For strain VH2 the chemotaxonomic properties were investigated, and DNA-DNA hybridization experiments with the type strain revealed the affiliation to the species G. polyisoprenivorans. The comparison of the 16S rDNA sequences, and especially hyper variable regions of these, led to the classification of strain Y2K to the same species. At present, the species G. polyisoprenivorans comprises three different isolates which share the ability to degrade isoprene rubber potently but which were obtained from different geographic regions.

Actinomycetales↗

Investigations into an unknown organism on the martian meteorite Allan Hills 84001.

Examination of fracture surfaces near the fusion crust of the martian meteorite Allan Hills (ALH) 84001 have been conducted using scanning electron microscopy (SEM) and atomic force microscopy (AFM) and has revealed structures strongly resembling mycelium. These structures were compared with similar structures found in Antarctic cryptoendolithic communities. On morphology alone, we conclude that these features are not only terrestrial in origin but probably belong to a member of the Actinomycetales, which we consider was introduced during the Antarctic residency of this meteorite. If true, this is the first documented account of terrestrial microbial activity within a meteorite from the Antarctic blue ice fields. These structures, however, do not bear any resemblance to those postulated to be martian biota, although they are a probable source of the organic contaminants previously reported in this meteorite.

Actinomycetales↗

Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments.

A major taxon of obligate marine bacteria within the order Actinomycetales has been discovered from ocean sediments. Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems. In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth. Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (Escherichia coli numbering). Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family Micromonosporaceae and suggests novelty at the genus level. This is the first evidence for the existence of widespread populations of obligate marine actinomycetes. Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.

Actinomycetales↗

Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr).

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear.

Actinomycetales↗

Bacterial parasite of a plant nematode: morphology and ultrastructure.

The life cycle of a bacterial endoparasite of the plant-parasitic nematode Meloidogyne incognita was examined by scanning and transmission electron microscopy. The infective stage begins with the attachment of an endospore to the surface of the nematode. A germ tube then penetrates the cuticle, and mycelil colonies form in the pseudocoelom. Sporulation is initiated when terminal cells of the mycelium enlarge to form sporangia. A septum within each sporangium divides the forespore from the basal or parasporal portion of the cell. The forespore becomes enclosed by several laminar coats. The parasporal cell remains attached to the forespore and forms the parasporal microfibers. After the newly formed spores are released into the soil, these microfibers apparently enable a mature spore to attach to the nematode. These results indicate that the endoparasite is a procaryotic organism having structural features that are more common to members of Actinomycetales and to the bacterium Pasteuria ramosa than to the sporozoans or to the family Bacillaceae, as previous investigatios have concluded.

Actinomycetales↗

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.

Actinomycetales↗