Kitasatospora acidiphila sp. nov., isolated from pine grove soil, exhibiting antimicrobial potential.

A polyphasic study was carried out to establish the taxonomic position of an acidophilic isolate designated MMS16-CNU292T (=JCM 32302T) from pine grove soil, and provisionally assigned to the genus Kitasatospora. On the basis of 16S rRNA gene sequence similarity, the strain formed a novel evolutionary lineage within Kitasatospora and showed highest similarities to Kitasatospora azatica KCTC 9699T (98.75 %), Kitasatospora kifunensis IFO 15206T (98.74 %), Kitasatospora purpeofusca NRRL B-1817T (98.61 %) and Kitasatospora nipponensis HKI 0315T (98.42 %), respectively. Strain MMS16-CNU292T possessed MK-9(H6) and MK-9(H8) as the major menaquinones, and a major amount of meso-diaminopimelic acid in the cell-wall peptidoglycan. The whole-cell hydrolysates were rich in galactose, glucose and mannose, and the polar lipids mainly consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannosides. The major fatty acids were anteiso-C15 : 1-A, anteiso-C15 : 0, and iso-C15 : 0, and the DNA G+C content was 71.5 mol%. The strain exhibited antibacterial activity against a number of bacterial strains, and the activity was generally greater when grown in acidic conditions. The phylogenetic, chemotaxonomic and phenotypic properties enabled distinction of MMS16-CNU292T from related species, and thus the isolate should be recognized as a new species of the genus Kitasatospora, for which the name Kitasatospora acidiphila sp. nov. (type strain=MMS16-CNU292T=KCTC 49011T=JCM 32302T) is proposed.

Streptacidiphilus bronchialis sp. nov., a ciprofloxacin-resistant bacterium from a human clinical specimen; reclassification of Streptomyces griseoplanus as Streptacidiphilus griseoplanus comb. nov. and emended description of the genus Streptacidiphilus.

The taxonomic position of strain 15-057AT, an acidophilic actinobacterium isolated from the bronchial lavage of an 80-year-old male, was determined using a polyphasic approach incorporating morphological, phenotypic, chemotaxonomic and genomic analyses. Pairwise 16S rRNA gene sequence similarities calculated using the GGDC web server between strain 15-057AT and its closest phylogenetic neighbours, Streptomyces griseoplanus NBRC 12779T and Streptacidiphilus oryzae TH49T, were 99.7 and 97.6 %, respectively. The G+C content of isolate 15-057AT was determined to be 72.6 mol%. DNA-DNA relatedness and average nucleotide identity between isolate 15-057AT and Streptomyces griseoplanus DSM 40009T were 29.2±2.5 % and 85.97 %, respectively. Chemotaxonomic features of isolate 15-057AT were consistent with its assignment within the genus Streptacidiphilus: the whole-cell hydrolysate contained ll-diaminopimelic acid as the diagnostic diamino acid and glucose, mannose and ribose as cell-wall sugars; the major menaquinone was MK9(H8); the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, glycophospholipid, aminoglycophospholipid and an unknown lipid; the major fatty acids were anteiso-C15 : 0 and iso-C16 : 0. Phenotypic and morphological traits distinguished isolate 15-057AT from its closest phylogenetic neighbours. The results of our taxonomic analyses showed that strain 15-057AT represents a novel species within the evolutionary radiation of the genus Streptacidiphilus, for which the name Streptacidiphilus bronchialis sp. nov. is proposed. The type strain is 15-057AT (=DSM 106435T=ATCC BAA-2934T).

Exclusivity offers a sound yet practical species criterion for bacteria despite abundant gene flow.

BACKGROUND: The question of whether bacterial species objectively exist has long divided microbiologists. A major source of contention stems from the fact that bacteria regularly engage in horizontal gene transfer (HGT), making it difficult to ascertain relatedness and draw boundaries between taxa. A natural way to define taxa is based on exclusivity of relatedness, which applies when members of a taxon are more closely related to each other than they are to any outsider. It is largely unknown whether exclusive bacterial taxa exist when averaging over the genome or are rare due to rampant hybridization. RESULTS: Here, we analyze a collection of 701 genomes representing a wide variety of environmental isolates from the family Streptomycetaceae, whose members are competent at HGT. We find that the presence/absence of auxiliary genes in the pan-genome displays a hierarchical (tree-like) structure that correlates significantly with the genealogy of the core-genome. Moreover, we identified the existence of many exclusive taxa, although individual genes often contradict these taxa. These conclusions were supported by repeating the analysis on 1,586 genomes belonging to the genus Bacillus. However, despite confirming the existence of exclusive groups (taxa), we were unable to identify an objective threshold at which to assign the rank of species. CONCLUSIONS: The existence of bacterial taxa is justified by considering average relatedness across the entire genome, as captured by exclusivity, but is rejected if one requires unanimous agreement of all parts of the genome. We propose using exclusivity to delimit taxa and conventional genome similarity thresholds to assign bacterial taxa to the species rank. This approach recognizes species that are phylogenetically meaningful, while also establishing some degree of comparability across species-ranked taxa in different bacterial clades.

Streptacidiphilus pinicola sp. nov., isolated from pine grove soil.

A moderately acidophilic actinobacterial strain, designated MMS16-CNU450T, was isolated from pine grove soil, and its taxonomic position was analysed using a polyphasic approach. The isolate showed best growth at 30 °C, pH 6 and 0.5 % (w/v) NaCl. On the basis of 16S rRNA gene sequence similarity, the isolate was assigned to the genus Streptacidiphilus, and the closest species were Streptacidiphilus rugosus AM-16T (sequence similarity, 98.61 %), Streptacidiphilus melanogenes NBRC 103184T (98.53 %), Streptacidiphilus jiangxiensis NBRC 100920T (98.19 %) and Streptacidiphilus anmyonensis NBRC 103185T (98.05 %). The isolate formed a distinct cluster of its own within the Streptacidiphilusclade in the phylogenetic tree. Based on whole-genome comparison between the strain MMS16-CNU450T and the type strains of related species, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values were in the range of 77.9-87.0 and 22.3-32.7 %, respectively. The DNA G+C content of the isolate was 68.6 mol%. The phylogenetic, phenotypic, chemotaxonomic and genomic data supported the affiliation of the strain to Streptacidiphilus, and the name Streptacidiphilus pinicola sp. nov. (type strain, MMS16-CNU450T=KCTC 49008T=JCM 32300T) is proposed accordingly.

Allostreptomyces indica sp. nov., isolated from India.

A novel actinobacterium, designated strain YIM 75704T, was isolated from a limestone quarry located at Gulbarga, Karnataka, India. The novel strain has showed typical morphological and chemotaxonomic characteristics of the family Streptomycetaceae. Comparison of 16S rRNA gene sequences indicated that this strain represents a novel member of the family Streptomycetaceae and exhibited 99.0% 16S rRNA gene sequence similarities with the type species of the recently described novel genus Allostreptomyces, that is, Allostreptomyces psammosilenae, whereas other species of Streptomyces were below 95% sequence similarity. The cell hydrolysates contained the LL-isomer of diaminopimelic acid and the predominant quinones were MK-9 (H6, H8 and H4). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylinositolmannosides and three unknown phospholipids. The DNA G+C content was 75.0 mol%. A polyphasic study of the strain with morphological, phenotypic, phylogenetic and with DNA-DNA hybridization evidence with related members showed that this strain represents novel species of Allostreptomyces for which the name Allostreptomyces indica sp. nov., is proposed. The type strain is YIM 75704T (= DSM 41985T=CCTCC AA 209051T= NCIM 5485T).

Genus Kitasatospora, taxonomic features and diversity of secondary metabolites.

The genus Kitasatospora was proposed in 1982. Although Kitasatospora strains resemble Streptomyces strains in morphology, they are clearly different in cell-wall composition, as they contain both LL- and meso-diaminopimelic acid. Aerial and submerged spores contain LL-, while vegetative and submerged mycelia contain mainly meso- in their cell walls. Currently, 23 species have been validly proposed. Members of the genus Kitasatospora form a tight cluster and represent a legitimate genus distinct from Streptomyces on the basis of phylogenetic analysis of 16S rRNA gene sequences. A variety of biologically active compounds have been found from Kitasatospora strains and structures of these compounds are extremely diverse. Genome sequences of 15 strains published so far are about 7-9 Mb in size and contain many genes governing secondary metabolites.

Allostreptomyces psammosilenae gen. nov., sp. nov., an endophytic actinobacterium isolated from the roots of Psammosilene tunicoides and emended description of the family Streptomycetaceae [Waksman and Henrici (1943)AL] emend. Rainey et al. 1997, emend. Kim et al. 2003, emend. Zhi et al. 2009.

A Gram-stain-positive actinobacterium, designated strain YIM DR4008T, was isolated from the root sample of Psammosilene tunicoides collected from Lijiang, Yunnan, China. Strain YIM DR4008T could grow at temperatures ranging from 10 to 50 °C (optimum 28-30 °C), at pH 5.0-11.0 (optimum pH 7.0) and in the presence of up to 4 % (w/v) NaCl. Sequence analysis of the 16S ribosomal RNA gene revealed that strain YIM DR4008T shared highest similarity (95.0 %) with Streptomyces griseoplanus NBRC 12779T and <95 % similarity with other known members of the genera Streptomyces, Kitasatospora and Streptacidiphilus. The diagnostic cell-wall diamino acid of strain YIM DR4008T was found to be ll-diaminopimelic acid. The whole-cell hydrolysates contained a major amount of galactose and mannose along with a small proportion of fucose, glucose, rhamnose and ribose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylinositol mannosides and three unidentified phospholipids. The respiratory menaquinones were MK-9(H6) and MK-9(H8), while the major cellular fatty acids (>10 %) were anteiso-C15 : 0, C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was determined to be 75.3 mol%. Based on the phenotypic, chemotaxonomic and molecular characteristics, strain YIM DR4008T is proposed to be recognized as a novel species of a new genus in the family Streptomycetaceae, with the name Allostreptomyces psammosilenae gen. nov., sp. nov. The type strain of the type species is YIM DR4008T (=DSM 42178T=CGMCC 4.7247T). An emended description of the family Streptomycetaceae is also provided.

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785.

A maltotriose-forming amylase (G3Amy) from Kitasatospora sp. MK-1785 was successfully isolated from a soil sample by inhibiting typical extracellular α-amylases using a proteinaceous α-amylase inhibitor. G3Amy was purified from the MK-1785 culture supernatant and characterized. G3Amy produced maltotriose as the principal product from starch and was categorized as an exo-α-amylase. G3Amy could also transfer maltotriose to phenolic and alcoholic compounds. Therefore, G3Amy can be useful for not only maltotriose manufacture but also maltooligosaccharide-glycoside synthesis. Further, the G3Amy gene was cloned and expressed in Escherichia coli cells. Analysis of its deduced amino acid sequence revealed that G3Amy consisted of an N-terminal GH13 catalytic domain and two C-terminal repeat starch-binding domains belonging to CBM20. It is suggested that natural G3Amy was subjected to proteolysis at N-terminal region of the anterior CBM20 in the C-terminal region. As with natural G3Amy, recombinant G3Amy could produce and transfer maltotriose from starch.

Analysis of novel kitasatosporae reveals significant evolutionary changes in conserved developmental genes between Kitasatospora and Streptomyces.

Actinomycetes are antibiotic-producing filamentous bacteria that have a mycelial life style. The members of the three genera classified in the family Streptomycetaceae, namely Kitasatospora, Streptacidiphilus and Streptomyces, are difficult to distinguish using phenotypic properties. Here we present biochemical and genetic evidence that helps underpin the case for the continued recognition of the genus Kitasatospora and for the delineation of additional Kitasatospora species. Two novel Kitasatospora strains, isolates MBT63 and MBT66, and their genome sequences are presented. The cell wall of the Kitasatospora strains contain a mixture of meso-and LL-diaminopimelic acid (A2pm), whereby a single DapF surprisingly suffices to incorporate both components into the Kitasatospora cell wall. The availability of two new Kitasatospora genome sequences in addition to that of the previously sequenced Kitasatospora setae KM-6054(T) allows better phylogenetic comparison between kitasatosporae and streptomycetes. This showed that the developmental regulator BldB and the actin-like protein Mbl are absent from kitasatosporae, while the cell division activator SsgA and its transcriptional activator SsgR have been lost from some Kitasatospora species, strongly suggesting that Kitasatospora have evolved different ways to control specific steps in their development. We also show that the tetracycline-producing strain "Streptomyces viridifaciens" DSM 40239 not only has properties consistent with its classification in the genus Kitasatospora but also merits species status within this taxon.

A novel taxonomic marker that discriminates between morphologically complex actinomycetes.

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

Streptacidiphilus hamsterleyensis sp. nov., isolated from a spruce forest soil.

Three acidophilic actinobacteria, isolates LSCA2, FGG8 and HSCA14(T), recovered from spruce litter were examined using a polyphasic approach. Chemotaxonomic and morphological properties of the isolates were found to be consistent with their classification in the genus Streptacidiphilus. The isolates were shown to have identical 16S rRNA gene sequences and were most closely related to Streptacidiphilus neutrinimicus DSM 41755(T) (99.9 % similarity). However, DNA:DNA relatedness between isolate HSCA14(T) and the type strain of S. neutrinimicus was found to be low at 44.0 (±14.1) %. A combination of phenotypic features, including degradative and nutritional characteristics were shown to distinguish the isolates from their nearest phylogenetic neighbours. Data from this study show that the isolates form a novel species in the genus for which the name S. hamsterleyensis sp. nov. is proposed. The type strain is HSCA 14(T) (=DSM 45900(T) = KACC 17456(T) = NCIMB 14865(T)).

Streptacidiphilus durhamensis sp. nov., isolated from a spruce forest soil.

The taxonomic position of three acidophilic actinobacteria, strains FGG38, FGG39 and FSCA67(T), isolated from the fermentation litter layer of a spruce forest soil was established using a polyphasic approach. The strains were shown to have chemotaxonomic and morphological properties consistent with their classification in the genus Streptacidiphilus and formed a distinct phyletic line in the Streptacidiphilus 16S rRNA gene tree being most closely related to Streptacidiphilus albus DSM 41753(T) (99.4 % similarity). DNA:DNA relatedness data showed that isolate FSCA67(T) and the type strain of S. albus belonged to markedly distinct genomic species. The isolates had many phenotypic properties in common and were distinguished readily from their closest phylogenetic neighbours in the Streptacidiphilus gene tree using a broad range of these features. Based on the combined genotypic and phenotypic data the three isolates are considered to represent a new Streptacidiphilus species. The name Streptacidiphilus durhamensis sp. nov. is proposed for this taxon with isolate FSCA67(T) (=DSM 45796(T) = KACC 17154(T) = NCIMB 14828(T)) [corrected] as the type strain.

Targeted search for actinomycetes from nearshore and deep-sea marine sediments.

Sediment samples collected off the coast of San Diego were analyzed for actinomycete diversity using culture-independent techniques. Eight new operational taxonomic units (OTUs) in the Streptomycetaceae were identified as well as new diversity within previously cultured marine OTUs. Sequences belonging to the marine actinomycete genus Salinispora were also detected, despite the fact that this genus has only been reported from more tropical environments. Independent analyses of marine sediments from the Canary Basin (3814 m) and the South Pacific Gyre (5126 and 5699 m) also revealed Salinispora sequences providing further support for the occurrence of this genus in deep-sea sediments. Efforts to culture Salinispora spp. from these samples have yet to be successful. This is the first report of Salinispora spp. from marine sediments > 1100 m and suggests that the distribution of this genus is broader than previously believed.

Phylogenetic study of the species within the family Streptomycetaceae.

Species of the genus Streptomyces, which constitute the vast majority of taxa within the family Streptomycetaceae, are a predominant component of the microbial population in soils throughout the world and have been the subject of extensive isolation and screening efforts over the years because they are a major source of commercially and medically important secondary metabolites. Taxonomic characterization of Streptomyces strains has been a challenge due to the large number of described species, greater than any other microbial genus, resulting from academic and industrial activities. The methods used for characterization have evolved through several phases over the years from those based largely on morphological observations, to subsequent classifications based on numerical taxonomic analyses of standardized sets of phenotypic characters and, most recently, to the use of molecular phylogenetic analyses of gene sequences. The present phylogenetic study examines almost all described species (615 taxa) within the family Streptomycetaceae based on 16S rRNA gene sequences and illustrates the species diversity within this family, which is observed to contain 130 statistically supported clades, as well as many unsupported and single member clusters. Many of the observed clades are consistent with earlier morphological and numerical taxonomic studies, but it is apparent that insufficient variation is present in the 16S rRNA gene sequence within the species of this family to permit bootstrap-supported resolution of relationships between many of the individual clusters.

Ribosomal and protein coding gene based multigene phylogeny on the family Streptomycetaceae.

The phylogenetic relationship among the three genera of the family Streptomycetaceae was examined using the small and large subunit ribosomal RNA genes, and the gyrB, rpoB, trpB, atpD and recA genes. The total stretches of the analyzed ribosomal genes were 4.2kb, and those of five protein coding genes were 4.5 kb. The resultant phylogenetic trees confirmed that each genus formed an independent clade in the majority of cases. The G+C contents of rRNA genes were 56.9-58.9 mol%, and those of protein coding genes were 65.4-72.4 mol%, the latter being closer to those of the genomic DNAs. The average nucleotide sequence identity between the organisms were 94.1-96.4% for rRNA genes and 85.7-90.6% for protein coding genes, thus indicating that protein coding genes can give higher resolution than rRNA genes. In addition, the protein coding gene trees were more stable than the rRNA gene trees, supported by higher bootstrap values and other treeing algorithms. Moreover, the genome data of six Streptomyces species indicated that many protein coding genes exhibited higher correlations with genome relatedness. The combined gene sequences were also shown to give a better resolution with higher stability than any single genes, though not necessarily more correlated with genome relatedness. It is evident from this study that the rRNA gene based phylogeny can be misleading, and also that protein coding genes have a number of advantages over the rRNA genes as the phylogenetic markers including a high correlation with the genome relatedness.

Genome sequence of Kitasatospora setae NBRC 14216T: an evolutionary snapshot of the family Streptomycetaceae.

Kitasatospora setae NBRC 14216(T) (=KM-6054(T)) 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 14216(T) 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.

Kitasatospora saccharophila sp. nov. and Kitasatospora kazusanensis sp. nov., isolated from soil and transfer of Streptomyces atroaurantiacus to the genus Kitasatospora as Kitasatospora atroaurantiaca comb. nov.

A polyphasic study was undertaken to establish the taxonomic positions of two isolates, SK15(T) and SK60(T), from soil samples that were found to have morphological and chemical properties consistent with Kitasatospora strains. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strains SK15(T) and SK60(T) form novel evolutionary lineages within the radiation of the genus Kitasatospora and share the highest 16S rRNA gene sequences similarities with their closest relatives, Kitasatospora setae IAM 15325(T) (97.8%) and Kitasatospora mediocidica IAM 15162(T )(97.5%), respectively. However, the results of DNA-DNA hybridization experiment and phenotypic data demonstrated that strains SK15(T) and SK60(T) are distinct from their closest phylogenetic neighbors and other Kitasatospora species. For chemotaxonomic characteristics, the cell-wall peptidoglycan of strains contained both meso- and LL-diaminopimelic acids as the diamino acids, the predominant quinone system was MK-9(H(6)) and MK-9(H(8)), whole-cell hydrolysates were rich in galactose, mannose and ribose, and the major fatty acids were C(16:0), anteiso-C(15:0), iso-C(15:0) and iso-C(16:0). On the basis of both phenotypic and phylogenetic evidence, strains SK15(T) and SK60(T) were assigned to represent two novel species of the genus Kitasatospora, for which the names Kitasatospora saccharophila sp. nov. (type strain SK15(T)=JCM 14559(T)=KCTC 19566(T)) and Kitasatospora kazusanensis sp. nov. (type strain SK60(T)=JCM 14560(T)=KCTC 19565(T)) are proposed. It is also proposed that Streptomyces atroaurantiacus should be transferred to the genus Kitasatospora as Kitasatospora atroaurantiaca comb. nov. (type strain NBRC 14327(T)=DSM 41649(T)).

Macroamphiphilic components of thermophilic actinomycetes: identification of lipoteichoic acid in Thermobifida fusca.

The cell envelopes of gram-positive bacteria contain structurally diverse membrane-anchored macroamphiphiles (lipoteichoic acids and lipoglycans) whose functions are poorly understood. Since regulation of membrane composition is an important feature of adaptation to life at higher temperatures, we have examined the nature of the macroamphiphiles present in the thermophilic actinomycetes Thermobifida fusca and Rubrobacter xylanophilus. Following hot-phenol-water extraction and purification by hydrophobic interaction chromatography, Western blotting with a monoclonal antibody against lipoteichoic acid strongly suggested the presence of a polyglycerophosphate lipoteichoic acid in T. fusca. This structure was confirmed by chemical and nuclear magnetic resonance analyses, which confirmed that the lipoteichoic acid is substituted with beta-glucosyl residues, in common with the teichoic acid of this organism. In contrast, several extraction methods failed to recover significant macroamphiphilic carbohydrate- or phosphate-containing material from R. xylanophilus, suggesting that this actinomycete most likely lacks a membrane-anchored macroamphiphile. The finding of a polyglycerophosphate lipoteichoic acid in T. fusca suggests that lipoteichoic acids may be more widely present in the cell envelopes of actinomycetes than was previously assumed. However, the apparent absence of macroamphiphiles in the cell envelope of R. xylanophilus is highly unusual and suggests that macroamphiphiles may not always be essential for cell envelope homeostasis in gram-positive bacteria.

In vitro antagonism of an actinobacterial Kitasatospora isolate against the plant pathogen Phytophthora citricola as elucidated with ultrahigh resolution mass spectrometry.

Many soil microorganisms antagonistic to soil borne plant pathogens are well known for their ability to control diseases in situ. A variety of substances, like lytic enzymes, siderophores and antibiotics, produced by these organisms have the potential to protect roots against pathogens. Understanding the ecology and a functional assessment of antagonistic microbial communities in soil requires in-depth knowledge of the mechanisms involved in these interactions, a challenging task in complex systems if low-resolution methods are applied. We propose an information-rich strategy of general relevance, composed of adequate preconcentration in conjunction with ultrahigh resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS) and nuclear magnetic resonance (NMR) spectroscopy to identify any bioactive substances in complex systems. This approach is demonstrated on the specific example of substance identification considered responsible for in vitro antagonism of an actinobacterial antagonist isolated from European beech (Fagus sylvatica) rhizosphere soil against the oomycetous root rot pathogen Phytophthora citricola. The isolate belonging to the genus Kitasatospora exhibited strong antibiosis against the oomycete in vitro. The bioactive substance was observed to exhibit a molar mass of 281.1699 g/mol in positive electrospray ionization mass spectra, and the high mass accuracy of the ICR-FT/MS measurements allowed a precise assignment of a molecular formula that was found identical to the macrolide polyketide cycloheximide C(15)H(23)NO(4)+H(+); its identity was then unequivocally confirmed by the information-rich atomic signature of proton NMR spectroscopy. In conclusion, the combination of the near orthogonal methods (pre)fractionation, ultrahigh-resolution ICR-FT mass spectrometry (yielding molecular and MS(n) fragment signatures) and nuclear magnetic resonance spectroscopy (providing atomic signatures) has been found capable of identifying a biocontrol active compound of Kitasatospora active against Phytophthora citricola expediently, quickly, and accurately. This straightforward approach is of general applicability to elucidate biocontrol mechanisms in any complex system with improved efficiency.

Electrical DNA-chip-based identification of different species of the genus Kitasatospora.

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 mug/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.