Pseudonocardia kongjuensis sp. nov., isolated from a gold mine cave.

The taxonomic position of an isolate that was recovered from a gold mine cave near Kongju, Republic of Korea, was determined by 16S rDNA sequence studies and chemotaxonomic characterization. Comparative studies of 16S rDNA sequences indicated that this organism was phylogenetically related to members of the genus Pseudonocardia, branching outside a cluster encompassing Pseudonocardia autotrophica and Pseudonocardia compacta. The affiliation to the genus was also supported by the cell chemistry, which was represented by a type IV cell wall, MK-8(H4) as the major menaquinone, a phospholipid type PIII pattern (phosphatidylcholine as a diagnostic phospholipid) and a DNA G+C content of 71 mol%. The fatty acid profile contained saturated, unsaturated and 10-methyl branched fatty acids, but tuberculostearic acid and hydroxy fatty acids were not present. The isolate differed from its phylogenetic neighbours in the presence of phosphatidylethanolamine, dodecanoate, 16-methylheptadecenoate and 16-methylheptadecanoate and the absence of phosphatidylinositol mannoside and phosphatidylmethylethanolamine. The unique combination of physiological properties, the cellular fatty acid profile and DNA-DNA hybridization data indicates that this organism is readily differentiated from the type strains of all of the validly published species of the genus Pseudonocardia. The name Pseudonocardia kongjuensis sp. nov. is proposed for the type strain, LM 157T (= IMSNU 50583T = KCTC 9990T = DSM 44525T).

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis.

The white-rot fungus Phellinus ribis produced a single form of laccase, which was purified to apparent electrophoretic homogeneity from cultures induced with 2,5-xylidine. This protein was a dimer, consisting of two subunits of 76 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that the enzyme contained about 28% carbohydrate content. The laccase appeared to be different from other known laccases by the UV-visible absorption spectrum analysis. One enzyme molecule contained one copper, one manganese, and two zinc atoms. The laccase showed optimal activity at pH 4.0-6.0, 5.0, and 6.0 with 2,6-dimethoxyphenol, ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], and syringaldazine, respectively. The enzyme preferably oxidized dimethoxyphenol and aromatic amine compounds. The stability of the laccase was low at acidic pH, whereas it showed high stability at neutral pH and mild temperature. The N-terminal amino acid sequence revealed a very low homology with other microbial laccases. With some substrates, the addition of manganese and H2O2 resulted in a remarkable increase in the oxidation rate. Without an appropriate phenolic substrate, the enzyme could not oxidize Mn(II) in the presence of H2O2 or pyrophosphate.

Amycolatopsis albidoflavus sp. nov.

The generic position of an actinomycete strain, 'Pseudonocardia sp.' IMSNU 22139, was investigated by the phylogenetic analysis of 16S rDNA sequence. Comparative studies of 16S rDNA sequences indicated that this organism consistently formed a distinct clade within the radiation of the genus Amycolatopsis of the family Pseudonocardiaceae. This organism was also found to have chemotaxonomic properties consistent with those of the genus Amycolatopsis, which were represented by a type IV cell wall (meso-diaminopimelic acid, arabinose and galactose), a major menaquinone of MK-9(H4), a predominant fatty acid of 14-methylpentadecanoic acid, phosphatidylmethylethanolamine as a diagnostic phospholipid (a phospholipid type PII pattern) and DNA base composition of 68.5 mol% G+C. On the basis of physiological properties, cellular fatty acid profiles and its unique phylogenetic position, this organism is readily differentiated from all of the validly described species of the genus Amycolatopsis, and the name Amycolatopsis albidoflavus sp. nov. is proposed for it. The type strain is IMSNU 22139T (= KCTC 9471T = ATCC 53205T).

Catellatospora koreensis sp. nov., a novel actinomycete isolated from a gold-mine cave.

A new actinomycete strain, LM 042T, which was isolated from a gold-mine cave in Kongju, Republic of Korea, is described by phenotypic and genotypic characters. The organism formed short chains of non-motile spores and globose bodies from substrate mycelium. An aerial mycelium was absent. This organism was chemotaxonomically characterized by the presence of meso-diaminopimelic acid, rhamnose, xylose, glucose, mannose and ribose in whole-cell hydrolysates (a type II cell wall and a variant of sugar pattern D), a glycolyl type of muramic acid, DNA G+C content of 70.4 mol%, a type PII phospholipid pattern (phosphatidylethanolamine as a diagnostic nitrogenous phospholipid), a tetrahydrogenated menaquinone with 10 isoprene units as a major menaquinone, and fatty acid profiles predominated by iso-branched hexadecanoic acid, iso-branched pentadecanoic acid and heptadcenoic acid. A comparative analysis of 16S rDNA sequences indicated that this organism formed a distinct clade within the evolutionary radiation of the family Micromonosporaceae and clustered with members of the genus Catellatospora. The 16S rDNA similarity values between the isolate and its phylogenetic neighbours, the two subspecies of Catellatospora citrea and Catellatospora tsunoense, were 95.0-95.2% and 94.9%, respectively. An equidistant relationship was observed among the isolate, Catellatospora ferruginea and all other members of the Micromonosporaceae genera (levels of similarity 93.0-94.0%). The combination of physiological, chemotaxonomic and DNA-DNA hybridization data supported that this organism is a novel species of the genus Catellatospora, for which the name Catellatospora koreensis sp. nov. is proposed. The type strain is LM 042T (= IMSNU 50729T).

Saccharothrix violacea sp. nov., isolated from a gold mine cave, and Saccharothrix albidocapillata comb. nov.

The generic position of two isolates from soils inside a gold mine cave in Kongju, Korea, was determined by 16S rDNA sequencing and chemotaxonomic characteristics. Phylogenetic analysis indicated that both of the isolates formed a clade with Lentzea albidocapillata and members of the genus Saccharothrix of the family Pseudonocardiaceae. The chemical composition of the isolates and of Lentzea albidocapillata was consistent with that of the genus Saccharothrix, which is characterized by a type III cell wall (the meso-isomer of diaminopimelic acid, and galactose and rhamnose as characteristic whole-cell sugars), MK-9(H4) as the major menaquinone, and a phospholipid type PII pattern (phosphatidylethanolamine as a diagnostic phospholipid). The combination of morphological features, chemotaxonomic characters and phylogenetic data supported the proposal that Lentzea albidocapillata, the only and type strain of the genus Lentzea, should be transferred to the genus Saccharothrix. On the basis of physiological properties, cellular fatty acid composition and DNA-DNA hybridization data, two new species within the genus Saccharothrix are proposed: Saccharothrix violacea sp. nov., type strain LM 036T (= IMSNU 50388T), and Saccharothrix albidocapillata comb. nov., type strain DSM 44073T (=IMSNU 21253T).

Hongia gen. nov., a new genus of the order Actinomycetales.

An aerobic, nocardioform actinomycete, named LM 161T, was isolated from a soil sample obtained from a gold mine in Kongiu, Republic of Korea. This organism formed well-differentiated aerial and substrate mycelia and produced branched hyphae that fragmented into short or elongated rods. The cell wall contains major amounts of LL-diaminopimelic acid, alanine, glycine, glutamic acid, mannose, glucose, galactose, ribose and acetyl muramic acid. The major phospholipids of this isolate are phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol, and the major isoprenologue is a tetrahydrogenated menaquinone with nine isoprene units. The whole-cell hydrolysate of strain LM 161T contains 12-methyltetradecanoic and 14-methylpentadecanoic acids as the predominant fatty acids, but does not contain mycolic acids. The G+C content of the DNA is 71.3 mol%. The phylogenetic position of the test strain was investigated using an almost complete 16S rDNA sequence. The isolate formed the deepest branch in the clade encompassing the members of the suborder Propionibacterineae Rainey et al. 1997. On the basis of chemical, phenotypic and genealogical data, it is proposed that this isolate be classified within a new genus as Hongia koreensis gen. nov., sp. nov. in the order Actinomycetales. The type strain is LM 161T (= IMSNU 50530T).

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences.

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.

Amycolatopsis thermoflava sp. nov., a novel soil actinomycete from Hainan Island, China.

A soil isolate, which had been assigned to the genus Nocardia, was shown to have properties consistent with its classification in the genus Amycolatopsis. An almost complete nucleotide sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for members of the family Pseudonocardiaceae and related taxa and phylogenetic trees were inferred using three tree-making algorithms. The organism consistently formed a distinct monophyletic clade with the type strain of Amycolatopsis methanolica, but DNA-DNA relatedness data showed that the two strains belonged to distinct genomic species. The organism was also distinguished from the type strains of all validly described species of Amycolatopsis using a battery of phenotypic properties. The genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Amycolatopsis. The name proposed for the new species is Amycolatopsis thermoflava sp. nov. The type strain is IFO 14333T.

A phylogenetic analysis of the genus Catellatospora based on 16S ribosomal DNA sequences, including transfer of Catellatospora matsumotoense to the genus Micromonospora as Micromonospora matsumotoense comb. nov.

Phylogenetic studies based on the 16S ribosomal gene sequences showed that members of the genus Catellatospora revealed phylogenetic heterogeneity within the family Micromonosporaceae as well as a heterogeneous menaquinone composition. Among them, Catellatospora matsumotoense was closely related to members of the genus Micromonospora, indicating that this organism should be excluded from the genus Catellatospora. On the basis of classical taxonomic characteristics and phylogenetic evidence, Catellatospora matsumotoense is proposed to be transferred to the genus Micromonospora as M. matsumotoense comb. nov.

Nocardia uniformis nom. rev.

A soil isolate representing the putatively novel species 'Nocardia uniformis' was found to have morphological, staining and chemotaxonomic properties consistent with its classification in the genus Nocardia. An almost complete sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organism was consistently associated with the type strain of Nocardia otitidiscaviarum albeit with a relatively low bootstrap value recorded for neighbour-joining analysis. The strain was also readily separated from representatives of all validly described Nocardia species using a set of phenotypic properties. The genotypic and phenotypic data indicate that the strain should be assigned to the genus Nocardia as a new species. The name proposed for the new species is Nocardia uniformis. The type strain is JCM 3224T.

Nocardia salmonicida nom. rev., a fish pathogen.

An almost complete gene sequence of 16S rDNA of 'Nocardia salmonicida' strain JCM 4826T was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees inferred using four tree-making algorithms. The organism and the type strain of Nocardia asteroides consistently formed a monophyletic clade with a distant sequence similarity of 97%. However, previous DNA relatedness experiments showed that strain JCM 4826T and Nocardia asteroides ATCC 19247T belong to different genomic species. The organism was also distinguished from representatives of all validly described species of Nocardia using a combination of phenotypic features. The polyphasic evidence showed that the strain merits recognition as a new species of the genus Nocardia. The name proposed for the new species is Nocardia salmonicida nom. rev.

Changes in the evolution of the antigenic profiles and morphology during coccoid conversion of Helicobacter pylori.

OBJECTIVES: The significance of the coccoid forms of H. pylori is still controversial and the questions of whether these forms are viable and infective or degenerative are still open. We induced conversion from rod to coccoid forms and studied morphological changes and antigenic evolutions during this conversion and, thereby, elucidated the viability of coccoid forms. METHODS: The H. pylori strain (C001) used for Western blotting was isolated from the patient with gastric cancer. The antigenic evolution during coccoid conversion of H. pylori was studied by Western blotting, using different sera from thirty patients known to be culture positive. These sera were used to reveal the total antigens of the strain cultured for 2 days (100% rod) and 15 days (> 99% coccoid). After SDS-PAGE, with 10% separating gel of total antigens (rod and coccoid), transblotting (Trans-Blot electrophoretic cell, Bio-Rad) was taken onto a nitrocellulose membrane (Bio-Rad). Then, the blots, with human sera diluted at 1/100, were developed with color reaction by goat serum anti-human IgG with alkaline phosphatase and BCIP. RESULTS: The antigenic profiles were not changed in 46.7% (14/30 cases) and were changed in 53.3% (16/30 cases) during coccoid conversion. Antigenic fractions changed during coccoid conversion were protein band at 120 kDa and band at 35 kDa, and were not detected in coccus forms. The rest of the profiles were identical between rod and coccoid forms. The protein which disappeared include CagA (120 kDa) and porin, or adhesin (35 kDa). The morphological changes during coccoid conversion were U shaped at day 7, doughnut shaped at day 9 and full coccoid at day 15. CONCLUSIONS: The results showed that coccoid forms of H. pylori retain cellular structures similar to rod form, and some of the antigens (CagA and porin) disappeared during coccoid conversion. Therefore, coccoid form might be viable and represent one of the stages of H. pylori biological cycle.

Lipoamide dehydrogenase from streptomyces seoulensis: biochemical and genetic properties.

Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9. 5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd. All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.

Nocardia flavorosea sp. nov.

An actinomycete strain, 'Nocardai flavorosea' JCM 3332, was found to have properties consistent with its classification in the genus Nocardia. An almost complete gene sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organisms consistently formed a distinct clade with the type strain of Nocardia carnea. However, DNA relatedness experiments showed that the strain and N. carnea DSM 43397T belonged to two distinct genomic species. The organism was also distinguished from representative of all of the validly described species of Nocardia using a combination of phenotypic properties. These genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Nocardia. The name proposed for the new species of is Nocardia flavorosea sp. nov. The type strain is JCM 3332T.

Expression and regulation of the sodF gene encoding iron- and zinc-containing superoxide dismutase in Streptomyces coelicolor Müller.

Streptomyces coelicolor Müller contains two superoxide dismutases (SODs), nickel-containing (NiSOD) and iron- and zinc-containing SOD (FeZnSOD). The sodF gene encoding FeZnSOD was isolated by using PCR primers corresponding to the N-terminal peptide sequence of the purified FeZnSOD and a C-terminal region conserved among known FeSODs and MnSODs. The deduced amino acid sequence exhibited highest similarity to Mn- and FeSODs from Propionibacterium shermanii and Mycobacterium spp. The transcription start site of the sodF gene was determined by primer extension. When the sodF gene was cloned in pIJ702 and introduced into Streptomyces lividans TK24, it produced at least 30 times more FeZnSOD than the control cells. We disrupted the sodF gene in S. lividans TK24 and found that the disruptant did not produce any FeZnSOD enzyme activity but produced more NiSOD. The expression of the cloned sodF gene in TK24 cells was repressed significantly by Ni, consistent with the regulation pattern in nonoverproducing cells. This finding suggests that the cloned sodF gene contains the cis-acting elements necessary for Ni regulation. When the sodF mRNA in S. coelicolor Muller cells was analyzed by S1 mapping of both 5' and 3' ends, we found that Ni caused a reduction in the level of monocistronic sodF transcripts. Ni did not affect the stability of sodF mRNA, indicating that it regulates transcription. S. lividans TK24 cells overproducing FeZnSOD became more resistant to oxidants such as menadione and lawsone than the control cells, suggesting the protective role of FeZnSOD. However, the sodF disruptant survived as well as the wild-type strain in the presence of these oxidants, suggesting the complementing role of NiSOD increased in the disruptant.

Transcriptional and post-transcriptional regulation by nickel of sodN gene encoding nickel-containing superoxide dismutase from Streptomyces coelicolor Müller.

A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp. The gene for NiSOD (sodN) was cloned from S. coelicolor Müller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E. coli, indicating that N-terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.

Streptomyces seoulensis sp. nov.

The taxonomic position of an actinomycete strain isolated from Korean soil was examined by a polyphasic approach. The isolate, designated IMSNU-1, was clearly assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic data. The test strain was the subject of a probabilistic identification study using the identification matrices generated by Langham et al. (J. Gen. Microbiol. 135:121-133, 1989) and found to be marginally close to clusters 19 and 39. An almost complete 16S rRNA gene (rDNA) sequence was obtained for the test strain and compared with those of representative streptomycetes. 16S rDNA sequence data not only support the strain's membership in the genus Streptomyces but also provide strong evidence that our isolate is genealogically distant from representatives of clusters 19 and 39, forming a separate phyletic line in a clade encompassed by streptomycetes. It is therefore proposed from the polyphasic evidence that strain IMSNU-1 be classified in the genus Streptomyces as Streptomyces seoulensis sp. nov.

Long-term identification of streptomycetes using pyrolysis mass spectrometry and artificial neural networks.

Sixteen reference strains and thirteen fresh isolates of three putatively novel Streptomyces species were examined six times over twenty months using pyrolysis mass spectrometry to examine the long-term reproducibility of the procedure. The reference strains and new isolates were correctly identified using information in each of the datasets and operational fingerprinting, but direct statistical comparison of the datasets for strain identification was unsuccessful between datasets. Artificial neural networks were also used to identify the strains held in the datasets. Neural networks trained with pyrolysis mass spectra from a single dataset were found to successfully identify the reference strains and fresh isolates in that dataset but were unable to identify many of the strains in the other datasets. However, a neural network trained on representative pyrolysis mass spectra from each of the first three datasets were found to identify the reference strains and fresh isolates in those three datasets and in the three subsequent datasets. Therefore, artificial neural network analysis of pyrolysis mass spectrometric data can provide a rapid, cost-effective, accurate and long-term reproducible way of identifying and typing microorganisms.

A proposal to reclassify Nocardia pinensis Blackall et al. as Skermania piniformis gen. nov., comb. nov.

The type strain of Nocardia pinensis was the subject of chemotaxonomic and 16S ribosomal DNA sequencing studies. The resultant nucleotide sequence was aligned with the sequences of representatives of the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella, and phylogenetic trees were generated by using the Fitch-Margoliash, maximum-parsimony, maximum-likelihood, and neighbor-joining methods. It was evident from the phylogenetic analyses that N. pinensis represents a distinct phyletic line that is most closely associated with the Gordona clade. This genealogical evidence, together with chemotaxonomic and phenotypic data derived from this and previous studies, indicates that N. pinensis merits generic status within the family Nocardiaceae. Therefore, we propose that N. pinensis Blackall et al. 1989 be reclassified as Skermania piniformis gen. nov., comb. nov. The type strain of Skermania piniformis cleaved an array of conjugated substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone.

Unique isozymes of superoxide dismutase in Streptomyces griseus.

Two unique isozymes of superoxide dismutase (EC 1.15.1.1) were purified to apparent homogeneity from Streptomyces griseus by a purification procedure consisting of ammonium sulfate precipitation and chromatographies on DEAE Sephacel, Sephacryl S-200, and DEAE 5PW. Superoxide dismutase I was composed of four identical subunits of 13.0 kDa. The absorption spectrum of superoxide dismutase I exhibited absorption bands at 276 and 378 nm and a broad shoulder at 530 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase I were g1 = 2.304, g2 = 2.248, and g3 = 2.012 and the resonance centered at g3 = 2.012 was split into triplet, indicating nickel-containing superoxide dismutase. Superoxide dismutase I contained 0.89 g-atom of nickel per mole of 13.0-kDa subunit. Superoxide dismutase II was composed of four identical subunits of 22.0 kDa. The absorption spectrum of superoxide dismutase II showed the featureless absorption band in the range of 300-500 nm. The g values of electron paramagnetic resonance spectrum of superoxide dismutase II were gz = 4.762, gx = 4.072, and gy = 3.742, indicating iron-containing superoxide dismutase. Superoxide dismutase II uniquely contains 0.40 g-atom of iron per mole of monomer as well as 0.43 g-atom of zinc per mole of monomer. The immunological cross-reactivity between two isozymes was not found. Nickel-containing superoxide dismutase was widely distributed within the genus Streptomyces; however, iron- and zinc-containing superoxide dismutase was not found in S. albus and S. longisporoflavus, on the basis of the immunological cross-reactivity.