Use of <Emphasis Type="Italic">rpoB</Emphasis> sequences and rep-PCR for phylogenetic study of <Emphasis Type="Italic">Anoxybacillus</Emphasis> species

This study was conducted to investigate the applicability of rpoB, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.     

Evaluation of the use of <Emphasis Type="Italic">recN</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial recN gene sequences (>1 kb) were obtained from 35 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of recN sequence analysis as an alternative to DNA–DNA hybridization (DDH) for distinguishing closely related species was also assessed. The recN based phylogeny mostly confirmed the conventional 16S rRNA and gyrB gene-based phylogenies and thus provides further support for these phylogenetic groupings. As is the case for the gyrB gene, pairwise recN sequence similarities cannot be used to predict the DNA relatedness between type strains but the recN genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. A recN genetic distance of >0.04 between two Amycolatopsis strains is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking).     

A newly constructed primer pair for the PCR amplification,cloningand sequencing of the flagellin (<Emphasis Type="Italic">flaA</Emphasis>) gene from isolatesof urease-negative <Emphasis Type="Italic">Campylobacter lari</Emphasis>

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564–572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70–75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

<Emphasis Type="Italic">Paenibacillus camelliae</Emphasis> sp. nov., isolated from fermented leaves of <Emphasis Type="Italic">Camellia sinensis</Emphasis>

A novel bacterium, strain blls-2^T was isolated from Pu’er tea. The isolate was Gram-positive, endospore-forming motile rod that grew at 15∼42°C and pH 6.0∼10.2. The DNA G+C content was 48.3 mol%, the predominant isoprenoid quinone was MK-7, and the predominant cellular fatty acid was anteiso-C15:0 (54.2%) followed by C16:0 (15.5%) and iso-C16:0 (8.2%). The polar lipid pattern of blls-2^T was characterized by the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phy-logenetic analysis based on 16S rRNA gene sequence showed that the strain was affiliated within the Paenibacillaceae. The strain was most closely related to Paenibacillus granivorans A30^T, with a similarity of 97.1%. Based on the phylogenetic and phenotypic characteristics of strain blls-2^T, the isolate is thought to represent a novel taxon in the genus Paenibacillus. The name Paenibacillus camelliae sp. nov. is proposed for the fermented tea isolate; the type strain is blls-2^T (= KCTC 13220^T= CECT 7361^T).     

<Emphasis Type="Italic">Pontibacter rhizosphera</Emphasis> sp. nov., isolated from rhizosphere soil of an Indian medicinal plant <Emphasis Type="Italic">Nerium indicum</Emphasis>

A gram-negative, motile, straight to curved rod shaped, pink pigmented bacterium was isolated from a soil sample collected from the rhizosphere of an Indian medicinal plant, Nerium indicum (Chuvanna arali) and subjected to a detailed polyphasic taxonomic study. The strain, designated as IMTB-1969^T, matched with most of the phenotypic and chemotaxonomic properties of the genus Pontibacter and represents a novel species. The major fatty acids of the strain were monounsaturated iso/anteiso branched C17 fatty acids (45.1%) and iso-C15:0 (16.5%). MK-7 was the predominant isoprenoid quinone. According to 16S rRNA gene sequence analysis, strain IMTB-1969^T was indicated to belonged to the phylum Bacteroidetes and further phylogenetic analysis revealed that the strain IMTB-1969^T belongs to the family Cytophagaceae and genus Pontibacter. The highest 16S rRNA gene sequence similarity was with Pontibacter korlensis CCTCC AB 206081^T (97.2%) and lower sequence similarity was observed with other species in the genus Pontibacter (95.9–94.0%). DNA–DNA relatedness study of the strain IMTB-1969^T confirmed that it represents a novel species. The G+C content of the genomic DNA was 52.2 (±0.5) mol%. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinction of strain IMTB-1969^T from its closest phylogenetic relatives. The strain IMTB-1969^T should be classified as novel species of the genus Pontibacter, for which the name Pontibacter rhizosphera sp. nov. is proposed. The type strain is IMTB-1969^T (=MTCC 10673^T = DSM 24399^T).     

<Emphasis Type="Italic">Bacillus berkeleyi</Emphasis> sp. nov., isolated from the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

A bacterial strain, designated KMM 6244^T, was isolated from the sea urchin Strongylocentrotus intermedius and subjected to a polyphasic taxonomic investigation. The bacterium was found to be heterotrophic, aerobic, non-motile and spore-forming. Comparative phylogenetic analysis based on 16S rRNA gene sequencing placed the marine isolate in the genus Bacillus. The nearest neighbor of strain KMM 6244^T was Bacillus decolorationis LMG 19507^T with a 16S rRNA gene sequence similarity of 98.0%. Sequence similarities with the other recognized Bacillus species were less than 96.0%. The results of the DNA–DNA hybridization experiments revealed a low relatedness (37%) of the novel isolate with the type strain of B. decolorationis LMG 19507^T. Strain KMM 6244^T grew at 4–45°C and with 0–12% NaCl. It produced catalase and oxidase and hydrolyzed aesculin, casein, gelatin and DNA. The predominant fatty acids were anteiso-C_15:0, iso-C_15:0, anteiso-C_17:0, C_15:0, iso-C_16:0 and iso-C_14:0. The DNA G + C content was 39.4 mol%. A combination of phylogenetic, genotypic and phenotypic data clearly indicated that strain KMM 6244^T represents a novel species in the genus Bacillus, for which the name Bacillus berkeleyi sp. nov. is proposed. The type strain is KMM 6244^T (KCTC 12718^T = LMG 26357^T).     

<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> in Poland and Japan is nodulated by <Emphasis Type="Italic">Mesorhizobium amorphae</Emphasis> strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91–98% and 90–98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51–75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61–64 mol% across M. amorphae strains.     

Phylogenetic,Inter, and Intraspecific Sequence Analysis of <Emphasis Type="Italic">spo0A</Emphasis> Gene of the Genus <Emphasis Type="Italic">Geobacillus</Emphasis>

In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species—Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus—could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%–92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high—above 99.0%. Similarity of spo0A sequences of G. subterraneus–G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus–G. thermoleovorans–G. kaustophilus–G. vulcani and G. thermocatenulatus–G. gargensis–G. caldoxylosilyticus–G. toebii–G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.     

<Emphasis Type="Italic">Sphingomonas jejuensis</Emphasis> sp. nov., isolated from marine sponge <Emphasis Type="Italic">Hymeniacidon flavia</Emphasis>

A Gram-negative, non-motile, rod shaped, and orange-pigmented chemoheterotrophic bacterium, strain MS-31^T was isolated from the marine sponge Hymeniacidon flavia, collected from near Jeju Island, Korea. The Strain MS-31^T was subjected to a polyphasic taxonomic study. The phylogenetic analysis based on the 16S rRNA gene sequences revealed that the novel isolate could be affiliated within the genus Sphingomonas. The strain MS-31^T showed 95.6% of 16S rRNA gene sequence similarity with the most closely related species Sphingomonas koreensis JSS26^T. The DNA G+C content of the strain MS-31^T was 69.4 mol%. The major isoprenoid quinone was ubiqunone 10 and predominant cellular fatty acids were summed feature 7 (comprising C_18:1 ω7c, C_18:1 Ω9t and/or C_18:1 ωl2t, 39.7%), C_16:0 (16.3%), C_14:0 2OH (15.9%) and summed feature 3 (comprising C_16:1 ω7c and/or C_15:0 iso 2OH, 11.7%). The polar lipids were sphingoglycolipid, phosphatidyletha-nolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified glycolipid. Based on the evidence from the polyphasic taxonomic study, the strain should be classified as a new species of the genus Sphingomonas. As a result, the name Sphingomonas jejuensis sp. nov. (type strain MS-31^T =KCTC 23321^T =NBRC 107775^T) is proposed.     

<Emphasis Type="Italic">Flavobacterium koreense</Emphasis> sp. nov., <Emphasis Type="Italic">Flavobacterium chungnamense</Emphasis> sp. nov., and <Emphasis Type="Italic">Flavobacterium cheonanense</Emphasis> sp. nov., isolated from a freshwater reservoir

Taxonomic studies were performed on three strains isolated from Cheonho reservoir in Cheonan, Korea. The isolates were Gram-negative, aerobic, rod-shaped, non-motile, catalase-positive, and oxidase-positive. Colonies on solid media were cream-yellow, smooth, shiny, and circular. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these strains belong to the genus Flavobacterium. The strains shared 98.6–99.4% sequence similarity with each other and showed less than 97% similarity with members of the genus Flavobacterium with validly published names. The DNA-DNA hybridization results confirmed the separate genomic status of strains ARSA-42^T, ARSA-103^T, and ARSA-108^T. The isolates contained menaqui-none-6 as the predominant menaquinone and iso-C_15:0, iso-C_15:0 3-OH, iso-Ci_15:1 G, and iso-C_16:0 3-OH as the major fatty acids. The genomic DNA G+C content of the isolates were 31.4–33.2 mol%. According to the phenotypic and genotypic data, these organisms are classified as representative of three novel species in the genus Flavobacterium, and the name Flavobacterium koreense sp. nov. (strain ARSA-42^T =KCTC 23182^T =JCM 17066^T =KACC 14969^T), Flavobacterium chungnamense sp. nov. (strain ARSA-103^T =KCTC 23183^T =JCM 17068^T =KACC 14971^T), and Flavobacterium cheonanense sp. nov. (strain ARSA-108^T =KCTC 23184^T =JCM 17069^T =KACC 14972) are proposed.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

Identification of nitrogen-fixing <Emphasis Type="Italic">Paenibacillus</Emphasis> from different plant rhizospheres and a novel <Emphasis Type="Italic">nifH</Emphasis> gene detected in the <Emphasis Type="Italic">P. stellifer</Emphasis>

A total of 534 isolates were selectively obtained from different plant rhizospheres based on their growth on nitrogen-free medium and their resistance to 80°C for 15 min. Of the 534 isolates, 23 isolates had nifH gene and exhibited nitrogenase activities. Based on 16S rDNA sequence, G + C content assay and DNA-DNA hybridization, the 23 isolates which divided into four monophyletic clusters were all belonged to the Paenibacillus genus. nifH gene deduced amino acid alignment aLnalysis revealed that cluster I, including 15 isolates, showed the highest NifH identity with Paenibacillus genus; while cluster II identified as P. stellifer by DNA-DNA hybridization was consistent with four uncultured bacterial clones. This study suggested that the nitrogen-fixing Paenibacillus were distributed in various ecosystems and prevalent in different plant rhizospheres. It was the first demonstration that nitrogen fixation existed in P. jamilae and P. stellifer. In eight isolates identified as P. stellifer species, a novel nifH gene was detected in Paenibacillus.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

<Emphasis Type="Italic">Streptomyces tunisialbus</Emphasis> sp. nov., a novel <Emphasis Type="Italic">Streptomyces</Emphasis> species with antimicrobial activity

A novel actinomycete strain designated S2^T was isolated from Tunisian rhizosphere soil of Lavandula officinalis. This isolate exhibited broad spectrum antibacterial activity against several Gram-positive and Gram-negative bacteria and also antifungal activity against yeast and filamentous fungi. The isolate S2^T presents morphological and chemotaxonomic characteristics typical of the members of the genus Streptomyces. Whole cell hydrolysates of S2^T were found to contain LL-diaminopimelic acid. The major fatty acids were identified as C16:0, anteiso-C15:0 and iso-C16:0 whereas the predominant menaquinones were found to be MK-9(H6) and MK-9(H8). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and three unidentified compounds. The G+C content of the genomic DNA was determined to be 71.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2^T belongs to the genus Streptomyces and is closely related to Streptomyces netropsis DSM 40259^T with 99.86% sequence similarity. Multi-locus sequence analysis (MLSA) based on four house-keeping gene alleles (gyrB, recA, trpB, rpoB) showed that isolate S2^T is closely related to S. netropsis, with an MLSA distance greater than 0.007. The DNA–DNA relatedness between strain S2^T and its near phylogenetic neighbour was 63.6 ± 2.3%, which is lower than the 70% threshold value for delineation of genomic prokaryotic species. This isolate was also distinguished from the type strain S. netropsis DSM 40259^T, using a combination of morphological and physiological features. Based on its phenotypic and molecular properties, strain S2^T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces tunisialbus sp. nov. is proposed. The type strain is S2^T (= JCM 32165^T = DSM 105760^T).     

<Emphasis Type="Italic">Methylibium </Emphasis><Emphasis Type="Italic">subsaxonicum</Emphasis> spec. nov., a Betaproteobacterium Isolated from a Hardwater Rivulet

A single strain, designated BF49^T, was isolated from a biofilm of a tufa deposit from the Westerhöfer rivulet, Lower Saxony, Germany. The G+C content of the genomic DNA of strain BF49^T was 69 mol% and the predominant ubiquinone was Q-8. Major fatty acids were C_16:1ω7c/15 iso 2OH and C_16:0. Comparative 16S rRNA gene sequence analysis indicated that the isolate was placed within the genus Methylibium, class Betaproteobacteria, distantly related to the type strain Methylibium petroleiphilum LMG 22953^T (97.4% similarity), Methylibium fulvum Gsoil 322^T (96%), and Methylibium aquaticum IMCC1728^T (95.7%). On the basis of phylogenetic and phenotypic distinctness we propose a novel species, Methylibium subsaxonicum sp. nov., with strain BF49^T (DSM 19570^T, CIP 109700^T) as the type strain.     

<Emphasis Type="Italic">Rhodoferax koreense</Emphasis> sp. nov,an obligately aerobic bacterium within the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>, and emended description of the genus <Emphasis Type="Italic">Rhodoferax</Emphasis>

Gram-staining-negative, uniflagellated, rod-shaped, designated as DCY110^T, was isolated from sludge located in Gangwon province, Republic of Korea. The phylogenetic tree of 16S rRNA gene sequence showed that the strain DCY110^T belonged to the genus Rhodoferax with a close similarity to Rhodoferax saidenbachensis DSM 22694^T (97.7%), Rhodoferax antarcticus DSM 24876^T (97.5%), Rhodoferax ferrireducens DSM 15236^T (97.3%), and Rhodoferax fermentans JCM 7819^T (96.7%). The predominant isoprenoid quinine was ubiquinone (Q-8). DNA G + C content was 62.8 mol%. The major polar lipids were phosphatidylethanolamine and two unidentified phospholipids. The major fatty acids (> 10%) were C_12:0, C_16:0, summed feature 3 (which comprised C_16:1 ω7c and/or C_16:1 ω6c). The DNA-DNA relatedness values between the strain DCY110^T and the closely related relatives used in this study were lower than 70%. Based on the following polyphasic analysis, the strain DCY110^T is considered as a novel species of the genus Rhodoferax, for which the name Rhodoferax koreense sp. nov. is proposed. The type strain is DCY-110^T (= KCTC 52288^T = JCM 31441^T).     

Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h. Mn-SOD activity required the ion Mn²⁺. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris.