- Volume 62, Issue 1, 2012

A thermophilic bacterium, designated strain RHA1T, was isolated from a sediment sample collected from a hot spring in Tengchong county, Yunnan province, south-west China, and was characterized by using a polyphasic approach. Based on its phenotypic and phylogenetic characteristics, strain RHA1T was affiliated to the genus Laceyella. The strain formed white aerial and yellow–white substrate mycelia, bearing single endospores on short sporophores. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Whole-cell hydrolysates contained ribose and glucose. The major fatty acids were iso-C15 : 0 (62.39 %) and anteiso-C15 : 0 (17.55 %). The predominant menaquinone was MK-9. The G+C content of the genomic DNA of strain RHA1T was 47.9 mol%. Based on DNA–DNA hybridization data, chemotaxonomic characteristics and differential physiological properties, strain RHA1T is considered to represent a novel species of the genus Laceyella, for which the name Laceyella sediminis sp. nov. is proposed; the type strain is RHA1T ( = DSM 45263T = CCTCC AA 208058T).

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n = 12) and a teat apex (n = 1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA–DNA hybridization with S. hyicus DSM 20459T and S. chromogenes DSM 20674T confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C15 : 0, ai-C15 : 0, i-C17 : 0 and C20 : 0 and the peptidoglycan type was A3α l-Lys–Gly5. Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4T ( = DSM 23656T = CCUG 59809T) is the type strain.

A mesophilic, aerotolerant, endospore-forming, fermentative bacterium, designated strain B1T, was isolated from soil polluted by crude oil in the Karamay Oil Field, China. Cells were Gram-positive, rod-shaped, 1.1–1.6 µm wide and 2.3–4.7 µm long, and were motile by means of peritrichous flagella. Growth occurred at 10–40 °C and pH 6.0–8.9; optimal growth occurred at 28–32 °C and pH 7.3. The optimal concentrations of NaCl and sea salts for growth were 0.5 and 1 % (w/v), respectively. The strain was halotolerant and grew in the presence of NaCl or sea salts up to a concentration of 9 % (w/v). Substrates utilized as sole carbon sources were beef extract, yeast extract, peptone, tryptone, casein, d-glucose, d-fructose, d-xylose, d-ribose, d-galactose, maltose, l-rhamnose, trehalose, l-valine, dl-alanine plus l-proline and dl-alanine plus l-glycine. The main products of glucose fermentation were ethanol and acetate. iso-C15 : 0, iso-C14 : 0, C16 : 0 and iso-C13 : 0 were the major fatty acids. 16S rRNA gene sequence analysis revealed that the isolate belongs to the genus Tepidibacter, showing 94.7 and 94.1 % similarity to the type strains of Tepidibacter formicigenes and Tepidibacter thalassicus, respectively. The genomic DNA G+C content of strain B1T was 29.8 mol%. On the basis of its phenotypic and genotypic properties, strain B1T is suggested to represent a novel species of the genus Tepidibacter, for which the name Tepidibacter mesophilus sp. nov. is proposed. The type strain is B1T ( = CGMCC 1.5148T  = JCM 16806T).

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22T, was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15–37 °C, at pH 7–10 and with <8 % (w/v) NaCl (optimum growth: 30 °C, pH 7–8 and 1–3 % NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22T was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162T (98.5 % 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2T (97.9 %), Bacillus infantis SMC 4352-1T (97.4 %), Bacillus firmus IAM 12464T (96.8 %) and Bacillus muralis LMG 20238T (96.8 %). DNA–DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22T from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22T ( = DSM 23494T = NRRL B-59432T = LMG 25783T).

A Gram-negative, non-sporulating, non-flagellated rod, designated BR-9T, was isolated from soil collected on the Korean peninsula. Strain BR-9T grew optimally at pH 6.0–7.0, at 30 °C and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BR-9T belonged to the genus Pedobacter and clustered with Pedobacter insulae DS-139T and Pedobacter koreensis WPCB189T. Strain BR-9T exhibited 98.2 and 97.5 % 16S rRNA gene sequence similarity with P. insulae DS-139T and P. koreensis WPCB189T, respectively, and <96.7 % sequence similarity with the type strains of other species in the genus Pedobacter. Strain BR-9T contained MK-7 as the predominant menaquinone and iso-C15 : 0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) as the major fatty acids. The DNA G+C content of strain BR-9T was 38.5 mol%. DNA–DNA relatedness between strain BR-9T and P. insulae DS-139T and P. koreensis KCTC 12536T was 3.4–4.2 %, which indicated that the isolate was genetically distinct from these type strains. Strain BR-9T was also distinguishable by differences in phenotypic properties. On the basis of the data presented, strain BR-9T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter boryungensis sp. nov. is proposed. The type strain is BR-9T ( = KCTC 23344T  = CCUG 60024T).

A rod-shaped, yellow and strictly aerobic marine bacterium, designated KYW382T, was isolated from seawater collected from the South Sea, Republic of Korea. Cells were Gram-negative and catalase- and oxidase-positive. The major fatty acids were iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 0 3-OH and anteiso-C15 : 0. The DNA G+C content was 32.4 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain KYW382T constituted an evolutionary lineage within the radiation enclosing the members of the genus Gaetbulibacter. The closest neighbour was Gaetbulibacter saemankumensis SMK-12T (96.1 % 16S rRNA gene sequence similarity). A number of phenotypic characteristics distinguished strain KYW382T from the described members of the genus Gaetbulibacter. On the basis of the data presented in this study, strain KYW382T represents a novel species, for which the name Gaetbulibacter aestuarii sp. nov. is proposed. The type strain is KYW382T ( = KCTC 23303T  = JCM 17455T). An emended description of the genus Gaetbulibacter is also given.

A Gram-negative, non-flagellated, non-spore-forming bacterium, designated YCS-6T, that was motile by gliding, was isolated from seawater on the southern coast of Korea. Strain YCS-6T grew optimally at 30 °C and with 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YCS-6T fell within the genus Tenacibaculum and was most closely associated with Tenacibaculum litopenaei B-IT, with which the isolate exhibited 95.9 % 16S rRNA gene sequence similarity. Sequence similarity between strain YCS-6T and other members of the genus Tenacibaculum was 93.8–95.7 %. Strain YCS-6T contained menaquinone-6 (MK-6) as the predominant respiratory quinone and iso-C15 : 0, summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C15 : 0 3-OH and iso-C15 : 1 G as the major fatty acids. The DNA G+C content was 32.7 mol%. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain YCS-6T from all other members of the genus Tenacibaculum. On the basis of our phenotypic, chemotaxonomic and phylogenetic data, strain YCS-6T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum geojense sp. nov. is proposed. The type strain is YCS-6T ( = KCTC 23423T  = CCUG 60527T).

A Gram-negative-staining, non-motile rod, designated GG-w14T, was isolated from the rhizosphere of Angelica polymorpha Maxim. Phylogenetic analysis of 16S rRNA gene sequences revealed that the isolate belonged to the genus Mucilaginibacter and exhibited 93.9–97.4 % 16S rRNA gene sequence similarity with recognized members of the genus Mucilaginibacter (closest relative Mucilaginibacter gossypii Gh-67T). DNA–DNA relatedness between strain GG-w14T and M. gossypii KCTC 22380T was <41 %. Strain GG-w14T grew at 4–35 °C, at pH 5.0–8.0 and with 0–1 % (w/v) NaCl. The isolate hydrolysed casein, CM-cellulose and starch and contained menaquinone 7 as the major menaquinone. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH; 39.9 %), iso-C15 : 0 (24.2 %) and iso-C17 : 0 3-OH (12.4 %). The DNA G+C content was 42.5 mol%. These data suggest that strain GG-w14T should be considered as a representative of a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter angelicae sp. nov. is proposed. The type strain is GG-w14T ( = KCTC 23250T = NCAIM B 02415T).

A pink-pigmented, Gram-negative, rod-shaped, motile, strictly aerobic bacterium, designated strain 311T, was isolated from desert sand in Xinjiang, China. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 311T was related closely to Cesiribacter andamanensis AMV16T (94.6 % similarity). The DNA G+C content of strain 311T was 47.1 mol% and the major respiratory quinone was menaquinone 7 (MK-7). The main cellular fatty acids were C16 : 1ω5c (29.9 %), iso-C15 : 0 (21.9 %), iso-C17 : 0 3-OH (13.3 %) and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B; 13.0 %). Based on phenotypic and chemotaxonomic data and phylogenetic analysis, strain 311T is considered to represent a novel species of the genus Cesiribacter, for which the name Cesiribacter roseus sp. nov. is proposed. The type strain is 311T ( = CCTCC AB 207142T  = KACC 15456T).

The taxonomic positions of two thermophilic actinomycetes isolated from an arid Australian soil sample were established based on an investigation using a polyphasic taxonomic approach. The organisms had chemical and morphological properties typical of members of the genus Amycolatopsis and formed distinct phyletic lines in the Amycolatopsis methanolica 16S rRNA subclade. The two organisms were distinguished from one another and from the type strains of related species of the genus Amycolatopsis using a range of phenotypic properties. Based on the combined genotypic and phenotypic data, it is proposed that the two isolates be classified in the genus Amycolatopsis as Amycolatopsis thermophila sp. nov. (type strain GY088T = NCIMB 14699T = NRRL B-24836T) and Amycolatopsis viridis sp. nov. (type strain GY115T = NCIMB 14700T = NRRL B-24837T).

A novel actinomycete, designated strain DLS-45T, was isolated from soil from the surface of a rock collected from the peak of Darangshi Oreum (Small Mountain) in Jeju, Republic of Korea. Substrate mycelium was well developed, but aerial mycelium was scant on most of the media tested. Spherical to subspherical spores on the aerial mycelium were in chains that were arranged in hooks and their surfaces were warty. The combination of morphological and chemical features supported the classification of the new isolate in the genus Actinomadura. The neighbour-joining tree based on 16S rRNA gene sequences showed that the strain belonged to the family Thermomonosporaceae and formed a coherent cluster with Actinomadura pelletieri IMSNU 22169T (98.0 % sequence similarity). The other closest relatives were Actinomadura bangladeshensis 3-46-b(3)T (98.2 %) and Actinomadura chokoriensis 3-25-a(11)T (97.9 %). Levels of DNA–DNA relatedness between strain DLS-45T and the type strains of the phylogenetic relatives were less than 17 %. A battery of phenotypic, genotypic and DNA–DNA relatedness data indicated that strain DLS-45T represented a novel species of the genus Actinomadura, for which the name Actinomadura meridiana sp. nov. is proposed. The type strain is strain DLS-45T ( = KCTC 19558T = DSM 45252T).

A novel actinomycete strain, designated TRM F103T, was isolated from a hypersaline habitat of the Tarim basin in Xinjiang province, north-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Amycolatopsis and was most closely related to Amycolatopsis halophila YIM 93223T (99.3 % 16S rRNA gene sequence similarity). However, DNA–DNA relatedness between these two strains, based on triplicate experiments, was only 31.6 %. The isolate contained meso-diaminopimelic acid and ribose, glucose and galactose as the major whole-cell sugars. The predominant menaquinone was MK-8(H4). The major fatty acids were iso-C16 : 0 and C16 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine and glucosamine-containing phospholipids. The G+C content of the genomic DNA was 66.4 mol%. The phenotypic data clearly distinguished the isolate from its closest relatives. The combined phylogenetic, chemotaxonomic and phenotypic data indicate that the isolate represents a novel species of the genus Amycolatopsis. The proposed name is Amycolatopsis salitolerans sp. nov., with TRM F103T ( = JCM 15899T = CCTCC AB 208326T) as the type strain.

A novel xylan-degrading actinomycete, strain YIM 61515T, was isolated from surface-sterilized leaves of the medicinal plant Maytenus austroyunnanensis. Cells were Gram-positive and non-spore-forming, produced primary branches and formed white to yellowish white colonies on the media tested. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 61515T was most similar to Promicromonospora aerolata V54AT and Promicromonospora vindobonensis V45T (99.4 and 99.1 % sequence similarity, respectively). The isolate formed a separate lineage in a cluster containing P. aerolata V54AT. 16S rRNA gene sequence similarity between the isolate and other members of the genus Promicromonospora ranged from 96.3 to 98.4 %. Chemotaxonomic data, including major menaquinones, fatty acid compositions and polar lipid profiles, supported the placement of strain YIM 61515T in the genus Promicromonospora. DNA–DNA relatedness, physiological and biochemical data showed that strain YIM 61515T could be distinguished from members of all known species of the genus Promicromonospora and therefore represented a novel species. The name Promicromonospora xylanilytica sp. nov. is proposed, with YIM 61515T ( = DSM 21603T = CCTCC AA 208046T) as type strain.

A novel Gram-positive, non-motile, rod-shaped bacterium, designated strain RB-62T, was isolated during a study of culturable bacteria from the gut of Moechotypa diphysis (Pascoe) and its taxonomic position was investigated. Strain RB-62T grew at 15–30 °C and pH 5.0–8.5. The isoprenoid quinones were menaquinones MK-11 (77.1 %), MK-10 (11.7 %) and MK-12 (11.2 %). The major cellular fatty acids were anteiso-C15 : 0 (34.6 %), anteiso-C17 : 0 (29.8 %), iso-C16 : 0 (17.0 %) and cyclohexyl-C17 : 0 (11.4 %). The diagnostic diamino acid of the cell-wall peptidoglycan was 2,4-diaminobutyric acid. The G+C content of the genomic DNA of strain RB-62T was 70.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RB-62T was affiliated with the genus Herbiconiux cluster within the family Microbacteriaceae, and was related most closely to Herbiconiux ginsengi wged11T (98.08 % similarity). The level of DNA–DNA relatedness between strain RB-62T and H. ginsengi wged11T was 43.2 % (reciprocal 66.7 %). Phenotypic and phylogenetic characteristics clearly distinguished strain RB-62T from recognized species of the genus Herbiconiux. Based on data from the present polyphasic study, strain RB-62T is considered to represent a novel species of the genus Herbiconiux, for which the name Herbiconiux moechotypicola sp. nov. is proposed. The type strain is RB-62T ( = KCTC 19653T = JCM 16117T).

Two xylose-utilizing yeast strains isolated from rotten wood collected in the rainforest in different mountains of Hainan province, southern China, were studied. Sequence analysis of the large subunit rDNA D1/D2 domain and internal transcribed spacer region revealed that the strains represent a novel anamorphic yeast species, for which the name Candida cellulosicola sp. nov. is proposed; the type strain is HNX16-2T ( = CGMCC 2.3503T = CBS 11952T). Phylogenetically, the novel species was closely related to a xylose-utilizing teleomorphic ascomycetous yeast species Spencermartinsiella europaea in the family Trichomonascaceae, but differed from the latter by 3.0 % mismatches in the D1/D2 domain.

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and ‘the rest’ of the present family Halobacteriaceae.

Pathogenic scuticociliates, which are common in the haemolymph or tissues of maricultured animals and often cause serious diseases, are a species-rich assemblage with mostly unresolved systematic relationships, especially in some less-studied groups. In the present study, we sequenced the small-subunit rRNA gene of six species of scuticociliates, Uronemella parafilificum, Metanophrys sinensis, Parauronema longum, Cohnilembus verminus, Porpostoma notata and Ancistrum crassum, the last two of which have not been studied previously using molecular analyses. Phylogenetic trees were constructed using Bayesian inference, maximum-likelihood and maximum-parsimony methods to assess the inter- and intra-generic relationships of scuticociliates. Results revealed the following: 1) Porpostoma did not cluster with the Philasteridae, Cohnilembidae or any other family of the order Philasterida; 2) sequences of Uronemella parafilificum and Uronemella filificum showed a difference of 1.02 % (15 nt sites), revealing a close relationship between them; 3) the approximately unbiased test rejected monophyly of both Metanophrys and Parauronema, indicating that the terminal position of the anterior end of the paroral membrane and the structure of membranelle 1 are unreliable characters for distinction of genera in this group of scuticociliates; 4) Ancistrum crassum grouped with Boveria subcylindrica, showing a close phylogenetic relationship between the orders Thigmotrichida and Pleuronematida; and 5) Parauronema longum, Cyclidium plouneouri and Cyclidium porcatum should be removed from their currently assigned genera.