- Volume 66, Issue 1, 2016

Two novel cell-wall-less, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains were isolated from acidic streamers formed on the surfaces of copper-ore-containing sulfidic deposits in south-west Spain and North Wales, UK. Cells of the strains varied from 0.1 to 2 μm in size and were pleomorphic, with a tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0–1.2 and 37–40 °C, with the optimal substrates for growth being beef extract (3 g l− 1) for strain S5T and beef extract with tryptone (3 and 1 g l− 1, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and small relative amounts of intact and core diether lipids were present. Strains S5T and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. The DNA G+C content was 37.3 mol% in strain S5T and 37.16 mol% for strain PM4. A similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales, with 73.9–86.3 % identity with sequences from members of the order with validly published names. The average nucleotide identity between genomes of the strains determined in silico was 98.75 %, suggesting, together with the 16S rRNA gene-based phylogenetic analysis, that the strains belong to the same species. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. are proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T and PM4 ( = JCM 30641 = VKM B-2940). The type strain of Cuniculiplasma divulgatum is S5T ( = JCM 30642T = VKM B-2941T).

Two extremely halophilic archaea, strains Cb34T and C170, belonging to the genus Halorubrum, were isolated from the brine of the hypersaline lake Aran-Bidgol in Iran. Cells of the two strains were motile, pleomorphic rods, stained Gram-variable and produced red-pigmented colonies. Strains Cb34T and C170 required 25 % (w/v) salts, pH 7.0 and 37 °C for optimal growth under aerobic conditions; 0.3 M Mg2+ was required. Cells of both isolates were lysed in distilled water and hypotonic treatment with < 10 % NaCl provoked cell lysis. Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that these two strains were closely related to Halorubrum cibi B31T (98.8 %) and other members of the genus Halorubrum. In addition, studies based on the rpoB′ gene revealed that strains Cb34T and C170 are placed among the species of Halorubrum and are closely related to Halorubrum cibi B31T, with rpoB′ gene sequence similarity less than or equal to 95.7 %. The polar lipid patterns of both strains consisted of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. The DNA G+C content was 62.1–62.4 mol%. DNA–DNA hybridization studies confirmed that strains Cb34T and C170 constitute a distinct species. Data obtained in this study show that the two strains represent a novel species, for which the name Halorubrum halodurans sp. nov. is proposed. The type strain is Cb34T ( = CECT 8745T = IBRC-M 10233T).

A novel streptomycete strain, designated XY25T, was isolated from the rhizosphere soil in an alfalfa field in Jingyang, Shanxi, China. The isolate showed optimal growth at 37 °C, and was capable of growing at pH 6–10 and in the presence of 0–6 % (w/v) NaCl. Mycelia of strain XY25T appeared spiral and developed into white spore chains with long-rod spores and a smooth surface. The 16S rRNA gene sequence of XY25T was determined and was found to be highly similar to those of species of the genus Streptomyces including Streptomyces silaceus DSM 41861T (99.11 % 16S rRNA gene sequence similarity), Streptomyces flavofungini DSM 40366T (98.49 %) and Streptomyces intermedius DSM 40372T (98.43 %), all of which were used for further characterization. Each of the four streptomycetes showed distinctive patterns of carbon usage and fatty acids composition. Analysis of cellular components of strain XY25T revealed ll-diaminopimelic acid as diagnostic diamino acid and xylose as the major sugar, whereas polar lipids were determined as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid, two unknown phosphatidylinositol mannosides and several unknown lipids. Menaquinones were dominated by MK-9(H6) and MK-9(H8), and the main fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. DNA–DNA hybridization studies indicated that strain XY25T showed relatedness values of 35.2–40.42 % with the closest related species. Based on these results, strain XY25T represents a novel species of the genus Streptomyces, for which the name Streptomyces alfalfae sp. nov. is proposed. The type strain is XY25T ( = KCTC 39571T = CCTCC AA2015019T).

A bacterial strain, THG-S15-4T, was isolated from garden soil taken from the Guro-gu district of Seoul, Republic of Korea. Strain THG-S15-4T was Gram-stain-positive, facultatively anaerobic, coccus-shaped and non-motile, forming white colonies. The strain grew optimally at 25–37 °C, at pH 7.0 and in the presence of 0–2.0 % (w/v) NaCl. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that strain THG-S15-4T was affiliated to species of the genus Brachybacterium, and the most closely related species were Brachybacterium rhamnosum KCTC 9917 T (98.5 % sequence similarity) and Brachybacterium squillarum KCTC 19899T (96.9 % sequence similarity). The DNA–DNA relatedness between strain THG-S15-4T and B. rhamnosum KCTC 9917 T was found to be below 20.0 %. The DNA G+C content was determined to be 69.5 mol%. The major isoprenoid quinone detected was MK-7. Strain THG-S15-4T was characterized chemotaxonomically as having meso-diaminopimelic acid in the cell-wall peptidoglycan. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, unidentified glycolipids and an unidentified polar lipid. The major fatty acids were found to be iso-C16 : 0 and anteiso-C15 : 0. The results of physiological and biochemical tests enabled strain THG-S15-4T to be differentiated phenotypically from species of the genus Brachybacterium with validly published names. Therefore, it is suggested that this newly isolated organism represents a novel species, for which the name Brachybacterium horti sp. nov. is proposed. The type strain is THG-S15-4T ( = KCTC 39563T = CCTCC AB 2015116T).

An alkalitolerant actinomycete strain, designated EGI 80425T, capable of producing ansamitocin P-3, was isolated from a saline–alkali soil sample of Xinjiang province, north-west China, and subjected to a polyphasic taxonomic characterization. Strain EGI 80425T formed non-fragmented substrate mycelia and white aerial hyphae with long spore chains. Whole-cell hydrolysates of the isolate contained meso-diaminopimelic acid as the diagnostic diamino acid and rhamnose as the major sugar. The major fatty acids were anteiso-C17 : 0, iso-C16 : 0 and C18 : 1ω9c. The predominant menaquinones were MK-10(H4), MK-10(H6), MK-10(H8) and MK-9(H4). The G+C content of the genomic DNA of strain EGI 80425T was 70.2 mol%. Strain EGI 80425T showed highest 16S rRNA gene sequence similarity to Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111T (96.44 %). Phylogenetic analysis showed that strain EGI 80425T clustered with the members of the genus Nocardiopsis. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain EGI 80425T represents a novel species of the genus Nocardiopsis, for which the name Nocardiopsis ansamitocini sp. nov. is proposed. The type strain is EGI 80425T ( = CGMCC 9969T = KCTC 39605T).

In a previous study on bifidobacterial distribution in New World monkeys, six strains belonging to the Bifidobacteriaceae were isolated from faecal samples of baby common marmosets (Callithrix jacchus L.). All the isolates were Gram-positive-staining, anaerobic, asporogenous and fructose-6-phosphate phosphoketolase-positive. Comparative analysis of 16S rRNA gene sequences revealed relatively low levels of similarity (maximum identity 96 %) to members of the genus Bifidobacterium, and placed the isolates in three independent clusters: strains of cluster I (MRM_5.9T and MRM_5.10) and cluster III (MRM_5.18T and MRM_9.02) respectively showed 96.4 and 96.7 % 16S rRNA gene sequence similarity to Bifidobacterium callitrichos DSM 23973T, while strains of cluster II (MRM_8.14T and MRM_9.14) showed 95.4 % similarity to Bifidobacterium stellenboschense DSM 23968T. Phylogenetic analysis of partial hsp60 and clpC gene sequences supported an independent phylogenetic position of each cluster from each other and from the related type strains B. callitrichos DSM 23973T and B. stellenboschense DSM 23968T. Clusters I, II and III respectively showed DNA G+C contents of 64.9–65.1, 56.4–56.7 and 63.1–63.7 mol%. The major cellular fatty acids of MRM_5.9T were C14 : 0, C16 : 0 and C18 : 1ω9c dimethylacetal, while C16 : 0 was prominent in strains MRM_5.18T and MRM_8.14T, followed by C18 : 1ω9c and C14 : 0. Biochemical profiles and growth parameters were recorded for all the isolates. Based on the data provided, the clusters represent three novel species, for which the names Bifidobacterium myosotis sp. nov. (type strain MRM_5.9T = DSM 100196T = JCM 30796T), Bifidobacterium hapali sp. nov. (type strain MRM_8.14T = DSM 100202T = JCM 30799T) and Bifidobacterium tissieri sp. nov. (type strain MRM_5.18T = DSM 100201T = JCM 30798T) are proposed.

An actinomycete strain, designated SA-246T, was isolated from a forest soil sample collected from Chungnam, South Korea. Applying a polyphasic approach, the isolate was identified as a member of the genus Catenulispora using morphological and chemotaxonomic characteristics, including the presence of ll-diaminopimelic acid, glutamic acid, alanine and glycine in the peptidoglycan. Whole-cell hydrolysates contained predominantly rhamnose, mannose, ribose, arabinose, galactose and glucose. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). 16S rRNA gene sequence analysis revealed that strain SA-246T belongs to the genus Catenulispora, showing the highest sequence similarity to Catenulispora yoronensis TT N02-20T (98.7 % 16S rRNA gene sequence similarity), Catenulispora subtropica TT 99-48T (98.2 %), Catenulispora graminis BR-34T (97.4 %), Catenulispora rubra Aac-30T (97.4 %) and Catenulispora acidiphila ID139908T (97.3 %). On the basis of polyphasic analysis from this study, strain SA-246T represents a novel species of the genus Catenulispora, for which the name Catenulispora fulva sp. nov. is proposed. The type strain is SA-246T ( = KACC 17878T = NBRC 110074T).

A novel obligately halophilic, facultatively alkaliphilic actinobacterium, designated EGI 80759T, was isolated from the rhizosphere of Tamarix hispida Willd, Karamay, Xinjiang province, north-west China. Cells of strain EGI 80759T were Gram-stain-positive, non-motile and non-endospore-forming rods. Strain EGI 80759T showed obligately halophilic growth with a tolerance to 8–25 % (w/v) NaCl (optimum growth at 10–12 %, w/v) and facultatively alkaliphilic growth within the pH range 7.0–11.0 (optimum growth at pH 9.0–10.0). Cell-wall hydrolysates of the isolate contained meso-diaminopimelic acid (peptidoglycan type A1γ), with glucose, glucosamine, ribose and mannose as the major sugars. The major fatty acids identified were 10-methyl-C17 : 0, C17 : 1ω8c and C17 : 0. The predominant menaquinone was MK-9(H4). The G+C content of the genomic DNA was 72.1 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, revealed that strain EGI 80759T clustered with members of the class Nitriliruptoria and showed highest 16S rRNA gene sequence similarities with Euzebya tangerina F10T (90.3 %) and Nitriliruptor alkaliphilus ANL-iso2T (88.1 %). On the basis of the data obtained from phenotypic and chemotaxonomic studies and the phylogenetic analysis, the isolate is proposed to be a representative of a novel genus and a novel species, Egibacter rhizosphaerae gen. nov., sp. nov., of a proposed novel family, Egibacteraceae fam. nov., and order, Egibacterales ord. nov., within the class Nitriliruptoria. The type strain of the type species, Egibacter rhizosphaerae, is EGI 80759T ( = CGMCC 1.14997T = KCTC 39588T).

A novel actinomycete, strain RCU-197T, was isolated from soil of a peat swamp forest in Rayong Province, Thailand. Using a polyphasic approach, the strain was classified in the genus Streptomyces. It contained ll-diaminopimelic acid in the cell-wall peptidoglycan. No diagnostic sugars were detected in whole-cell hydrolysates and there was a lack of mycolic acids. The major menaquinones were MK-9(H6) and MK-9(H8). The predominant cellular fatty acids were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. The polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside, an unknown aminolipid and two unknown phospholipids. Phylogenetic analysis of 16S rRNA gene sequences showed the strain formed distinct clade within the genus Streptomyces and was closely related to Streptomyces echinatus NBRC 12763T (98.78 % 16S rRNA gene sequence similarity). According to the polyphasic approach as well as DNA–DNA relatedness, the strain could be clearly differentiated from closely related species and represents a novel species of the genus Streptomyces, for which the name Streptomyces actinomycinicus sp. nov. is proposed. The type strain is RCU-197T ( = JCM 30864T = TISTR 2208T = PCU 342T).

A novel bacterial strain, designated THG-S11.7T, was isolated from garden soil in Incheon, South Korea. Cells of the strain were Gram-stain-positive, aerobic, non-motile cocci, and were catalase- and oxidase-positive. Colonies of the strain were white. Strain THG-S11.7T grew optimally at 28 °C, at pH 7.0 and in the presence of 2.0 % NaCl. 16S rRNA gene sequence analysis indicated that the strain was a member of the genus Nocardioides. Strain THG-S11.7T showed a 16S rRNA gene sequence similarity of 98.2 % to Nocardioides kongjuensis KCTC 19054T, 98.0 % to Nocardioides caeni KCTC 19600T, 97.9 % to Nocardioides daeguensis KCTC 19799T, 97.8 % to Nocardioides nitrophenolicus KCTC 047BPT, 97.6 % to Nocardioides aromaticivorans KACC 20613T, 97.5 % to Nocardioides simplex KACC 20620T and 97.0 % to Nocardioides ginsengisoli KCTC 19135T. DNA–DNA relatedness values between strain THG-S11.7T and the closest phylogenetic neighbours were below 45.0 % and the DNA G+C content of strain THG-S11.7T was 72.2 mol%. Strain THG-S11.7T was characterized chemotaxonomically as having ll-diaminopimelic acid in the cell-wall peptidoglycan and menaquinone MK-8(H4) as the predominant isoprenoid quinone. The major phospholipid was determined to be diphosphatidylglycerol. The major cellular fatty acids of strain THG-S11.7T were iso-C15 : 0, C16 : 0 and iso-C16 : 0. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolate represents a novel species of the genus Nocardioides, for which the name Nocardioides albidus sp. nov. is proposed. The type strain is THG-S11.7T ( = KCTC 39607T = CCTCC AB 2015297T).

Three bacterial strains, HKU51T, HKU52T and HKU53, were isolated from a conjunctival swab, corneal scraping and blood culture of three patients in Hong Kong with conjunctivitis, keratitis and catheter-related bacteraemia, respectively. Cells were Gram-stain-positive, aerobic, catalase-positive, non-sporulating and non-motile bacilli. The three strains had unique biochemical profiles that were distinguishable from those of closely related species of the genus Tsukamurella. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. 16S rRNA gene sequence analysis revealed 100 % sequence identity between HKU52T and HKU53, and the two strains shared 99.5 % sequence identity with Tsukamurella sunchonensis JCM 15929T and Tsukamurella pseudospumae JCM 13375T; HKU51T shared 99.6 % sequence identity with Tsukamurella pulmonis CCUG 35732T. The DNA G+C contents of strains HKU51T, HKU52T and HKU53 were 70.9 ± 2.2, 71.3 ± 2.1 and 71.2 ± 2.3 mol% (mean ± sd; n = 3), respectively. DNA–DNA hybridization confirmed that the novel strains were distinct from other known species of the genus Tsukamurella ( ≤ 50.1 ± 3.7 % DNA–DNA relatedness); two of the isolates, HKU52T and HKU53, represented the same species ( ≥ 94.6 ± 5.6 % DNA–DNA relatedness), while the third isolate, HKU51T, represented another species. The novel species Tsukamurella hongkongensis sp. nov. is proposed to accommodate strains HKU52T and HKU53, with HKU52T ( = JCM 30715T = DSM 100208T) as the type strain; whilst another novel species, Tsukamurella sinensis sp. nov., is proposed to accommodate the third isolate, HKU51T ( = JCM 30714T = DSM 100207T), which is designated the type strain.

The name ‘Mycobacterium alsiense’, although reported in 2007, has not been validly published. Polyphasic characterization of three available strains of this species led us to the conclusion that they represent a distinct species within the genus Mycobacterium. The proposed novel species grows slowly and presents pale yellow-pigmented colonies. Differentiation from other mycobacteria is not feasible on the basis of biochemical and cultural features alone while genetic analysis, extended to eight housekeeping genes and one spacer region, reveals its clear distinction from all other mycobacteria. Mycobacterium asiaticum is the most closely related species on the basis of 16S rRNA gene sequences (similarity 99.3 %); the average nucleotide identity between the genomes of the two species is 80.72 %, clearly below the suggested cut-off (95–96 %). The name Mycobacterium alsense sp. nov. is proposed here for the novel species and replaces the name ‘M. alsiense’, ex Richter et al. 2007 , given at the time of isolation of the first strain. The type strain is TB 1906T ( = DSM 45230T = CCUG 56586T).

A novel species of the genus Actinoplanes, strain LDG1-22T, for which we propose the name Actinoplanes lichenis sp. nov., was isolated from a lichen sample collected from tree bark in Thailand. The taxonomic position of the species has been described based on a polyphasic approach. Strain LDG1-22T produced irregular sporangia on agar media. It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. The major menaquinone was MK-9(H4); the polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and phosphatidylglycerol. Whole-cell hydrolysates contained ribose, glucose, mannose and small amounts of arabinose and xylose. The major cellular fatty acids were anteiso-C15 : 0 (31.2 %) and iso-C16 : 0 (14.2 %). Mycolic acids were absent. The G+C content was 73.6 %. 16S rRNA gene sequence analysis of strain LDG1-22T showed highest similarity (98.8 %) to Actinoplanes friuliensis DSM 45797T and it clustered with Actinoplanes nipponensis JCM 3264T and Actinoplanes missouriensis JCM 3121T in phylogenetic tree analysis. On the basis of the phenotypic characteristics and DNA–DNA relatedness, strain LDG1-22T could be distinguished from related species of the genus Actinoplanes and so represents a novel species of this genus. The type strain of Actinoplanes lichenis sp. nov. is LDG1-22T ( = JCM 30485T = TISTR 2343T = PCU 344T).

A Gram-stain-positive, aerobic, coccoid, non-motile, non-spore-forming bacterium, designated strain S3Af-1T, was isolated from surface-sterilized bark of Bruguiera sexangula collected from Dongzhaigang National Nature Reserve in Hainan, China, and examined using a polyphasic approach to clarify its taxonomic position. This bacterium did not produce substrate mycelia or aerial hyphae, and no diffusible pigments were observed on the media tested. Strain S3Af-1T grew optimally without NaCl, at 28–30 °C and at pH 7.0.Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S3Af-1T belonged to the genus Microlunatus and shared highest similarity with ‘Microlunatus terrae’ BS6 (97.43 %) and Microlunatus soli CC-12602T (97.08 %). DNA–DNA hybridization results indicated that the level of relatedness between strain S3Af-1T and M. soli CC-12602T was less than 70 %. The DNA G+C content of strain S3Af-1T was 67.1 mol%. The cell-wall peptidoglycan contained ll-2,6-diaminopimelic acid. MK-9(H6) and MK-9(H4) were the predominant menaquinones. Phosphatidylglycerol, diphosphatidylglycerol, an unidentified glycolipid, two unidentified phospholipids and other lipids were detected in the polar lipid extracts. The major fatty acids were iso-C16 : 0, anteiso-C17 : 0 and anteiso-C15 : 0. On the basis of phylogenetic analysis, and phenotypic and chemotaxonomic characteristics, strain S3Af-1T represents a novel species of the genus Microlunatus, for which the name Microlunatus endophyticus sp. nov. is proposed. The type strain is S3Af-1T ( = DSM 100019T = CGMCC 4.7306T).

A novel bacterial strain, designated S5T, was isolated from Pingaleshwar beach, in India. Cells were Gram-stain-positive, rod-shaped, non-motile and non-endospore-forming. Based on 16S rRNA gene sequence analysis, the strain was identified as belonging to the class Firmibacteria and was related most closely to Amphibacillus fermentum DSM 13869T (97.6 % sequence similarity). However, it shared only 93.1 % 16S rRNA gene sequence similarity with Amphibacillus xylanus NBRC 15112T, the type species of the genus, indicating that strain S5T might not be a member of the genus Amphibacillus. The DNA–DNA relatedness between strain S5T and Amphibacillus fermentum DSM 13869T was 39 %. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol and two phospholipids. Isoprenoid quinones were absent from strain S5T. Fatty acid analysis revealed that anteiso-C15 : 0, C16 : 0 and iso-C15 : 0 were the predominant fatty acids present. The results of phylogenetic, chemotaxonomic and biochemical tests allowed the clear differentiation of strain S5T, which is considered to represent a novel species of a new genus in the family Bacillaceae, for which the name Pelagirhabdus alkalitolerans gen. nov., sp. nov. is proposed. The type strain of Pelagirhabdus alkalitolerans is S5T ( = KCTC 33632T = CGMCC 1.15177T). Based on the present study, it is also suggested to transfer Amphibacillus fermentum to this new genus, as Pelagirhabdus fermentum comb. nov. The type strain of Pelagirhabdus fermentum is Z-7984T = (DSM 13869T = UNIQEM 210T).