- Volume 64, Issue Pt_1, 2014

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3–8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4–0.5×7.5–25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5–37 °C (optimum at 20–30 °C), at pH 6.0–10 (optimum 7.0–7.5) and with 0–0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1T and 94 % sequence similarity to Methanospirillum lacunae Ki8-1T. DNA–DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1T revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum , for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1T ( = DSM 26304T = VKM B-2808T).

A previously undescribed, slowly growing, scotochromogenic mycobacterial strain (49061T) was isolated from a patient with pulmonary infections during the hsp65-sequence-based identification of Korean clinical isolates. Its 16S rRNA gene sequence was unique and the phylogenetic analysis based on 16S rRNA gene sequence (1393 bp) placed the organism into the slow-growing Mycobacterium group close to Mycobacterium gordonae (99.0 % sequence similarity). Growth characteristics and acid-fastness also supported the placement of this species into the genus Mycobacterium . Phenotypically, this strain was generally similar to Mycobacterium gordonae ; however, of particular interest, the optimal growth temperature of strain 49061T was 25–30 °C, and it was not able to grow at 37 °C on 7H10 agar slants. Unique MALDI-TOF MS profiles of lipids, phylogenetic analysis based on another two gene sequences (hsp65 and rpoB) and a low DNA–DNA relatedness (46.52±0.7) strongly supported the taxonomic status of this strain as a representative of a distinct species from M. gordonae . It was concluded that the strain represents a novel species for which the name Mycobacterium paragordonae is proposed with the type strain 49061T ( = JCM 18565T = KCTC 29126T).

A filamentous actinomycete strain, designated GW9-2T, was isolated from a soil sample collected from the Fildes Peninsula, King George Island, West Antarctica. The strain was identified using a polyphasic taxonomic approach. The strain grew slowly on most media tested, producing small amounts of aerial mycelia and no diffusible pigments on most media tested. The strain grew in the presence of 0–12 % (w/v) NaCl (optimum, 2–4 %), at pH 9.0–11.0 (optimum, pH 9.0) and 10–37 °C (optimum, 28 °C). The isolate contained meso-diaminopimelic acid, no diagnostic sugars and MK-9(H4) as the predominant menaquinone. The major phospholipids were phosphatidylglycerol, phosphatidylcholine and phosphatidylmethylethanolamine. The major fatty acids were iso-C16 : 0, anteiso-C17 : 0, C18 : 1ω9c, iso-C15 : 0 and iso-C17 : 0. DNA–DNA relatedness was 37.6 % with Nocardiopsis lucentensis DSM 44048T, the nearest phylogenetic relative (97.93 % 16S rRNA gene sequence similarity). On the basis of the results of a polyphasic study, a novel species, Nocardiopsis fildesensis sp. nov., is proposed. The type strain is GW9-2T ( = CGMCC 4.7023T = DSM 45699T = NRRL B-24873T).

A Gram-positive, endospore-forming, rod-shaped bacterium, designated isolate J4T, was isolated from a neutral saline lake sample from Xinjiang Uyghur Autonomous Region, China, and subjected to a polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain J4T is most closely related to Salinibacillus aidingensis 25-7T (with 96.7 % similarity), Salinibacillus kushneri 8-2T (96.5 %), Ornithinibacillus scapharcae TW25T (96.4 %), Salirhabdus euzebyi CVS-14T (96.4 %) and Ornithinibacillus californiensis MB-9T (96.2 %). Chemotaxonomic analysis showed menaquinone-7 (MK-7) to be the major isoprenoid quinone of strain J4T; diphosphatidylglycerol and phosphatidylglycerol were the major cellular polar lipids and the cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. The major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The genomic DNA G+C content of strain J4T was determined to be 36.2 mol%. Strain J4T was positive for catalase activity and negative for oxidase activity. Strain J4T was observed to grow at 25–50 °C (optimal 35–42 °C), pH 6.5–8.0 (optimal 7.0–7.5) and in media containing 1–21 % (w/v) NaCl (optimal 9–12 %). Based on these data, strain J4T represents a novel species of the genus Salinibacillus and the name Salinibacillus xinjiangensis sp. nov. is proposed. The type strain is J4T ( = CGMCC 1.12331T = JCM 18732T).

Genomic, taxonomic and biochemical studies were performed on two strains of α-haemolytic streptococci that showed them to be clustered with major members of the Streptococcus mitis group. These Gram-stain-positive strains were isolated from tooth surfaces of caries-free humans and showed the classical spherical shape of streptococcal species growing in chains. Sequence analysis from concatenated 16S and 23S rRNA gene and sodA genes showed that these strains belonged to the mitis group, but both of them clustered into a new phylogenetic branch. The genomes of these two isolates were sequenced, and whole-genome average nucleotide identity (ANI) demonstrated that these strains significantly differed from any streptococcal species, showing ANI values under 91 % even when compared with the phylogenetically closest species such as Streptococcus oralis and S. mitis . Biochemically, the two isolates also showed distinct metabolic features relative to closely related species, like α-galactosidase activity. From the results of the present study, the name Streptococcus dentisani sp. nov. is proposed to accommodate these novel strains, which have been deposited in open collections at the Spanish type Culture Collection (CECT) and Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures (DSMZ), being respectively identified as Streptococcus dentisani Str. 7746 ( = CECT 8313 = DSM 27089) and Streptococcus dentisani Str. 7747T ( = CECT 8312T = DSM 27088T).

A bacterial strain designated RLAHU4BT was isolated from root nodules of Lupinus albus in León (Spain). The 16S rRNA gene sequence of this strain showed similarities lower than 97 % with respect to species of the genus Cohnella . The strain was a Gram-variable, sporulating rod, motile by means of peritrichous flagella, and facultatively anaerobic. It was positive for oxidase, catalase and β-galactosidase production but negative for urease, amylase and gelatinase. Strain RLAHU4BT grew in the presence of 5 % NaCl. MK-7 was the predominant menaquinone and meso-diaminopimelic acid was present in the peptidoglycan. anteiso-C15 : 0, iso-C16 : 0, iso-C15 : 0 and C16 : 0 were the major fatty acids. Major polar lipids of strain RLAHU4BT were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids, two unknown aminophospholipids and one unknown lipid. The DNA G+C content was 57.8 mol%. Strain RLAHU4BT presented phenotypic differences from all recognized species of the genus Cohnella . The phylogenetic, chemotaxonomic and phenotypic data indicated that strain RLAHU4BT belongs to a novel species of the genus Cohnella , for which the name Cohnella lupini sp. nov. is proposed, with strain RLAHU4BT ( = LMG 27416T = CECT 8236T) as the type strain.

Two strains, 8-4-E12T and 8-4-E13T, were isolated from a biowaste composting reactor. Based on 16S rRNA gene sequences, both strains belong to the genus Bacillus . Strain 8-4-E12T was most closely related to the type strains of Bacillus shackletonii , B. acidicola , B. sporothermodurans and B. oleronius (96.4, 96.3, 96.0 and 95.6 % 16S rRNA gene similarity, respectively), whereas strain 8-4-E13T was most closely related to the type strain of Bacillus humi (96.5 % sequence similarity). Strains 8-4-E12T and 8-4-E13T shared 94 % 16S rRNA gene sequence similarity. The fatty acid profile of strain 8-4-E12T was dominated by saturated iso- and anteiso-branched fatty acids (iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0), and also contained considerable amounts of C16 : 0. The fatty acid profile of strain 8-4-E13T showed a predominance of iso-C15 : 0 (65 %), with smaller amounts of other saturated branched-chain fatty acids along with an unsaturated alcohol. Both strains contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as major polar lipids. Additionally, strain 8-4-E12T contained an unknown lipid and strain 8-4-E13T two unknown (amino-)phospholipids. The diagnostic diamino acid found in the cell-wall peptidoglycan of 8-4-E12T and 8-4-E13T was meso-diaminopimelic acid. The predominant menaquinone was MK-7. The results of physiological and biochemical tests also allowed phenotypic differentiation of the two strains from each other and from related Bacillus species. On the basis of their phylogenetic, phenotypic and chemotaxonomic properties, strains 8-4-E12T and 8-4-E13T represent novel species of the genus Bacillus , for which the names Bacillus pervagus sp. nov. (type strain 8-4-E12T = DSM 23947T = LMG 27601T) and Bacillus andreesenii sp. nov. (type strain 8-4-E13T = DSM 23948T = LMG 27602T) are proposed.

A coccal-shaped organism, designated 516T, was isolated from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 516T had 98.9 % sequence similarity to that of the type strain Lactococcus garvieae NBRC 100934T. Comparison of three housekeeping genes, rpoA, rpoB and pheS, revealed that strain 516T was well separated from Lactococcus garvieae NBRC 100934T. DNA–DNA hybridization studies indicated that strain 516T had low DNA relatedness with Lactococcus garvieae NBRC 100934T (46.1 %). The DNA G+C content of strain 516T was 38.1 mol% and the major fatty acids were C16 : 0 (22.7 %), C19 : 0 cyclo ω8c (17.9 %) and summed feature 7 (29.0 %). Based on the evidence, strain 516T represents a novel species of the genus Lactococcus , for which the name Lactococcus formosensis sp. nov. is proposed. The type strain is 516T ( = NBRC 109475T = BCRC 80576T).

A taxonomic study was performed on Gram-stain-positive, catalase-negative and regular rod-shaped bacterial strains R4BT and R4C, isolated from the stomachs of honeybees. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strains was within the genus Lactobacillus ; the highest sequence similarity to R4BT was shown by Lactobacillus acidophilus BCRC 10695T (93.6 %). Lower sequence similarities were found to other obligately homofermentative lactobacilli. A PCR–DGGE method could detect the sequence of the 16S rRNA gene of strain R4BT at different developmental stages of honeybees occurring in two different locations in the Czech Republic. The distinctiveness of the strains from other lactobacilli was also confirmed by analysis of sequences of other phylogenetic markers applicable to the taxonomy of the genus Lactobacillus , ribotyping and rep-PCR analysis. The DNA G+C content of strain R4BT was 41.3 mol%. The predominant cellular fatty acids of strain R4BT were C18 : 1ω9c, summed C19 : 1ω6c/C19 : 0 cyclo ω10c, C16 : 0, summed C18 : 1ω7c/C18 : 1ω6c and summed C16 : 1ω7c/C16 : 1ω6c. The major polar lipids of strain R4BT were glycolipids, lipids and phospholipids. Phenotypic and phylogenetic characteristics also confirmed the independent status of the strains at the species level. Interestingly, strain R4BT was able to inhibit growth in vitro of Paenibacillus larvae subsp. larvae (causal agent of American foulbrood in honeybees) and Melissococcus plutonius (causal agent of European foulbrood). The name Lactobacillus apis sp. nov. is proposed for this novel taxon; the type strain is R4BT ( = CCM 8403T = LMG 26964T).

Two novel endospore-forming, aerobic bacilli, strains E173aT and E265T, were isolated from soil and sediment samples from Kozakli and Altinsu hot springs, Nevsehir (Turkey). Their young cells in the exponential phase of growth were motile, Gram-stain-positive, straight rods, 0.6–1.1×3.0–8.0 µm in size, but they became strikingly long, approximately 0.6–1.2 by 9.0–35.0 µm, after the stationary phase of growth. Cells varied in tests for oxidase, and had a weakly positive reaction for catalase. Both strains could grow between 40 and 70 °C, with optimal growth at 60 °C (E173aT) and 55 °C (E265T). Growth occurred within the range pH 5.0–11.0 with optimal growth at pH 9.0 (E173aT) and pH 8.5 (E265T). Strain E173aT grew within a salinity range from 0 to1.5 % (w/v) NaCl with optimal growth at 0.5 %, while strain E265T grew within the range 0–5.0 % (w/v), with an optimum at 3.0 %. The new isolates differed from each other in some phenotypic and chemotaxonomic characters as well as repetitive extragenic palindromic element PCR (rep-PCR) fingerprints. 16S rRNA gene sequence similarities suggested distant relationships with other members of the family Bacillaceae (<95.8 %), although the two strains showed 97.5 % sequence similarity between them, and had 55 % relatedness by DNA–DNA hybridization. The DNA G+C contents were 44.8 (E173aT) and 43.5 mol% (E265T). Moreover, the chemotaxonomic data of E173aT and E265T [presence of low amounts of meso-diaminopimelic acid, A1γ to A1γ′ cross-linkage types in peptidoglycan, fatty acids including iso-C15 : 0 (>60 %), iso-C17 : 0 and C16 : 0] supported the consideration of these isolates as members of a novel genus. Based upon phenotypic, phylogenetic and chemotaxonomic characteristics, it is proposed that new isolates represent a novel genus, Thermolongibacillus gen. nov., with two novel species: Thermolongibacillus altinsuensis sp. nov. (type strain E265T = DSM 24979T = NCIMB 14850T) and Thermolongibacillus kozakliensis sp. nov. (type strain E173aT = DSM 24978T = NCIMB 14849T).

A taxonomic study employing a polyphasic approach was performed on a novel anaerobic bacterium isolated from natural gas production-water. The bacterium stained Gram-negative and consisted of non-motile, non-spore-forming, rod-shaped cells. Products of glucose or starch fermentation were ethanol, CO2, formate, acetate and H2. The predominant fatty acids were C16 : 0 ALDE and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c. The DNA G+C content was 45.5 mol%. 16S rRNA gene sequence analysis demonstrated that the nearest phylogenetic neighbours of the novel strain were Acetivibrio multivorans DSM 6139T (98.5 %) and Proteiniclasticum ruminis JCM 14817T (95.4 %). The DNA–DNA hybridization value between the novel organism and Acetivibrio multivorans PeC1 DSM 6139T was determined to be only 30.2 %, demonstrating the separateness of the two species. Based on phylogenetic, phenotypic and chemotaxonomic evidence that clearly distinguished strain 232.1T from Proteiniclasticum ruminis and other close relatives, it is proposed that the novel isolate be classified as representing a novel species of a new genus within the family Clostridiaceae , Youngiibacter fragilis gen. nov., sp. nov. The type strain of the type species is 232.1T ( = ATCC BAA-2257T = DSM 24749T). In addition, Acetivibrio multivorans is proposed to be reclassified as Youngiibacter multivorans comb. nov.

Daqu is a fermentation starter used in the production of Chinese liquors. A thermophilic bacterium, designated strain H-18T, was isolated from a high-temperature Daqu sample collected from the manufacturing process of a sesame-flavoured liquor in Shandong province, China. It was investigated in a taxonomic study using a polyphasic approach. Strain H-18T formed white aerial mycelium and greyish-yellow substrate mycelium, bearing single endospores on aerial and substrate hyphae or on unbranched short sporophores. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids were iso-C15 : 0 and iso-C17 : 0. The predominant menaquinone was MK-7. These chemotaxonomic properties are similar to those of members of the genus Thermoactinomyces . The G+C content of the genomic DNA was 49.1 mol%. 16S rRNA gene sequence comparisons indicated that strain H-18T was most closely related to Thermoactinomyces vulgaris KCTC 9076T (96.42 % similarity), Thermoactinomyces intermedius KCTC 9646T (96.06 %), Laceyella putida KCTC 3666T (96.32 %) and Laceyella sacchari KCTC 9790T (95.55 %). Strain H-18T showed low DNA–DNA relatedness (40.8, 33.4, 20.0 and 14.4 %) with the above strains. Based on morphological and chemotaxonomic characteristics, DNA–DNA hybridization data and physiological properties, strain H-18T represents a novel species of the genus Thermoactinomyces , for which the name Thermoactinomyces daqus sp. nov. is proposed. The type strain is H-18T ( = DSM 45914T = CICC 10681T).

A novel thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming, motile, rod-shaped bacterium, strain C161abT, was isolated from a soil sample collected near Kizildere, Saraykoy-Buharkent power plant in Denizli. The isolate could grow at temperatures between 35 and 70 °C (optimum 55 °C), at pH 6.5–9.0 (optimum pH 8.0–8.5) and with 0–2.5 % NaCl (optimum 0.5 %, w/v). The strain formed cream-coloured, circular colonies and tolerated up to 70 mM boron. Its DNA G+C content was 37.8 mol%. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. Strain C161abT contained menaquinones MK-7 (96 %) and MK-6 (4 %). The major cellular fatty acids were iso-branched fatty acids: iso-C15 : 0 (52.2 %) and iso-C17 : 0 (28.0 %,) with small amounts of C16 : 0 (7.4 %). Phylogenetic analysis based on the 16S rRNA gene revealed 94.6–96.8 % sequence similarity with all recognized species of the genus Anoxybacillus . Strain C161abT showed the greatest sequence similarity to Anoxybacillus rupiensis DSM 17127T and Anoxybacillus voinovskiensis DSM 17075T, both had 96.8 % similarity to strain C161abT, as well as to Anoxybacillus caldiproteolyticus DSM 15730T (96.6 %). DNA–DNA hybridization revealed low levels of relatedness with the closest relatives of strain C161abT, A. rupiensis (21.2 %) and A. voinovskiensis (16.5 %). On the basis of the results obtained from phenotypic, chemotaxonomic, genomic fingerprinting, phylogenetic and hybridization analyses, the isolate is proposed to represent a novel species, Anoxybacillus calidus sp. nov. (type strain C161abT = DSM 25520T = NCIMB 14851T).

A bacterial consortium that accumulated more than 90 % (w/w) polyhydroxybutyrate (PHB) from lactate was selected in a laboratory-scale bioreactor with a ‘feast–famine’ regime. Bacterial strain YDT, representing a dominant species in this enrichment, was isolated and characterized. Analysis of the 16S rRNA gene sequence revealed that the isolate is a member of the class Gammaproteobacteria , forming an independent phylogenetic lineage. The closest relative of the isolate was Plasticicumulans acidivorans TUD-YJ37T, with 94 % 16S rRNA gene sequence similarity. Strain YDT was an obligate aerobe with large, ovoid, Gram-negative cells, motile by means of a polar flagellum. It utilized a relatively broad spectrum of substrates (e.g. carbohydrates, fatty acids) as carbon and energy sources. The temperature range for growth was 20–45 °C, with an optimum at 40 °C; the pH range was pH 6.0–8.0, with an optimum at pH 7.0. The major respiratory lipoquinones were Q-8 (91 %) and Q-7 (9 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and an unidentified aminolipid. The predominant fatty acids in the membrane polar lipids were C16 : 1ω7c, C16 : 0 and C18 : 1ω7c. The G+C content of the genomic DNA was 68.5 mol%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, the isolate is proposed to represent a novel species in the genus Plasticicumulans , for which the name Plasticicumulans lactativorans sp. nov. is proposed. The type strain is YDT ( = DSM 25287T = NCCB 100398T).

A moderately halophilic, Gram-stain-negative, non-sporulating bacterium designed as strain TYRC17T was isolated from olive-processing effluents. The organism was a straight rod, motile by means of peritrichous flagella and able to respire both oxygen and nitrate. Growth occurred with 0–25 % (w/v) NaCl (optimum, 7 %), at pH 5–11 (optimum, pH 7.0) and at 4–50 °C (optimally at 35 °C). It accumulated poly-β-hydroxyalkanoate granules and produced exopolysaccharides. The predominant fatty acids were C18 : 1ω7c, C16 : 1ω7c and C16 : 0. Ubiquinone 9 (Q-9) was the only respiratory quinone. The DNA G+C content of TYRC17T was 53.9 mol%. Phylogenetic analyses of 16S rRNA gene sequences revealed that the strain represents a member of the genus Halomonas and more precisely of the subgroup containing Halomonas sulfidaeris , H. titanicae , H. variabilis , H. zhanjiangensis , H. alkaliantarctica , H. boliviensis and H. neptunia . TYRC17T showed high 16S-rRNA sequence identities in particular with the three last species listed (99.4–99.5 %). A multilocus sequence analysis (MLSA) using the 23S rRNA, gyrB, rpoD and secA genes allowed clarifying the phylogenetic position of TYRC17T. This, combined with the level of DNA–DNA hybridization between TYRC17T and its closest relatives ranging from 21.6 % to 48.4 %, indicated that TYRC17T did not represent any of these species. On the basis of phenotypic and genotypic characteristics, and also genomic and phylogenetic evidence, it was concluded that strain TYRC17T represented a novel species of the genus Halomonas . The name Halomonas olivaria sp. nov. is proposed with TYRC17T ( = DSM 19074T = CCUG 53850BT) as the type strain.

A Gram-stain-negative, non-motile and aerobic bacterium, designated CF17T, was isolated from coastal planktonic seaweeds, East China Sea. The isolate grew at 18–37 °C (optimum 25–28 °C), pH 6.5–9.0 (optimum 7.0–8.0) and with 0–5 % NaCl (optimum 1–2 %, w/v) and 0.5–10 % sea salts (optimum 2–3 %, w/v). Growth of strain CF17T could be stimulated prominently by supplementing the growth medium with the autoclaved supernatant of a culture of strain CF5, which was isolated from the same sample along with strain CF17T. The cell morphology of strain CF17T was a bean-shaped rod consisting of a swollen end and a long prostheca. The phylogenetic analysis of 16S rRNA gene sequences indicated that strain CF17T clustered with Gemmobacter nectariphilus DSM 15620T within the genus Gemmobacter . The DNA G+C content of strain CF17T was 61.4 mol%. The respiratory quinone was ubiquinone Q-10. The major fatty acids included C18 : 1ω7c and C18 : 0. The polar lipids of strain CF17T consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two uncharacterized phospholipids, one uncharacterized aminolipid, three uncharacterized glycolipids and one uncharacterized lipid. On the basis of phenotypic, phylogenetic and chemotaxonomic data, strain CF17T ( = CGMCC 1.11024T = JCM 18498T) is considered to represent a novel species of the genus Gemmobacter , for which the name Gemmobacter megaterium sp. nov. is proposed.