- Volume 58, Issue 1, 2008

Two novel Gram-negative bacterial strains, designated CL-GR15T and CL-GR35, were isolated from coastal seawater of the east coast of Korea. Identical 16S rRNA gene sequences were found in the two strains, and it was found that the strains represented a distinct and deep evolutionary lineage of descent in the order Rhizobiales, and clustered with yet-uncultured marine bacteria. This lineage could not be associated with any of 12 known families in the order Rhizobiales. The most closely related established genus was Ochrobactrum (90.7–92.5 % sequence similarity) in the family Brucellaceae. Cells of the strains were facultatively anaerobic, weakly motile rods which occasionally exhibited cohesion with each other. The strains were catalase-, oxidase- and gelatinase-positive, and accumulated intracellular poly-β-hydroxybutyrate granules. Ubiquinone 10 was the major quinone. The major polar lipids comprised phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, an unidentified aminolipid and an unidentified glycolipid. The cellular fatty acids mainly consisted of C18 : 1 ω7c, summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 1 ω7c) and C20 : 1 ω7c. The DNA G+C content was 52.8–53.0 mol%. DNA–DNA hybridization experiments revealed high values (>97 %) for relatedness between strains CL-GR15T and CL-GR35 and suggested that these two strains constituted a single species. The distinct phylogenetic position and combinations of genotypic, phenotypic and chemotaxonomic characteristics support the proposal of Cohaesibacter gen. nov., with the type species Cohaesibacter gelatinilyticus sp. nov. (type strain, CL-GR15T=KCCM 42319T=DSM 18289T). Cohaesibacteraceae fam. nov. is also proposed.

A moderately halophilic Gram-negative bacterium, strain DS2T, was isolated from seawater from the East Sea in Korea. Strain DS2T grew at salinities of 0–14 % (w/v) NaCl and at temperatures in the range 10–38 °C. The cells were non-motile short rods (1.0–1.4 μm in width and 1.6–2.6 μm in length) and the major fatty acids were C18 : 1 ω7c and 11-methyl C18 : 1 ω7c. The genomic DNA G+C content was 72.0 mol% and the predominant lipoquinone was Q-10. The major cellular phospholipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS2T formed a distinct phyletic line from the genus Rubrimonas within the Alphaproteobacteria. The levels of 16S rRNA gene sequence similarity with respect to the type strains of related genera were below 94 %. On the basis of physiological and phylogenetic properties, strain DS2T represents a novel genus and species of the Alphaproteobacteria, for which the name Albimonas donghaensis gen. nov., sp. nov. is proposed. The type strain is DS2T (=KCTC 12586T=DSM 17890T).

A non-spore-forming, Gram-negative, motile, rod-shaped, boron-accumulating bacterium isolated from soil was characterized by polyphasic taxonomy. The strain, designated BAM-48T, was positive for catalase and oxidase activities and grew at 4–37 °C, 0–1 % NaCl and pH 5–9. Comparative analysis of the 16S rRNA gene sequence demonstrated that the novel isolate BAM-48T was closely related to Variovorax paradoxus IAM 12373T (99.3 % sequence similarity), Variovorax soli GH9-3T (98.3 %) and Variovorax dokdonensis DS-43T (97.0 %). DNA–DNA hybridization values between strain BAM-48T and V. paradoxus NBRC 15149T, V. soli KACC 11579T and V. dokdonensis KCTC 12544T were only 49.1, 25.4 and 24.6 %, respectively. The major respiratory quinone was ubiquinone 8 (Q-8). The DNA G+C content was 71.2 mol%. Strain BAM-48T contained C16 : 0 (36.1 %), C16 : 1 ω7c (21.4 %) and C17 : 0 cyclo (19.0 %) as the major fatty acids. Based on phenotypic, chemotaxonomic and phylogenetic data, strain BAM-48T should be classified as a member of a novel species in the genus Variovorax, for which the name Variovorax boronicumulans sp. nov. is proposed. The type strain is BAM-48T (=NBRC 103145T =KCTC 22010T).

Two thermotolerant, methylotrophic yeast strains, PT44T and S051T, were respectively isolated from a tree exudate and soil collected in Thailand. They were categorized as thermotolerant strains on the basis of their good growth below 20 °C and up to a relatively high temperature (37 °C). The major characteristics of the two strains that place them in the genus Ogataea are the formation of four helmet- or hat-shaped ascospores in a deliquescent ascus that may be produced parthenogenetically or by conjugation between a cell and its bud or between independent cells; multilateral budding; assimilation of nitrate; the presence of ubiquinone Q7; negative for Diazonium blue B colour and urease reactions; and the absence of arthroconidia and ballistoconidia. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that strain PT44T was differentiated from the strain S051T by 25 nucleotide substitutions and 1 gap in 554 nt, which was sufficient to justify the description of two separate species. The closest recognized species in terms of pairwise sequences similarity to PT44T was Pichia (Ogataea) dorogensis, with 13 nucleotide substitutions and 1 gap in 554 nt. Strain S051T was closest to Pichia thermomethanolica, with 7 nucleotide substitutions in 566 nt. Phenotypic characteristics of strains PT44T and S051T allowed them to be differentiated from each other and from the closest related species. On the basis of the above finding, the two strains represent two novel species of the genus Ogataea, for which the names Ogataea chonburiensis sp. nov. (type strain PT44T =BCC 21227T =NBRC 101965T =CBS 10363T) and Ogataea nakhonphanomensis sp. nov. (type strain S051T =BCC 21228T =NBRC 101966T =CBS 10362T) are proposed. We also propose the transfer of two thermotolerant methylotrophic members of the genus Pichia described previously to the genus Ogataea: Pichia siamensis is renamed Ogataea siamensis (Limtong, Srisuk, Yongmanitchai, Kawasaki, Yurimoto, Nakase & Kato) Limtong, Srisuk, Yongmanitchai, Yurimoto & Nakase comb. nov. (type strain JCM 12264T =TISTR 5818T) and Pichia thermomethanolica is renamed Ogataea thermomethanolica (Limtong, Srisuk, Yongmanitchai, Yurimoto, Nakase & Kato) Limtong, Srisuk, Yongmanitchai, Yurimoto & Nakase comb. nov. (type strain CBS 10098T =JCM 12984T =BCC 16875T).

A novel anaerobic, mesophilic, protein-utilizing bacterial strain, GWT, was isolated from the mesophilic hydrogen-producing granular sludge used to treat food industry wastewater. The strain was a Gram-positive, non-spore-forming and non-motile rod. Growth of the strain was observed at 20–48 °C and at pH 6.4–10.0. The strain used yeast extract and peptone as carbon and energy sources. Weak growth was also observed with tryptone and Casamino acids as carbon and energy sources. The strain used none of the tested carbohydrates, alcohols or fatty acids. The fermentation products in peptone-yeast broth included ethanol, acetic acid, hydrogen and carbon dioxide. Gelatin was not hydrolysed. Nitrate was reduced. Indole was produced. NH3 and H2S were not produced. The DNA G+C content of strain GWT was 38.0 mol%. The predominant cellular fatty acids were the saturated fatty acids C14 : 0 (15.58 %), C16 : 0 (25.40 %) and C18 : 0 (12.03 %). Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain GWT represented a new branch within cluster XII of the Clostridium subphylum, with <89.6 % 16S rRNA gene sequence similarities to all described species. On the basis of polyphasic evidence from this study, strain GWT represents a new genus and novel species, for which the name Proteiniborus ethanoligenes gen. nov., sp. nov. is proposed. The type strain is GWT (=CGMCC 1.5055T=JCM 14574T).

A Gram-positive, motile, moderately halophilic and rod-shaped bacterium, strain AHS-1T, was isolated from a marine solar saltern in Korea, and its exact taxonomic position was investigated by use of a polyphasic study. Strain AHS-1T grew optimally at pH 6.5–7.0 and 37–40 °C in the presence of 10–12 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain AHS-1T belonged to the genus Lentibacillus. Strain AHS-1T exhibited a 16S rRNA gene sequence similarity value of 97.0 % to the type strain of Lentibacillus salarius and values of 95.0–96.7 % to the type strains of other recognized Lentibacillus species. Strain AHS-1T had cell-wall peptidoglycan based on meso-diaminopimelic acid. It contained MK-7 as the predominant menaquinone. The major fatty acids (>10 % of total fatty acids) were anteiso-C15 : 0 and anteiso-C17 : 0, and the major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and two unidentified glycolipids. The DNA G+C content was 49.0 mol%. Strain AHS-1T shared a mean level of DNA–DNA relatedness of 8.5 % with L. salarius KCTC 3911T. Strain AHS-1T could also be distinguished from recognized Lentibacillus species based on several phenotypic properties. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain AHS-1T is considered to represent a novel species of the genus Lentibacillus, for which the name Lentibacillus salinarum sp. nov. is proposed. The type strain is AHS-1T (=KCTC 13162T=CCUG 54822T).

A moderately halophilic, strictly anaerobic bacterium, designated 37HS60T, was isolated from an olive mill wastewater in southern Morocco (Marrakesh). The cells were straight, motile and stained Gram-positive, forming spherical and terminal spores and with an atypical thick and stratified multilayered cell wall. Major fatty acid components were iso-C17 : 1ω10c or anteiso-C17 : 1ω3c (19.3 %), C14 : 0 (14.3 %), C16 : 1ω7c (9.9 %), C16 : 1ω7c DMA (8.5 %) and C16 : 0 (7.6 %). Strain 37HS60T grew from 20 to 50 °C with an optimum at 40–45 °C, and growth was observed from pH 5.5 to 8.5 with an optimum of 6.8. The salinity range for growth was 10–100 g NaCl l−1 with an optimum at 50 g NaCl l−1. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 37HS60T fell within the evolutionary radiation enclosed by cluster XII of the Clostridium subphylum. Strain 37HS60T exhibited highest 16S rRNA gene sequence similarity of 92.0 % with Caloranaerobacter azorensis and 90.6 % with Clostridium purinilyticum. Moreover, 37HS60T did not grow on basal medium with hexose or pentose sugars as carbon and energy sources. Pyruvate, fumarate and succinate were the best substrates for 37HS60T growth with 1.0 g yeast extract l−1. The DNA G+C content was 33.0 mol%. Due to its phenotypic and genotypic characteristics, isolate 37HS60T is proposed as a novel species of a new genus, Clostridiisalibacter paucivorans gen. nov., sp. nov. The type strain is 37HS60T (=JCM 14354T=CCUG 53849T).

A moderately halophilic, obligate alkaliphile (growth range pH 8–12), designated strain YN-1T, was isolated from indigo balls obtained from Ibaraki, Japan. The cells of the isolate stained Gram-positive, and were aerobic, non-motile, sporulating rods which grew optimally at pH 9. The strain grew in 3–14 % NaCl with optimum growth in 5 % NaCl. It hydrolysed casein and Tweens 20, 40 and 60, but not gelatin, starch, DNA or pullulan. Its major isoprenoid quinone was MK-7 and its cellular fatty acid profile mainly consisted of anteiso-C15 : 0, anteiso-C17 : 0 and anteiso-C17 : 1. 16S rRNA phylogeny suggested that strain YN-1T was a member of group 7 (alkaliphiles) of the genus Bacillus, with the closest relative being Bacillus clarkii DSM 8720T (similarity 99.5 %). However, DNA–DNA hybridization showed a low DNA–DNA relatedness (7 %) of strain YN-1T with B. clarkii DSM 8720T. Owing to the significant differences in phenotypic and chemotaxonomic characteristics, and phylogenetic and DNA–DNA relatedness data, the isolate merits classification as a new species, for which the name Bacillus polygoni is proposed. The type strain of this species is YN-1T (=JCM 14604T=NCIMB 14282T).

A moderately halophilic, spore-forming, Gram-positive, short-rod-shaped bacterium, designated strain MS10T, was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain MS10T was able to grow at NaCl concentrations in the range 5–20 % (w/v) with optimum growth at 10 % (w/v) NaCl. Growth occurred at temperatures of 10–50 °C (optimal growth at 33–35 °C) and pH 6.0–9.0 (optimal growth at pH 7.0). Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MS10T fell within the branch encompassing members of the genus Halobacillus and was most closely related to Halobacillus dabanensis JCM 12772T (99.2 % 16S rRNA gene sequence similarity). The DNA G+C content of strain MS10T was 45.7 mol%, the major respiratory isoprenoid quinone was MK-7 and the cell-wall peptidoglycan was of the l-Orn–d-Asp type, characteristics consistent with its affiliation to the genus Halobacillus. Strain MS10T showed a level of DNA–DNA hybridization with H. dabanensis JCM 12772T of 29 % and levels below 70 % were also obtained with respect to other recognized members of the genus Halobacillus. The major fatty acids of strain MS10T were iso-C16 : 0, anteiso-C15 : 0, iso-C14 : 0 and iso-C15 : 0. Overall, the phenotypic, genotypic and phylogenetic results presented in this study demonstrate that strain MS10T represents a novel species of the genus Halobacillus, for which the name Halobacillus mangrovi sp. nov. is proposed. The type strain is MS10T (=CECT 7206T=CCM 7397T).

Seventy-three strains of streptococci were isolated from the bat oral cavity. The colonies of strains grown on mitis salivarius agar were similar in morphology to those of mutans-like streptococci. The novel strains were analysed biochemically using the Rapid ID32 Strep microsystem and were subjected to DNA–DNA hybridization with other oral streptococci and to 16S rRNA gene sequence analysis. Based on phylogenetic and phenotypic evidence, it is proposed that these isolates be classified as Streptococcus dentirousetti sp. nov. The type strain of Streptococcus dentirousetti sp. nov. is NUM 1303T (=JCM 14596T=DSM 18963T).

A novel moderately halophilic, Gram-positive bacterium, designated strain QW6T, was isolated from textile industry wastewater in Qom, Iran. Strain QW6T was strictly aerobic, non-motile, non-sporulating and oxidase- and catalase-positive. It grew at salinities of 1–25 % (w/v) NaCl, showing optimal growth at 7.5–10.0 % (w/v). Growth occurred at 5.0–45.0 °C and over the pH range 6.5–10.0; growth was optimal at 35 °C and pH 7.5. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain QW6T is a member of the genus Salinicoccus. The organism possessed Lys- and Gly-containing peptidoglycan. Strain QW6T had iso-C15 : 0 and anteiso-C15 : 0 as the predominant fatty acids and MK-6 as the major respiratory lipoquinone. The chemotaxonomic profile of this organism was consistent with its classification in the genus Salinicoccus. The DNA G+C content of strain QW6T was 54.4 mol%. On the basis of phenotypic characteristics, 16S rRNA gene sequence analysis and DNA–DNA relatedness of less than 50 % to species of the genus Salinicoccus, it is proposed that strain QW6T (=DSM 18903T=CCM 7448T) should be placed in the genus Salinicoccus as the type strain of a novel species, Salinicoccus iranensis sp. nov.

Gram-positive, rod-shaped, endospore-forming, thermophilic bacteria were isolated from a geothermal soil collected on the north-west slope of Mount Melbourne in Antarctica. They grew aerobically at 42–60 °C (optimum 55 °C) and at pH 4.5–7.5 (optimum pH 5.5). Phylogenetic analysis of 16S rRNA gene sequences showed that these isolates were related most closely to the type strain of Alicyclobacillus pomorum (91 % similarity). Growth occurred in the presence of ferrous iron at micromolar concentrations and acid was produced from various sugars. Iso-branched fatty acids C15 : 0 (45.56 %) and C17 : 0 (35.81 %) were the most abundant cellular fatty acids. The DNA G+C content was 55.1 mol%. On the basis of phenotypic and phylogenetic characteristics, it is concluded that these strains represent a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus pohliae sp. nov. is proposed. The type strain is MP4T (=CIP 109385T =NCIMB 14276T).

A low-G+C, Gram-positive isolate, designated strain CVS-8T, was isolated from a sea salt evaporation pond on the Island of Sal in the Cape Verde Archipelago. This organism was found to be a catalase- and oxidase-positive, non-motile, spore-forming, aerobic, curved rod-shaped organism with an optimum growth temperature of about 35–37 °C and an optimum pH between 7.5 and 8.0. Optimal growth occurred in media containing 4–6 % (w/v) NaCl and no growth occurred in medium without NaCl. The cell-wall peptidoglycan was of the A1γ type with meso-diaminopimelic acid, the major respiratory quinone was MK-7, the major fatty acids were iso-15 : 0, 16 : 0, anteiso-15 : 0 and iso-16 : 0 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminoglycophospholipid. The G+C content of the DNA was 37.9 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-8T represented a novel species of the genus Bacillus, the highest levels of sequence similarity (mean pairwise similarity values of ∼97.5 %) being found with respect to the type strains of Bacillus shackletonii and Bacillus acidicola. On the basis of the phylogenetic, physiological and biochemical data, strain CVS-8T represents a novel species of the genus Bacillus, for which the name Bacillus isabeliae sp. nov. is proposed. The type strain is CVS-8T (=LMG 22838T=CIP 108578T).

Four Gram-positive, catalase-negative, coccoid-shaped isolates were obtained from the caecum and rectum of horses with oligofructose-induced equine laminitis. Phenotypic and phylogenetic studies were performed on these isolates. Initial biochemical profiling assigned two of the isolates to Streptococcus bovis. The other two isolates, however, could not be assigned conclusively on the basis of their biochemical profiles. Gene sequence analysis demonstrated that the four new isolates were related most closely to Streptococcus suis based on the 16S rRNA gene and to Streptococcus orisratti based on the manganese-dependent superoxide dismutase gene (sodA). Sequence divergence values from recognized Streptococcus species based on these two genes were >3 and >13 %, respectively, for all four isolates. Phylogenetic and phenotypic analyses demonstrated that the four isolates formed two distinct clonal groups that are suggested to represent two novel species of the genus Streptococcus. The names proposed for these organisms are Streptococcus henryi sp. nov. (type strain 126T =ATCC BAA-1484T =DSM 19005T) and Streptococcus caballi sp. nov. (type strain 151T =ATCC BAA-1485T =DSM 19004T).

There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA–DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (atpD, dnaK, gap, glnA, gltA, gyrB, pnp, recA, rpoB and thrC) was performed on 34 representatives of the genus Ensifer. Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA–DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA–DNA hybridization for the assessment of genetic relatedness between Ensifer species. Our MLSA data, confirmed by DNA–DNA hybridizations, support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.