- Volume 58, Issue 3, 2008

The taxonomic positions of four aerobic, obligately halo(alkali)philic/-tolerant, methanotrophic bacteria previously affiliated with the genera Methylobacter (‘Methylobacter alcaliphilus’ strains 20Z and 5Z) and Methylomicrobium (Methylomicrobium strains AMO1 and NI) were investigated. Phylogenetic analysis of 16S rRNA gene sequences indicated that the strains form a separate branch within the type I methanotrophic bacteria and are closely related to Methylomicrobium pelagicum. DNA–DNA hybridization data revealed relatively low levels of relatedness of Methylomicrobium sp. AMO1 and Methylomicrobium sp. N1 with each other and with previously described species of the genera Methylomicrobium and Methylobacter (<55 %), indicating that they may represent novel species. Based on the results presented here and on previously reported morphological and physiological characteristics, we classify these halotolerant and halophilic methanotrophic strains as representing novel species within the genus Methylomicrobium: Methylomicrobium alcaliphilum sp. nov. (type strain 20ZT =VKM B-2133T =NCIMB 14124T; reference strain 5Z =VKM B-2180), Methylomicrobium japanense sp. nov. (type strain NIT =VKM B-2462T =FERM BP-5633T =NBRC 103677T) and Methylomicrobium kenyense sp. nov. (type strain AMO1T =NCCB 97157T =NCIMB 13566T =VKM B-2464T). The genus Methylomicrobium has been emended in its description.

A Gram-negative, strictly aerobic, non-spore-forming bacterium, designated strain BD-c194T, was isolated from diesel-contaminated soil in Geoje, Korea. The cells were short, motile rods with single polar flagella. Growth of strain BD-c194T was observed between 15 and 45 °C (optimally at 30–35 °C) and between pH 6.0 and 9.5 (optimally at pH 7.5–9.0). The predominant fatty acids were 11-methyl C18 : 1 ω7c, C16 : 0, C18 : 1 ω7c, C18 : 0 and an unknown fatty acid (equivalent chain-length 18.814); a large amount of phosphatidylglycerol and a small amount of diphosphatidylglycerol were present as polar lipids. The G+C content of the genomic DNA was 60.8 mol% and the major isoprenoid quinone was Q-10. A comparative 16S rRNA gene sequence analysis showed that strain BD-c194T formed a well-defined phyletic lineage within the genus Devosia (with 100 % bootstrap support). The levels of 16S rRNA gene sequence similarity with respect to the type strains of other Devosia species ranged from 95.0 to 96.1 %. On the basis of chemotaxonomic data and molecular properties, strain BD-c194T represents a novel species within the genus Devosia, for which the name Devosia geojensis sp. nov. is proposed. The type strain is BD-c194T (=KCTC 22082T =DSM 19414T).

A Gram-negative, aerobic, neutrophilic, moderately halophilic bacterial strain, HS225T, was isolated from seawater samples around the Zhoushan Archipelago, Zhejiang Province, China. The isolate grew optimally in media with 5.0 % NaCl, at pH 7.0–8.0 and at 25–30 °C. The predominant fatty acids were C16 : 0 ω9c, C16 : 0, C12 : 0 3-OH and C18 : 1 ω9c. The genomic DNA G+C content was 59.0 mol%. Based on 16S rRNA gene sequence analysis, the isolate was found to be affiliated to the genus Marinobacter. Strain HS225T exhibited closest phylogenetic affinity to Marinobacter koreensis DD-M3T (98.1 % 16S rRNA gene sequence similarity). DNA–DNA relatedness data and DNA G+C contents as well as physiological and biochemical test results allowed the genotypic and phenotypic differentiation of strain HS225T from closely related species. Therefore, it is proposed that strain HS225T represents a novel species of the genus Marinobacter, for which the name Marinobacter pelagius sp. nov. is proposed. The type strain is HS225T (=CGMCC 1.6775T =JCM 14804T)

A detailed classification of a novel bacterial strain, designated F11T, capable of degrading fluorobenzene as a sole carbon and energy source, was performed by using a polyphasic approach. This Gram-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium was isolated from a sediment sample collected from an industrially contaminated site in northern Portugal. The predominant whole-cell fatty acids were C19 : 0 cyclo ω8c, C16 : 0, C18 : 1 ω7c, C18 : 0, C18 : 0 3-OH and C16 : 0 3-OH. The G+C content of the DNA was 62.9 mol% and the major respiratory quinone was ubiquinone 10 (UQ-10). 16S rRNA gene sequence analysis revealed that strain F11T was a member of the class Alphaproteobacteria and was phylogenetically related to the genus Labrys, having sequence similarities of 95.6 and 93.1 % to the type strains of Labrys monachus and Labrys methylaminiphilus, respectively. DNA–DNA hybridization experiments revealed levels of relatedness of <70 % between strain F11T and the type strains of L. monachus and L. methylaminiphilus (38.6 and 34.1 %, respectively), justifying the classification of strain F11T as representing a novel species of the genus Labrys. The name Labrys portucalensis sp. nov. is proposed for this organism. The type strain is F11T (=LMG 23412T=DSM 17916T).

An aerobic, Gram-negative, motile, non-pigmented bacterium, strain KMM 3042T, isolated from a deep-sea brittle star in the Fiji Sea, was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed marine Mn(II)-oxidizing isolate S185-2B as the closest neighbour of strain KMM 3042T (99.9 % sequence similarity). The two strains formed a distinct lineage within the genus Pseudomonas adjacent to the members of the Pseudomonas borbori cluster, sharing highest sequence similarity of 97.4 and 97.0 %, respectively, with P. borbori DSM 17834T and Pseudomonas flavescens DSM 12071T. The DNA–DNA hybridization value (71 %) between strains KMM 3042T and S185-2B confirmed their assignment to the same species. On the basis of phylogenetic analysis, DNA–DNA hybridization and physiological and biochemical characterization, strains KMM 3042T and S185-2B should be assigned to a novel species of the genus Pseudomonas, for which the name Pseudomonas marincola sp. nov. is proposed. The type strain is KMM 3042T (=NRIC 0729T =JCM 14761T).

A Gram-negative, mesophilic bacterial strain, designated 1-1BT, which degrades polycyclic aromatic hydrocarbons, was isolated from petroleum-contaminated seawater during a bioremediation experiment. A 16S rRNA gene sequence analysis indicated that the isolate was affiliated with the genus Thalassospira in the Alphaproteobacteria; the sequence was found to be most similar to those of Thalassospira profundimaris WP0211T (99.8 %), Thalassospira xiamenensis M-5T (98.2 %) and Thalassospira lucentensis DSM 14000T (98.1 %). However, the levels of DNA–DNA relatedness between strain 1-1BT and these type strains were 50.7±17.2, 35.7±17.8 and 32.0±21.1 %, respectively. In addition, strain 1-1BT was found to be distinct from the other described species of the genus Thalassospira in terms of some taxonomically important traits, including DNA G+C content, optimum growth temperature, salinity tolerance, utilization of carbon sources and fatty acid composition. Furthermore, strain 1-1BT and T. profundimaris were also different with regard to motility and denitrification capacities. On the basis of physiological and DNA–DNA hybridization data, strain 1-1BT represents a novel species within the genus Thalassospira, for which the name Thalassospira tepidiphila sp. nov. is proposed. The type strain is 1-1BT (=JCM 14578T =DSM 18888T).

Two novel species from Aspergillus section Flavi from different species of Arachis (peanuts) in Argentina are described as Aspergillus arachidicola sp. nov. and Aspergillus minisclerotigenes sp. nov. Their novel taxonomic status was determined using a polyphasic taxonomic approach with phenotypic (morphology and extrolite profiles) and molecular (β-tubulin and calmodulin gene sequences) characters. A. minisclerotigenes resembles Aspergillus flavus and Aspergillus parvisclerotigenus in producing aflatoxins B1 and B2, cyclopiazonic acid, kojic acid and aspergillic acid, but in addition it produces aflatoxins G1 and G2, aflavarins, aflatrem, aflavinines, parasiticolides and paspaline. This species also includes several isolates previously assigned to A. flavus group II and three Australian soil isolates. A. arachidicola produces aflatoxins B1, B2, G1 and G2, kojic acid, chrysogine and parasiticolide, and some strains produce aspergillic acid. The type strain of A. arachidicola is CBS 117610T =IBT 25020T and that of A. minisclerotigenes is CBS 117635T =IBT 27196T. The Mycobank accession numbers for Aspergillus minisclerotigenes sp. nov. and Aspergillus arachidicola sp. nov. are respectively MB 505188 and MB 505189 (http://www.mycobank.org).

Here, we investigate a group of red to pinkish ballistoconidia-forming yeasts that were preliminarily identified as Sporobolomyces roseus or Sporidiobolus pararoseus. Detailed molecular and micromorphological studies revealed that the sexual strains and several conspecific anamorphic isolates belonged to a novel teleomorph that represents the sexual stage of Sporobolomyces roseus. Consequently, a new taxon in the genus Sporidiobolus is here described as Sporidiobolus metaroseus sp. nov. (type strain CBS 7683T). The main characteristics of Sporidiobolus metaroseus are presented and compared with those of the more closely related species. Our studies also led to the clarification of the life cycle of Sporidiobolus pararoseus. We confirm that the teliospores of this species germinate by forming short branches of hyphae, instead of basidia.

Strains of a novel anaerobic, Gram-negative coccus were isolated from the supra-gingival plaque of children. Independent strains from each of six subjects were shown, at a phenotypic level and based on 16S rRNA gene sequencing, to be members of the genus Veillonella. Analysis revealed that the six strains shared 99.7 % similarity in their 16S rRNA gene sequences and 99.0 % similarity in their rpoB gene sequences. The six novel strains formed a distinct group and could be clearly separated from recognized species of the genus Veillonella of human or animal origin. The novel strains exhibited 98 and 91 % similarity to partial 16S rRNA and rpoB gene sequences of Veillonella parvula ATCC 10790T, the most closely related member of the genus. The six novel strains could be differentiated from recognized species of the genus Veillonella based on partial 16S rRNA and rpoB gene sequencing. The six novel strains are thus considered to represent a single novel species of the genus Veillonella, for which the name Veillonella rogosae sp. nov. is proposed. The type strain is CF100T (=CCUG 54233T=DSM 18960T).

A novel xylan-degrading bacterium, YB-45T, was isolated from forest soil. The organism is a facultatively anaerobic, Gram-variable, motile, endospore-forming, rod-shaped bacterium. It grew optimally at 37 °C and pH 7.5 in the presence of 3 % (w/v) NaCl. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. The DNA G+C content was 51.7 mol% and the predominant menaquinone was MK-7. Growth was observed with many carbohydrates, including xylan, as sole carbon sources. Strain YB-45T produces a wide variety of hydrolytic enzymes, such as xylanase, cellulase, amylase, β-mannanase, β-mannosidase, β-xylosidase, α-galactosidase, β-galactosidase and β-glucosidase. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain YB-45T belongs to the genus Paenibacillus, sharing sequence similarity that was <96 %. It was related most closely to Paenibacillus jamilae DSM 13815T, with 95.7 % sequence similarity. On the basis of morphological, chemotaxonomic, physiological and phylogenetic properties, strain YB-45T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus woosongensis sp. nov. is proposed. The type strain is YB-45T (=KCTC 3953T=DSM 16971T).

A Gram-positive or Gram-variable, motile, moderately halophilic, rod-shaped bacterial strain, ISL-50T, was isolated from a marine solar saltern in Korea and subjected to a polyphasic taxonomic study. Strain ISL-50T grew optimally at pH 7.0–8.0 and 37 °C in the presence of 8 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ISL-50T belonged to the genus Halobacillus. Strain ISL-50T exhibited 16S rRNA gene sequence similarity values of 95.3–98.2 % with respect to the type strains of recognized Halobacillus species. The cell-wall peptidoglycan was based on meso-diaminopimelic acid, the predominant menaquinone was MK-7 and the major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content was 39.3 mol%. DNA–DNA relatedness data and differential phenotypic properties revealed that strain ISL-50T was distinguishable from the recognized Halobacillus species. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain ISL-50T represents a novel species of the genus Halobacillus, for which the name Halobacillus seohaensis sp. nov. is proposed. The type strain is ISL-50T (=KCTC 13145T=CCUG 54358T).

A Gram-positive, moderately halophilic, motile, strictly aerobic, endospore-forming, oxidase- and catalase-positive, rod-shaped bacterium, strain YIM kkny16T, was isolated from a saline mud sample collected from the Keke salt lake in the Qaidam Basin, north-west China. This isolate grew in the presence of 0–25 % (w/v) NaCl and at pH 6.0–10.0 and 10–50 °C; optimum growth was observed with 10 % (w/v) NaCl and at pH 7.0 and 37 °C. Strain YIM kkny16T had meso-diaminopimelic acid as the diagnostic diamino acid, MK-7 as the predominant respiratory quinone, with a significant amount of MK-6, and anteiso-C15 : 0, iso-C14 : 0 and C16 : 1 ω7c alcohol as major fatty acids. Major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content was 41.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain YIM kkny16T was a member of the genus Virgibacillus, exhibiting sequence similarities of 94.9–97.3 % to the type strains of recognized Virgibacillus species. Strain YIM kkny16T could be differentiated from recognized Virgibacillus species based on phenotypic characteristics, chemotaxonomic differences, phylogenetic analysis and DNA–DNA hybridization data. On the basis of evidence from this polyphasic study, strain YIM kkny16T is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus kekensis sp. nov. is proposed. The type strain is YIM kkny16T (=DSM 17056T=CGMCC 1.6298T).

An obligately anaerobic, spore-forming, Gram-type-positive but Gram-staining-negative thermophilic bacterium, strain JW/YJL-F3T, was isolated from a Fijian hot spring sediment sample. Cells of strain JW/YJL-F3T were straight to slightly curved rods, 0.5–1.2 μm in diameter and 1.5–19 μm long. The temperature range for growth was between 40 and 67 °C, with an optimum at 60–63 °C. The pH25 °C range for growth was 4.5–8.4 with an optimum of 6.8. The salinity range for growth was 0–0.5 %. Strain JW/YJL-F3T utilized a range of substrates including arabinose, cellobiose, galactose, glucose, inulin, lactose, maltose, mannose, raffinose, ribose, trehalose, xylose and yeast extract as carbon and energy sources. The major fermentation end products from glucose were ethanol, acetate and formate. Strain JW/YJL-F3T converted thiosulfate to elemental sulfur, producing sulfur globules. The DNA G+C content was 37.6 mol% as determined by HPLC. Phylogenetic analysis using the 16S rRNA gene sequence indicated that the isolate is distantly related to the clade of the genus Thermoanaerobacterium. However, Thermoanaerobacterium polysaccharolyticum (96.7 % similarity to the type strain) and Thermoanaerobacterium zeae were the closest relatives, forming a separate, well-supported clade together with the novel isolate. Because Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae and strain JW/YJL-F3T have different features from other Thermoanaerobacterium species, including a higher G+C content and formate production, and are placed distantly from the remaining species of Thermoanaerobacterium (greater than 10 % distance) in the 16S rRNA gene sequence analysis, we propose to place the new isolate JW/YJL-F3T and Thermoanaerobacterium polysaccharolyticum and Thermoanaerobacterium zeae into the novel genus Caldanaerobius gen. nov. as Caldanaerobius fijiensis gen. nov., sp. nov. (the type species), Caldanaerobius polysaccharolyticus comb. nov. and Caldanaerobius zeae comb. nov., respectively. The type strain of Caldanaerobius fijiensis is JW/YJL-F3T (=ATCC BAA-1278T =DSM 17918T).

Strain BCRC 14193, isolated from soil, shared more than 99 % 16S rRNA gene sequence similarity with Bacillus amyloliquefaciens BCRC 11601T and Bacillus velezensis BCRC 17467T. This strain was previously identified as B. amyloliquefaciens, based on DNA–DNA hybridization, but its DNA relatedness value with B. velezensis BCRC 17467T was 89 %. To investigate the relatedness of strain BCRC 14193, B. amyloliquefaciens and B. velezensis, the partial sequence of the gene encoding the subunit B protein of DNA gyrase (gyrB) was determined. B. velezensis BCRC 17467T shared high gyrB gene sequence similarity with B. amyloliquefaciens BCRC 14193 (98.4 %) and all of the B. amyloliquefaciens strains available (95.5–95.6 %). DNA–DNA hybridization experiments revealed high relatedness values between B. velezensis BCRC 17467T and B. amyloliquefaciens BCRC 11601T (74 %) and the B. amyloliquefaciens reference strains (74–89 %). Based on these data and the lack of phenotypic distinctive characteristics, we propose Bacillus velezensis as a later heterotypic synonym of Bacillus amyloliquefaciens.

Gram-negative, spore-forming rods were isolated from a human urine sample (strain 5402403T) and a human cerebrospinal fluid sample (strain 4401170T). Based on genotypic characteristics, these strains belonged to the genus Paenibacillus and were closely related. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that they clustered with Paenibacillus massiliensis 2301065T (95.9 and 94.3 % 16S rRNA gene sequence similarity, respectively, for strains 5402403T and 4401170T), Paenibacillus illinoisensis NRRL NRS-61356T (90.6 and 93.8 %), Paenibacillus xylanilyticus XIL14T (95.3 and 95.4 %), Paenibacillus barcinonensis BP-23T (94.3 and 94.0 %), Paenibacillus pabuli NCIMB 12781T (89.1 and 92.3 %) and Paenibacillus amylolyticus NRRL NRS-290T (94.2 and 93.8 %). The predominant fatty acids were 15 : 0 anteiso (49.0 and 55.3 %, respectively, for strains 5402403T and 4401170T), 16 : 0 iso (15.4 and 13.5 %), 16 : 0 (7.6 and 3.6 %), 15 : 0 (6.3 and 2.8 %), 17 : 0 anteiso (5.7 and 7.5 %), 14 : 0 iso (4.1 and 2.7 %) and 15 : 0 iso (4.1 and 3.4 %). 16S rRNA gene sequence similarity between strain 4401170T and strain 5402403T was 98.4 %, but the DNA–DNA reassociation rate between the two strains was 53.2 %. So, considering the recommendations of the ad hoc committee, they do not belong to the same species. On the basis of phenotypic data and genotypic inference, it is proposed that the strains should be assigned to the novel species Paenibacillus urinalis sp. nov. (type strain 5402403T =CIP 109357T =CCUG 53521T) and Paenibacillus provencensis sp. nov. (type strain 4401170T =CIP 109358T =CCUG 53519T).

Vibrio cholerae and Vibrio mimicus have similar phenotypes and genomes making rapid differentiation of these two species difficult. The first standard multilocus sequence analysis (MLSA) scheme for the identification of these species is described. A collection of 45 representative isolates from different geographical regions and hosts was examined using segments of the housekeeping genes pyrH, recA and rpoA. Overall, the closest phylogenetic neighbours of these species were Vibrio furnissii and Vibrio fluvialis. V. cholerae and V. mimicus formed separate species clusters on the basis of each gene, suggesting that these genes are useful as identification markers. These species clusters arose by the accumulation of point mutations. The pyrH gene showed the highest resolution for differentiating V. cholerae and V. mimicus. The maximum interspecies pyrH gene sequence similarity was 91 %. Clearly, V. mimicus strains were more heterogeneous than V. cholerae strains at the three loci. It is suggested that vibrio species may be defined on the basis of MLSA data. A vibrio species was defined as a group of strains forming a monophyletic group on the basis of these loci and with an intraspecific sequence similarity of at least 95 %. V. cholerae and V. mimicus isolates can be readily identified through the open database resource ‘The Taxonomy of Vibrios’ (http://www.taxvibrio.lncc.br/).

Many nanoflagellate morphospecies comprise an enormous variation of genotypes, probably indicating cryptic species. One of the best-investigated morphospecies with respect to molecular and ecophysiological variation are flagellates of the Spumella morphotype. Here, we have phylogenetically analysed three protein-coding genes (actin, alpha-tubulin, beta-tubulin), internal transcribed spacers (ITS1, ITS2) and the 5.8S rDNA of 17 Spumella-like strains isolated from soil, freshwater and marine samples in order to (i) test the validity of the current Spumella-like phylogenetic classification system based exclusively on small subunit (SSU) rDNA, (ii) elucidate the phylogenetic associations of SSU rDNA-unresolved strains and (iii) evaluate the validity of the assignment of ecophysiological adaptations to previously identified SSU rDNA sequence clades. All single-gene analyses show different patterns of support, are incongruent and identify a number of conflicting nodes. Likewise, a concatenation of all protein genes fails to recover specific SSU rDNA clades. However, a combined analysis of all genes confidently resolved the conflicts of the single genes and the protein-gene concatenations and resulted in a tree topology that is identical to the SSU rDNA analysis, but with enhanced phylogenetic resolution and decisively greater support. We conclude that, depending on the genes concatenated, a ‘supergene’ analysis minimizes artefactual effects of single genes and may be superior in its performance in phylogenetically analysing cryptic species. We confirm the validity of the SSU rDNA Spumella-like phyloclades and support the suggestion that these clades indeed seem to reflect certain ecophysiological adaptations.