Chthonobacter rhizosphaerae sp. nov., a bacterium isolated from rhizosphere soil of Citrus sinenesis.

A Gram-staining negative, facultative anaerobic, motile and short rod-shaped bacterium, designated strain yh7-1T, was isolated from rhizosphere soil of Citrus sinenesis collected from the garden of Citrus sinenesis in Ailao Mountain, south-west China. Cells grew at 15-45 °C, pH 5.0-9.0 and were able to tolerate up to 1% (w/v) NaCl on R2A medium. The respiratory lipoquinone was Q-10 and the major cellular fatty acids contained summed feature 8 (C18:1 ω7c or C18:1 ω6c) and C18:0. Polar lipids in the cellular membrane were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and one unidentified aminophospholipid. The genomic DNA G+C content was 69.9 mol%. On basis of 16S rRNA gene sequence analysis, strain yh7-1T showed the highest similarities with Chthonobacter albigriseus KCTC 42450T (97.6%), Mongoliimonas terrestris KCTC 42635T (97.0%) and lower than 97.0% to other species. Phylogenetic trees based on 16S rRNA gene sequences indicated that strain yh7-1T clustered with C. albigriseus KCTC 42450T. The ANI values ranged between 78.1 and 82.7% for C. albigriseus KCTC 42450T, M. terrestris KCTC 42635T and strain yh7-1T, which were lower than the prokaryotic species delineation threshold of 95.0-96.0%. The digital DNA-DNA hybridization values between C. albigriseus KCTC 42450T, M. terrestris KCTC 42635T and strain yh7-1T indicated that the new isolate represents a novel genomic species. According to the phenotypic and genotypic characteristics, strain yh7-1T should belong to the genus Chthonobacter, for which the name Chthonobacter rhizosphaerae sp. nov. (type strain yh7-1T = CGMCC 1.17236T = CCTCC AB 2019258T = KCTC 82185T) is proposed.

Methylocystis silviterrae sp.nov., a high-affinity methanotrophic bacterium isolated from the boreal forest soil.

A novel species is proposed for a high-affinity methanotrophic representative of the genus Methylocystis. Strain FST was isolated from a weakly acidic (pH 5.3) mixed forest soil of the southern Moscow area. Cells of FST are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type-II methanotrophs. Only methane and methanol are used as carbon sources. FST grew at a temperature range of 4-37 °C (optimum 25-30 °C) and a pH range of 4.5 to 7.5 (optimum pH 6.0-6.5). The major fatty acids were C18  :  1ω8c, C18  :  1ω7c and C18  :  0; the major quinone as Q-8. FST displays 16S rRNA gene sequences similarity to other taxonomically recognized members of the genus Methylocystis, with Methylocystis hirsuta CSC1T (99.6 % similarity) and Methylocystis rosea SV97T (99.3 % similarity) as its closest relatives. The genome comprises 3.85 Mbp and has a DNA G+C content of 62.6 mol%. Genomic analyses and DNA-DNA relatedness with genome-sequenced members of the genus Methylocystis demonstrated that FST could be separated from its closest relatives. FST possesses two particulate methane monooxygenases (pMMO): low-affinity pMMO1 and high-affinity pMMO2. In laboratory experiments, it was demonstrated that FST might oxidize methane at atmospheric concentration. The genome contained various genes for nitrogen fixation, polyhydroxybutyrate synthesis, antibiotic resistance and detoxification of arsenic, cyanide and mercury. On the basis of genotypic, phenotypic and chemotaxonomic characteristics, it is proposed that the isolate represents a novel species, Methylocystis silviterrae sp. nov. The type strain is FST (=KCTC 82935T=VKM B-3535T).

Hansschlegelia quercus sp. nov., a novel methylotrophic bacterium isolated from oak buds.

Novel aerobic, restricted facultatively methylotrophic bacteria were isolated from buds of English oak (Quercus robur L.; strain DubT) and northern red oak (Quercus rubra L.; strain KrD). The isolates were Gram-negative, asporogenous, motile short rods that multiplied by binary fisson. They utilized methanol, methylamine and a few polycarbon compounds as carbon and energy sources. Optimal growth occurred at 25 °C and pH 7.5. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and phoshatidylglycerol. The major cellular fatty acids of cells were C18 : 1 ω7c, 11-methyl C18 : 1 ω7c and C16 : 0. The major ubiquinone was Q-10. Analysis of 16S rRNA gene sequences showed that the strains were closely related to the members of the genus Hansschlegelia: Hansschlegelia zhihuaiae S113T(97.5-98.0 %), Hansschlegelia plantiphila S1T (97.4-97.6 %) and Hansschlegelia beijingensis PG04T(97.0-97.2 %). The 16S rRNA gene sequence similarity between strains DubT and KrD was 99.7 %, and the DNA-DNA hybridization (DDH) result between the strains was 85 %. The ANI and the DDH values between strain DubT and H. zhihuaiae S113T were 80.1 and 21.5  %, respectively. Genome sequencing of the strain DubT revealed a genome size of 3.57 Mbp and a G+C content of 67.0 mol%. Based on the results of the phenotypic, chemotaxonomic and genotypic analyses, it is proposed that the isolates be assigned to the genus Hansschlegelia as Hansschlegelia quercus sp. nov. with the type strain DubT (=VKM B-3284T=CCUG 73648T=JCM 33463T).

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).

Terasakiella salincola sp. nov., a marine alphaproteobacterium isolated from seawater, and emended description of the genus Terasakiella.

A Gram-reaction-negative, S-shaped, motile, poly-ß-hydroxybutyrate-accumulating, facultatively anaerobic, beige-pigmented bacterium, designated strain KMU-80T, was isolated from seawater collected from the Republic of Korea. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Methylocystaceae, of the class Alphaproteobacteria, and that it possessed the greatest sequence similarity (96.7 %) to Terasakiella pusilla NBRC 13613T. The DNA G+C content of KMU-80T was 48.3 mol%, and ubiquinone 10 was the sole respiratory quinone. The predominant cellular fatty acids consisted of C18 : 1ω7c (60.2 %), C16 : 0 (13.4 %) and C16 : 1ω7c and/or C16 : 1ω6c (11.1 %). Strain KMU-80T had phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid and four unidentified lipids as polar lipids. Based on its distinct phylogenetic position and the combination of genotypic and phenotypic characteristics, this strain is considered to represent a novel species of the genus Terasakiella, for which the name Terasakiella salincola sp. nov. is proposed. The type strain of T. salincola sp. nov. is KMU-80T (= KCCM 90274T = NBRC 112846T). An amended description of the genus Terasakiella is also provided.

Chthonobacter albigriseus gen. nov., sp. nov., isolated from grass-field soil.

A novel aerobic, Gram-stain-negative and non-motile bacterial strain, designated ED7T, was isolated from grass-field soil in Cheonan, Korea. Strain ED7T utilized methanol and methylamine, but not formate, as carbon and energy sources. The strain was able to grow at 20-42 °C (optimum 30-35 °C), at pH 7.0-8.5 (optimum pH 7.5-8.0), and in the absence of NaCl. According to the similarities of the 16S rRNA gene sequences, strain ED7T was most closely related to the genera Labrenzia (≤93.3 % 16S rRNA gene sequence similarity), Pleomorphomonas (≤93.1 %) and Prosthecomicrobium (≤93.1 %). A phylogenetic analysis based on the 16S rRNA gene sequence of strain ED7T revealed that it was affiliated with the family Methylocystaceae, being most closely related to the genus Pleomorphomonas. In contrast to Pleomorphomonas koreensis and Pleomorphomonas oryzae, strain ED7T did not contain the nifH gene. The DNA G+C content of the genomic DNA of strain ED7T was 71.8 mol%. The predominant fatty acids of strain ED7T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 2 (C14 : 0 3-OH, and/or C16 : 1 iso I), 11-methyl C18 : 1ω7c and C16 : 0 3-OH. The major isoprenoid quinone was ubiquinone 10 (Q-10). The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unknown aminophospholipid. Based on phenotypic, chemotaxonomic and genotypic characteristic data, strain ED7T could be differentiated from other genera, suggesting that strain ED7T represents a novel species of a new genus in the family Methylocystaceae, for which the name Chthonobacter albigriseus gen. nov., sp. nov. is proposed. The type strain of the type species is ED7T (=JCM 30603T=KCTC 42450T).

Oharaeibacter diazotrophicus gen. nov., sp. nov., a diazotrophic and facultatively methylotrophic bacterium, isolated from rice rhizosphere.

A novel facultatively methanol-utilizing bacterial strain, SM30T, was isolated from rice rhizosphere. Strain SM30T was Gram-stain-negative, aerobic, motile, short rods, and grew optimally at pH 7 and at 28 °C. It could tolerate 0 to 2 % (w/v) NaCl. Based on 16S rRNA gene sequence comparisons, strain SM30T was most closely related to Pleomorphomonas oryzae DSM 16300T, with a low similarity of 94.17 %. One of the lanthanide metals, lanthanum, could enhance its growth slightly on methanol. Phylogenetic trees, based on the mxaF, xoxF and cpn60 genes of SM30T showed its distinct phylogenetic position with respect to species with validly published names. Polymerase chain reaction (PCR) amplification of the nifH and growth on nitrogen-free medium indicated that strain SM30T is a diazotroph. The major cellular fatty acids were summed feature 8 (containing 18 : 1ω7c and 18 : 1ω6c) and cyclo 19 : 0ω8c. The major quinone was ubiquinone 10. The DNA G+C content was 74.6 mol%. Based on the genotypic and phenotypic characteristics, strain SM30T represents a novel genus and species, for which the name Oharaeibacter diazotrophicus gen. nov., sp. nov. is proposed with the type strain SM30T (=NBRC 111955T=DSM 102969T).

Salinity Affects the Composition of the Aerobic Methanotroph Community in Alkaline Lake Sediments from the Tibetan Plateau.

Lakes are widely distributed on the Tibetan Plateau, which plays an important role in natural methane emission. Aerobic methanotrophs in lake sediments reduce the amount of methane released into the atmosphere. However, no study to date has analyzed the methanotroph community composition and their driving factors in sediments of these high-altitude lakes (>4000 m). To provide new insights on this aspect, the abundance and composition in the sediments of six high-altitude alkaline lakes (including both freshwater and saline lakes) on the Tibetan Plateau were studied. The quantitative PCR, terminal restriction fragment length polymorphism, and 454-pyrosequencing methods were used to target the pmoA genes. The pmoA gene copies ranged 104-106 per gram fresh sediment. Type I methanotrophs predominated in Tibetan lake sediments, with Methylobacter and uncultivated type Ib methanotrophs being dominant in freshwater lakes and Methylomicrobium in saline lakes. Combining the pmoA-pyrosequencing data from Tibetan lakes with other published pmoA-sequencing data from lake sediments of other regions, a significant salinity and alkalinity effect (P = 0.001) was detected, especially salinity, which explained ∼25% of methanotroph community variability. The main effect was Methylomicrobium being dominant (up to 100%) in saline lakes only. In freshwater lakes, however, methanotroph composition was relatively diverse, including Methylobacter, Methylocystis, and uncultured type Ib clusters. This study provides the first methanotroph data for high-altitude lake sediments (>4000 m) and shows that salinity is a driving factor for the community composition of aerobic methanotrophs.

Chenggangzhangella methanolivorans gen. nov., sp. nov., a member of the family Methylocystaceae, transfer of Methylopila helvetica Doronina et al. 2000 to Albibacter helveticus comb. nov. and emended description of the genus Albibacter.

A Gram-stain-negative, rod-shaped, non-motile and aerobic bacterial strain, designated CHL1T, was isolated from a sludge sample collected from a sewage treatment tank of an agricultural chemical factory. The strain grew at salinities of 0.5-5 % (w/v) NaCl (optimum 2.5 %). Growth occurred at pH 6.0-8.0 (optimum pH 7.0) and 5-40 °C (optimum 28-30 °C). The genomic DNA G+C content was determined to be 70.4 mol%. Q-10 was detected as the respiratory quinone. The major fatty acids (>10 %) were C18 : 1ω7c and/or C18 : 1ω6c and C16 : 0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified phospholipids and two unidentified aminophospholipids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CHL1T formed a distinct clade with Albibacter methylovorans DSM 22840T and Methylopila helvetica DM9T within the family Methylocystaceae. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, the strain merits recognition as a representative of a novel species of a new genus within the family Methylocystaceae, for which the name Chenggangzhangella methanolivorans gen. nov., sp. nov. is proposed. The type strain of the type species is CHL1T (=KCTC 42661T=CCTCC AB 2015175T). In addition, the species Methylopila helveticaDoronina et al. (2000) is proposed to be transferred to the genus Albibacter as Albibacterhelveticus comb. nov. (type strain DM9T=CIP 106788=VKM B-2189) on the basis of the phylogenetic analysis. An emended description of the genus Albibacter is also provided.

Terasakiella brassicae sp. nov., isolated from the wastewater of a pickle-processing factory, and emended descriptions of Terasakiella pusilla and the genus Terasakiella.

A Gram-stain-negative, motile, polyhydroxybutyrate-accumulating, aerobic, S-shaped bacterium, designated B3T, was isolated from the wastewater of a pickle-processing factory. 16S rRNA gene sequence similarity analysis showed that it was most closely related to the type strain, Terasakiella pusilla (96.6% similarity). Strain B3T was able to grow at 4-40 °C (optimum 32-37 °C), pH 5.5-9.0 (optimum 6.5-7.5) and with 0.5-8% (w/v) NaCl present (optimum 1-2%, w/v). Chemotaxonomic analysis showed that the respiratory quinone was ubiquinone Q-10, the major fatty acids included C16:0, C18:1ω7c and C16:1ω7c and/or iso-C15:2-OH. The major polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, aminophospholipid and three uncharacterized phospholipids. The genomic DNA G+C content of strain B3T was 42.3 mol%. The DNA-DNA relatedness value between B3T and T. pusilla DSM 9263T was 23.9%. On the basis of the phenotypic, chemotaxonomic and genotypic characteristics of strain B3T, it represents a novel species of the genus Terasakiella, for which the name Terasakiella brassicae sp. nov. is proposed. The type strain is B3T (=KCTC 42652T=CGMCC 1.15254T). Emended descriptions of T. pusilla and the genus Terasakiella are also presented.

Methylopila henanense sp. nov., a novel methylotrophic bacterium isolated from tribenuron methyl-contaminated wheat soil.

A bacterial strain, designated LYBFD3-16A2(T), was isolated from tribenuron methyl contaminated wheat soil. Cells were observed to be Gram-negative short rods with a single flagellum. The strain was found to utilize methanol, glucose, maltose and mannitol as carbon and energy sources, and utilized glutamate, leucine, phenylalanine as organic nitrogen sources. Strain LYBFD3-16A2(T) was found to be aerobic, to form urease, produce hydrogen sulfide and reduce nitrate to nitrite. The indole test in tryptone broth was observed to be positive. The major cellular fatty acids were identified as C18:1ω7c (81.3 %), 11-methylC18:1ω7c (7.9 %), C18:0 (3.0 %) and C16:0 (3.0 %). The major phospholipids were identified as phosphatidylcholine, phosphatidylethanolamine, phosphatidyglycerol and diphosphatidylglycerol. The main ubiquinone was identified as Q-10. The DNA G+C content was determined to be between 70.2 and 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated the affiliation of strain LYBFD3-16A2 to members of the genus Methylopila. The DNA-DNA hybridization values of the novel strain with the type strains of the most closely related species Methylopila musalis MUSA(T) and Methylopila jiangsuensis JZL-4(T) were 35.4 % and 31.4 % respectively. The genotypic and phenotypic characterization, along with chemotaxonomic properties of strain LYBFD3-16A2(T), showed that the strain represents a novel species of the genus Methylopila for which the name Methylopila henanense sp. nov. is proposed. The type strain is LYBFD3-16A2(T) (=CGMCC1.10703(T) = LMG 25959(T)).

Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria.

Methane-oxidizing bacteria are nature's primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10(21) M(-1). The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the iron-containing soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methane-oxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms.

Methylocystis bryophila sp. nov., a facultatively methanotrophic bacterium from acidic Sphagnum peat, and emended description of the genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993.

A novel species is proposed for two facultatively methanotrophic representatives of the genus Methylocystis, strains H2s(T) and S284, which were isolated from an acidic (pH 4.3) Sphagnum peat-bog lake (Teufelssee, Germany) and an acidic (pH 3.8) peat bog (European North Russia), respectively. Cells of strains H2s(T) and S284 are aerobic, Gram-negative, non-motile, curved coccoids or short rods that contain an intracytoplasmic membrane system typical of type-II methanotrophs. They possess both a soluble and a particulate methane monooxygenase (MMO); the latter is represented by two isozymes, pMMO1 and pMMO2. The preferred growth substrates are methane and methanol. In the absence of C1 substrates, however, these methanotrophs are capable of slow growth on acetate. Atmospheric nitrogen is fixed by means of an aerotolerant nitrogenase. Strains H2s(T) and S284 grow between pH 4.2 and 7.6 (optimum pH 6.0-6.5) and at 8-37 °C (optimum 25-30 °C). The major fatty acids are C18 : 1ω8c, C18 : 1ω7c and C16 : 1ω7c; the major quinone is Q-8. The DNA G+C content is 62.0-62.3 mol%. Strains H2s(T) and S284 share identical 16S rRNA gene sequences, which displayed 96.6-97.3 % similarity to sequences of other taxonomically characterized members of the genus Methylocystis. Therefore, strains H2s(T) and S284 are classified as members of a novel species, for which the name Methylocystis bryophila sp. nov. is proposed; strain H2s(T) ( = DSM 21852(T)  = VKM B-2545(T)) is the type strain.

Methylopila musalis sp. nov., an aerobic, facultatively methylotrophic bacterium isolated from banana fruit.

A newly isolated, facultatively methylotrophic bacterium (strain MUSA(T)) was investigated. The isolate was strictly aerobic, Gram-stain-negative, asporogenous, motile, rod-shaped and multiplied by binary fission. The strain utilized methanol, methylamine and an apparently narrow range of multi-carbon compounds, but not methane, dichloromethane or CO2/H2, as the carbon and energy sources. Growth occurred at pH 5.5-9.5 (optimum, pH 7.0) and 16-40 °C (optimum, 28-30 °C). The major fatty acids of methanol-grown cells were C18 : 1ω7c, C18 : 0 and 11-methyl-C18 : 1ω7c . The predominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine. The major ubiquinone was Q-10. The strain had methanol and methylamine dehydrogenases as well as the enzymes of the N-methylglutamate pathway (lyases of γ-glutamylmethylamide and N-methylglutamate). C1 assimilation occurs via the isocitrate lyase-negative serine pathway. Ammonium was assimilated by glutamate dehydrogenase and the glutamate cycle (glutamate synthase/glutamine synthetase). The DNA G+C content of the strain was 64.5 mol% (determined from the melting temperature). Based on 16S rRNA gene sequence similarity (97.0-98.9 %) and DNA-DNA relatedness (36-38 %) with representatives of the genus Methylopila (Methylopila capsulata IM1(T) and Methylopila jiangsuensis JZL-4(T)) the isolate was classified as a novel species of the genus Methylopila, for which the name Methylopila musalis sp. nov. is proposed. The type strain is MUSA(T) ( = VKM B-2646(T) = DSM 24986(T) = CCUG 61696(T)).

Hansschlegelia beijingensis sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic bacterium isolated from watermelon rhizosphere soil.

A novel Gram-stain-negative, aerobic, rod-shaped strain designated PG04(T) was isolated from the rhizosphere of watermelon plants cultivated in Beijing, China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, isolate PG04(T) belonged clearly to the genus Hansschlegelia and was most closely related to Hansschlegelia zhihuaiae (97.3 % similarity to the type strain). The predominant respiratory quinone was ubiquinone 10 (Q-10) and the polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major fatty acids were C18 : 1ω7c (41.3 %), C19 : 0 cyclo ω8c (30.6 %) and C16 : 0 (19.1 %). The G+C content of the DNA was about 64.4 mol%. DNA-DNA hybridization experiments showed 34.4 % relatedness between strain PG04(T) and H. zhihuaiae DSM 18984(T). The results of physiological and biochemical tests and differences in fatty acid profiles allowed clear phenotypic differentiation of strain PG04(T) from the most closely related species in the genus, H. zhihuaiae. Strain PG04(T) therefore represents a novel species within the genus Hansschlegelia, for which the name Hansschlegelia beijingensis sp. nov. is proposed, with the type strain PG04(T) ( = DSM 25481(T) = ACCC 05759(T)).

Characterization of Methylocystis strain JTA1 isolated from aged refuse and its tolerance to chloroform.

To accelerate the efficiency of methane biodegradation in landfills, a Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, JTA1, which can utilize methane as well as acetate, was isolated from the Laogang MSW landfills, Shanghai, China. Strain JTA1 was a member of genus Methylocystis on the basis of 16S rRNA and pmoA gene sequence similarity. The maximum specific cell growth rates (micro(max) = 0.042 hr(-1), R2 = 0.995) was derived through Boltzmann simulation, and the apparent half-saturation constants (K(m(app)) = 7.08 mmol/L, R2 = 0.982) was calculated according to Michaelis-Menton hyperbolic model, indicating that Methylocystis strain JTA1 had higher-affinity potential for methane oxidation than other reported methanotrophs. By way of adding the strain JTA1 culture, the methane consumption of aged refuse reached 115 mL, almost two times of control experiment. In addition, high tolerance of Methylocystis strain JTA1 to chloroform could facilitate the methane oxidation of aged refuse bio-covers. At the chloroform concentration of 50 mg/L, the methane-oxidation rate of bio-cover reached 0.114 mL/(day x g), much higher than the highest rate, 0.0135 mL/(day x g), of reported bio-covers. In conclusion, strain JTA1 opens up a new possibility for environmental biotechnology, such as soil or landfills bioremediation and wastewater decontamination.

Composition of methane-oxidizing bacterial communities as a function of nutrient loading in the Florida everglades.

Agricultural runoff of phosphorus (P) in the northern Florida Everglades has resulted in several ecosystem level changes, including shifts in the microbial ecology of carbon cycling, with significantly higher methane being produced in the nutrient-enriched soils. Little is, however, known of the structure and activities of methane-oxidizing bacteria (MOB) in these environments. To address this, 0 to 10 cm plant-associated soil cores were collected from nutrient-impacted (F1), transition (F4), and unimpacted (U3) areas, sectioned in 2-cm increments, and methane oxidation rates were measured. F1 soils consumed approximately two-fold higher methane than U3 soils; additionally, most probable numbers of methanotrophs were 4-log higher in F1 than U3 soils. Metabolically active MOB containing pmoA sequences were characterized by stable-isotope probing using 10 % (v/v) (13)CH(4). pmoA sequences, encoding the alpha subunit of methane monooxygenase and related to type I methanotrophs, were identified from both impacted and unimpacted soils. Additionally, impacted soils also harbored type II methanotrophs, which have been shown to exhibit preferences for high methane concentrations. Additionally, across all soils, novel pmoA-type sequences were also detected, indicating presence of MOB specific to the Everglades. Multivariate statistical analyses confirmed that eutrophic soils consisted of metabolically distinct MOB community that is likely driven by nutrient enrichment. This study enhances our understanding on the biological fate of methane being produced in productive wetland soils of the Florida Everglades and how nutrient-enrichment affects the composition of methanotroph bacterial communities.

Distribution of methanotrophs in the phyllosphere.

Plants have been reported to emit methane as well as methanol originating in their cell-wall constituents. We investigated methanotrophs in the phyllosphere by the enrichment culture method with methane as sole carbon source. We enriched methanotrophs from the leaves, flowers, bark, and roots of various plants. Analysis of the pmoA and mxaF genes retrieved from the enrichment cultures revealed that methanotrophs closely related to the genera Methylomonas, Methylosinus, and Methylocystis inhabit not only the rhizosphere but also the phyllosphere, together with methanol-utilizing bacteria.

Methylopila jiangsuensis sp. nov., an aerobic, facultatively methylotrophic bacterium.

The taxonomic status was determined of an aerobic, facultatively methylotrophic strain, JZL-4(T), isolated from activated sludge. The cells were gram-negative, asporogenous, colourless, motile, short rods. The strain utilized methanol, methylamine, formate and a variety of polycarbon compounds, but not methane, dichloromethane or CO(2)/H(2), as carbon and energy sources. C(1) compounds were assimilated via the isocitrate lyase-negative serine pathway. Optimal growth occurred at 30 °C, pH 6.5-7.5 and 0.5 % (w/v) NaCl. The major cellular fatty acids were C(18 : 1)ω7c and C(18 : 0). The major phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine (PME); PME, the main phospholipid of strain JZL-4(T), was absent or present in only minor amounts in Methylopila capsulata IM1(T), Methylopila helvetica DM9(T) and Albibacter methylovorans DM10(T). The major ubiquinone was Q-10. The DNA G+C content of strain JZL-4(T) was 70.4 mol% (T(m)). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain showed high sequence similarities to M. capsulata IM1(T) (97.2 %), A. methylovorans DM10(T) (94.9 %) and M. helvetica DM9(T) (94.1 %), and showed less than 94 % similarity to strains of other species with validly published names. Strain JZL-4(T) had a low level of DNA-DNA relatedness (34 %) with M. capsulata IM1(T). On the basis of phenotypic, genetic and phylogenetic data, strain JZL-4(T) is proposed to represent a novel species of the genus Methylopila, with the name Methylopila jiangsuensis sp. nov. The type strain is strain JZL-4(T) ( = ACCC 05406(T)  = DSM 22718(T)  = VKM B-2555(T)).

Hansschlegelia zhihuaiae sp. nov., isolated from a polluted farmland soil.

A novel Gram-negative, aerobic, coccoid-shaped strain designated S 113(T) was isolated from a polluted-soil sample collected in Jiangsu Province, China. A polyphasic taxonomic study including phylogenetic analysis based on the 16S rRNA gene sequence and determination of phenotypic characteristics was performed on the new isolate. The highest 16S rRNA gene sequence similarity was 96.8 %, with Hansschlegelia plantiphila S(1)(T). The predominant respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids were C(18 : 1)ω7c and C(16 : 0). The G+C content of the DNA was about 65.7 mol%. DNA-DNA hybridization experiments showed 44.9 % relatedness for strain S 113(T) with its closest relative, H. plantiphila NCIMB 14035(T). The dominant phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and phosphatidylcholine. The results of our polyphasic taxonomic analysis indicate that strain S 113(T) represents a novel species within the genus Hansschlegelia, for which the name Hansschlegelia zhihuaiae sp. nov. is proposed. The type strain is S 113(T) ( = DSM 18984(T)  = CCTCC AB 206143(T)  = KCTC 12880(T)).