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).

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.

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).

Genome sequence of Methylocystis hirsuta CSC1, a polyhydroxyalkanoate producing methanotroph.

Polyhydroxyalkanoates (PHAs) are biodegradable plastics that can be produced by some methanotrophic organisms such as those of the genus Methylocystis. This allows the conversion of a detrimental greenhouse gas into an environmentally friendly high added-value bioproduct. This study presents the genome sequence of Methylocystis hirsuta CSC1 (a high yield PHB producer). The genome comprises 4,213,043 bp in 4 contigs, with the largest contig being 3,776,027 bp long. Two of the other contigs are likely to correspond to large size plasmids. A total of 4,664 coding sequences were annotated, revealing a PHA production cluster, two distinct particulate methane monooxygenases with active catalytic sites, as well as a nitrogen fixation operon and a partial denitrification pathway.

Anaerobic carbon monoxide metabolism by Pleomorphomonas carboxyditropha sp. nov., a new mesophilic hydrogenogenic carboxydotroph.

Carbon monoxide (CO)-metabolism and phenotypic and phylogenetic characterization of a novel anaerobic, mesophilic and hydrogenogenic carboxydotroph are reported. Strain SVCO-16 was isolated from anaerobic sludge and grows autotrophically and mixotrophically with CO. The genes cooS and cooF, coding for a CO dehydrogenase complex, and genes similar to hycE2, encoding a CO-induced hydrogenase, were present in its genome. The isolate produces H2 and CO2 from CO, and acetate and formate from organic substrates. Based on the 16S rRNA sequence, it is an Alphaproteobacterium most closely related to the genus Pleomorphomonas (98.9%-99.2% sequence identity). Comparison with other previously characterized Pleomorphomonas showed that P. diazotrophica and P. oryzae do not metabolize CO, and P. diazotrophica does not grow anaerobically with organic substrates. Average nucleotide identity values between strain SVCO-16 and P. diazotrophica, P. oryzae or P. koreensis were 86.66 ± 0.21%. These values are below the boundary to define species (95%-96%). Digital DNA-DNA hybridization estimates between strain SVCO-16 and reference strains were also below the 70% threshold for species delineation: 29.1%-34.5%. Based on the differences in CO metabolism, genome analyses and cellular fatty acid composition, the isolate should be classified into the genus Pleomorphomonas as a representative of a novel species, Pleomorphomonas carboxyditropha. The type strain of Pleomorphomonas carboxyditropha is SVCO-16T (strain deposit numbers, DSM 106132T and TSD-119T).

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.

[Decline of Activity and Shifts in the Methanotrophic Community Structure of an Ombrotrophic Peat Bog after Wildfire].

This study examined potential disturbances of methanotrophic communities playing a key role in reducing methane emissions from the peat bog Tasin Borskoye, Vladimir oblast, Russia as a result of the 2007 wildfire. The potential activity of the methane-oxidizing filter in the burned peatland site and the abundance of indigenous methanotrophic bacteria were significantly reduced in comparison to the undisturbed site. Molecular analysis of methanotrophic community structure by means of PCR amplification and cloning of the pmoAgene encoding particulate methane monooxygenase revealed the replacement of typical peat-inhabiting, acidophilic type II methanotrophic bacteria with type I methanotrophs, which are less active in acidic environments. In summary, both the structure and the activity of the methane-oxidizing filter in burned peatland sites underwent significant changes, which were clearly pronounced even after 7 years of the natural ecosystem recovery. These results point to the long-term character of the disturbances caused by wildfire in peatlands.

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)).

Diversity and activity of methanotrophs in landfill cover soils with and without landfill gas recovery systems.

Aerobic CH4 oxidation plays an important role in mitigating CH4 release from landfills to the atmosphere. Therefore, in this study, oxidation activity and community of methanotrophs were investigated in a subtropical landfill. Among the three sites investigated, the highest CH4 concentration was detected in the landfill cover soil of the site (A) without a landfill gas (LFG) recovery system, although the refuse in the site had been deposited for a longer time (∼14-15 years) compared to the other two sites (∼6-11 years) where a LFG recovery system was applied. In April and September, the higher CH4 flux was detected in site A with 72.4 and 51.7gm(-2)d(-1), respectively, compared to the other sites. The abundance of methanotrophs assessed by quantification of pmoA varied with location and season. A linear relationship was observed between the abundance of methanotrophs and CH4 concentrations in the landfill cover soils (R=0.827, P<0.001). The key factors influencing the methanotrophic diversity in the landfill cover soils were pH, the water content and the CH4 concentration in the soil, of which pH was the most important factor. Type I methanotrophs, including Methylococcus, Methylosarcina, Methylomicrobium and Methylobacter, and type II methanotrophs (Methylocystis) were all detected in the landfill cover soils, with Methylocystis and Methylosarcina being the dominant genera. Methylocystis was abundant in the slightly acidic landfill cover soil, especially in September, and represented more than 89% of the total terminal-restriction fragment abundance. These findings indicated that the LFG recovery system, as well as physical and chemical parameters, affected the diversity and activity of methanotrophs in landfill cover soils.

[Sequence analysis of 16S rDNA and pmoCAB gene cluster of trichloroethylene-degrading methanotroph].

Methanotrophs could degrade methane and various chlorinated hydrocarbons. The analysis on methane monooxygenase gene cluster sequence would help to understand its catalytic mechanism and enhance the application in pollutants biodegradation. The methanotrophs was enriched and isolated with methane as the sole carbon source in the nitrate mineral salt medium. Then, five chlorinated hydrocarbons were selected as cometabolic substrates to study the biodegradation. The phylogenetic tree of 16S rDNA using MEGE5.05 software was constructed to identify the methanotroph strain. The pmoCAB gene cluster encoding particulate methane monooxygenase (pMMO) was amplified by semi-nested PCR in segments. ExPASy was performed to analyze theoretical molecular weight of the three pMMO subunits. As a result, a strain of methanotroph was isolated. The phylogenetic analysis indicated that the strain belongs to a species of Methylocystis, and it was named as Methylocystis sp. JTC3. The degradation rate of trichloroethylene (TCE) reached 93.79% when its initial concentration was 15.64 µmol/L after 5 days. We obtained the pmoCAB gene cluster of 3 227 bp including pmoC gene of 771 bp, pmoA gene of 759 bp, pmoB gene of 1 260 bp and two noncoding sequences in the middle by semi-nested PCR, T-A cloning and sequencing. The theoretical molecular weight of their corresponding gamma, beta and alpha subunit were 29.1 kDa, 28.6 kDa and 45.6 kDa respectively analyzed using ExPASy tool. The pmoCAB gene cluster of JTC3 was highly identical with that of Methylocystis sp. strain M analyzed by Blast, and pmoA sequences is more conservative than pmoC and pmoB. Finally, Methylocystis sp. JTC3 could degrade TCE efficiently. And the detailed analysis of pmoCAB from Methylocystis sp. JTC3 laid a solid foundation to further study its active sites features and its selectivity to chlorinated hydrocarbon.

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.

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)).

The relative contribution of methanotrophs to microbial communities and carbon cycling in soil overlying a coal-bed methane seep.

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH4 and also a likely source of ancient (i.e. (14) C-dead) carbon to soil microbial communities. Natural abundance (13) C and (14) C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO2 and CH4 were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison of PLFA and 16s rDNA data suggested type I and II methanotroph populations were well estimated and overestimated by their PLFA biomarkers, respectively. The δ(13) C values of PLFAs common in type I and II methanotrophs were as negative as -67‰ and consistent with the assimilation of CBM. PLFAs more indicative of nonmethanotrophic bacteria had δ(13) C values that were intermediate indicating assimilation of both plant- and CBM-derived carbon. Δ(14) C values of select PLFAs (-351 to -936‰) indicated similar patterns of CBM assimilation by methanotrophs and nonmethanotrophs and were used to estimate that 35-91% of carbon assimilated by nonmethanotrophs was derived from CBM depending on time of sampling and soil depth.

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)).