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).

Unusual Genomic Traits Suggest Methylocystis bryophila S285 to Be Well Adapted for Life in Peatlands.

The genus Methylocystis belongs to the class Alphaproteobacteria, the family Methylocystaceae, and encompasses aerobic methanotrophic bacteria with the serine pathway of carbon assimilation. All Methylocystis species are able to fix dinitrogen and several members of this genus are also capable of using acetate or ethanol in the absence of methane, which explains their wide distribution in various habitats. One additional trait that enables their survival in the environment is possession of two methane-oxidizing isozymes, the conventional particulate methane monooxygenase (pMMO) with low-affinity to substrate (pMMO1) and the high-affinity enzyme (pMMO2). Here, we report the finished genome sequence of Methylocystis bryophila S285, a pMMO2-possessing methanotroph from a Sphagnum-dominated wetland, and compare it to the genome of Methylocystis sp. strain SC2, which is the first methanotroph with confirmed high-affinity methane oxidation potential. The complete genome of Methylocystis bryophila S285 consists of a 4.53 Mb chromosome and one plasmid, 175 kb in size. The genome encodes two types of particulate MMO (pMMO1 and pMMO2), soluble MMO and, in addition, contains a pxmABC-like gene cluster similar to that present in some gammaproteobacterial methanotrophs. The full set of genes related to the serine pathway, the tricarboxylic acid cycle as well as the ethylmalonyl-CoA pathway is present. In contrast to most described methanotrophs including Methylocystis sp. strain SC2, two different types of nitrogenases, that is, molybdenum-iron and vanadium-iron types, are encoded in the genome of strain S285. This unique combination of genome-based traits makes Methylocystis bryophila well adapted to the fluctuation of carbon and nitrogen sources in wetlands.

Isolation of methanotrophic bacteria from termite gut.

The guts of termites feature suitable conditions for methane oxidizing bacteria (MOB) with their permanent production of CH4 and constant supply of O2 via tracheae. In this study, we have isolated MOB from the gut contents of the termites Incisitermes marginipennis, Mastotermes darwiniensis, and Neotermes castaneus for the first time. The existence of MOB was indicated by detecting pmoA, the gene for the particulate methane monooxygenase, in the DNA of gut contents. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction supported those findings. The MOB cell titer was determined to be 10(2)-10(3) per gut. Analyses of the 16S rDNA from isolates indicated close similarity to the genus Methylocystis. After various physiological tests and fingerprinting methods, no exact match to a known species was obtained, indicating the isolation of new MOB species. However, MALDI-TOF MS analyses revealed a close relationship to Methylocystis bryophila and Methylocystis parvus.

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)).

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.

[Methanotrophic bacteria in cold seeps of the floodplains of northern rivers].

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are a potentially important, although poorly studied sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3-5 degrees C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with predomination of type I methanotrophs. Among the latter, microorganisms related to Methylobacterpsychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two isolates were determined. Methylobactersp. CMS7 exhibited active growth at 4-10 degrees C, while Methylocystis sp. SB12 grew better at 20 degrees C. Experimental results confirmed the major role ofmethanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

Complete genome sequence of Methylocystis sp. strain SC2, an aerobic methanotroph with high-affinity methane oxidation potential.

Methylocystis sp. strain SC2 is an aerobic type II methanotroph isolated from a highly polluted aquifer in Germany. A specific trait of the SC2 strain is the expression of two isozymes of particulate methane monooxygenase with different methane oxidation kinetics. Here we report the complete genome sequence of this methanotroph that contains not only a circular chromosome but also two large plasmids.

Genome sequence of the methanotrophic alphaproteobacterium Methylocystis sp. strain Rockwell (ATCC 49242).

Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.

Chloride-associated adaptive response in aerobic methylotrophic dichloromethane-utilising bacteria.

Aerobic methylotrophic bacteria able to grow with dichloromethane (DCM) as the sole carbon and energy source possess a specific glutathione S-transferase, DCM dehalogenase, which transforms DCM to formaldehyde, used for biomass and energy production, and hydrochloric acid, which is excreted. Evidence is presented for chloride-specific responses for three DCM-degrading bacteria, Methylobacterium extorquens DM4, Methylopila helvetica DM6 and Albibacter methylovorans DM10. Chloride release into the medium was inhibited by sodium azide and m -chlorophenylhydrazone, suggesting an energy-dependent process. In contrast, only nigericin affected chloride excretion in Mb. extorquens DM4 and Mp. helvetica DM6, while valinomycin had the same effect in A. methylovorans DM10 only. Chloride ions stimulated DCM-dependent induction of DCM dehalogenase expression for Mp. helvetica DM6 and A. methylovorans DM10, and shortened the time for onset of chloride release into the medium. Striking chloride-containing structures were observed by electron microscopy and X-ray microanalysis on the cell surface of Mp. helvetica DM6 and A. methylovorans DM10 during growth with DCM, and with methanol in medium supplemented with sodium chloride. Taken together, these data suggest the existence of both general and specific chloride-associated adaptations in aerobic DCM-degrading bacteria.

Methylocystis rosea sp. nov., a novel methanotrophic bacterium from Arctic wetland soil, Svalbard, Norway (78 degrees N).

A Gram-negative, rod-shaped, non-motile, non-spore-forming, pink-pigmented bacterium, SV97T, was isolated from a wetland soil near Ny-Alesund, Svalbard Islands, Norway (78 degrees N). On the basis of 16S rRNA gene sequence similarity, strain SV97T was shown to belong to the Alphaproteobacteria and was highly related to a number of non-characterized Methylocystis strains with GenBank accession nos AJ458507 and AJ458502 (100 %) and AF177299, AJ458510, AJ458467, AJ458471, AJ431384, AJ458475, AJ458484, AJ458501 and AJ458466 (99 %). The most closely related type strains were Methylocystis parvus OBBP(T) (97.2 %) and Methylocystis echinoides IMET 10491T (97%). The closest related recognized species within the genus Methylosinus was Methylosinus sporium NCIMB 11126T (96.0% similarity). Chemotaxonomic and phenotypic data (C(18:1)omega8 as the major fatty acid, non-motile, no rosette formation) supported the affiliation of strain SV97T to the genus Methylocystis. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV97(T) from the two recognized Methylocystis species. Strain SV97T therefore represents a novel species, for which the name Methylocystis rosea sp. nov. is proposed, with the type strain SV97T (= DSM 17261T = ATCC BAA-1196T).

Amplification of marine methanotrophic enrichment DNA with 16S rDNA PCR primers for type II alpha proteobacteria methanotrophs.

Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.