Methylotroph Quorum Sensing Signal Identification by Inverse Stable Isotopic Labeling.

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a 13C-carbon source and then identifying 12C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies N-acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive 13C-carbon sources.

A Novel Cold-adapted Methylovulum species, with a High C16:1ω5c Content, Isolated from an Arctic Thermal Spring in Spitsbergen.

A novel cold-adapted methane-oxidizing bacterium, termed TFB, was isolated from the thermoglacial Arctic karst spring, Trollosen, located in the South Spitsbergen National Park (Norway). The source water is cold and extremely low in phosphate and nitrate. The isolate belongs to the Methylovulum genus of gammaproteobacterial methanotrophs, with the closest phylogenetic affiliation with Methylovulum miyakonense and Methylovulum psychrotolerans (96.2 and 96.1% 16S rRNA gene sequence similarities, respectively). TFB is a strict aerobe that only grows in the presence of methane or methanol. It fixes atmospheric nitrogen and contains Type I intracellular membranes. The growth temperature range was 2-22°C, with an optimum at 13-18°C. The functional genes pmoA, mxaF, and nifH were identified by PCR, whereas mmoX and cbbL were not. C16:1ω5c was identified as the major fatty acid constituent, at an amount (>49%) not previously found in any methanotrophs, and is likely to play a major role in cold adaptation. Strain TFB may be regarded as a new psychrotolerant or psychrophilic species within the genus Methylovulum. The recovery of this cold-adapted bacterium from a neutral Arctic thermal spring increases our knowledge of the diversity and adaptation of extremophilic gammaproteobacterial methanotrophs in the candidate family "Methylomonadaceae".

Native top-down mass spectrometry provides insights into the copper centers of membrane-bound methane monooxygenase.

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.

A novel surface-enhanced Raman scattering (SERS) detection for natural gas exploration using methane-oxidizing bacteria.

Methane-oxidizing bacteria (MOB), a unique group of Gram-negative bacteria utilizing methane as a sole source of carbon and energy, have been proved to be a biological indicator for gas prospecting. Field and cultivation-free detection of MOB is important but still challenging in current microbial prospecting of oil and gas (MPOG) system. Herein, SERS was used for the first time to our knowledge to investigate two species of methanotrophs and four closely relevant bacteria that universally coexisted in the upper soil of natural gas. A special but very simple approach was utilized to make silver nanoparticles (Ag NPs) sufficiently contact with every single bacterial cell, and highly strong and distinct Raman signals free from any native fluorescence have been obtained, and successfully utilized for distinguishing MOB from other species. A more convincing multi-Raman criterion based on single Raman bands, and further the entire Raman spectrum in combination with statistical analysis (e.g., principal component analysis (PCA)), which were found capable of classifying MOB related bacterial cells in soil with an accuracy of 100%. This study therefore demonstrated sensitive and rapid SERS measurement technique accompanied by complete Raman database of various gas reservoirs related bacteria could aid field exploration of natural gas reservoir.

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z.

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.

Spectral and thermodynamic properties of methanobactin from γ-proteobacterial methane oxidizing bacteria: a case for copper competition on a molecular level.

Methanobactin (mb) is a low molecular mass copper-binding molecule analogous to iron-binding siderophores. The molecule is produced by many methanotrophic or methane oxidizing bacteria (MOB), but has only been characterized to date in one MOB, Methylosinus trichosporium OB3b. To explore the potential molecular diversity in this novel class of metal binding compound, the spectral (UV-visible, fluorescent, and electron paramagnetic resonance) and thermodynamic properties of mb from two γ-proteobacterial MOB, Methylococcus capsulatus Bath and Methylomicrobium album BG8, were determined and compared to the mb from the α-proteobacterial MOB, M. trichosporium OB3b. The mb from both γ-proteobacterial MOB differed from the mb from M. trichosporium OB3b in molecular mass and spectral properties. Compared to mb from M. trichosporium OB3b, the extracellular concentrations were low, as were copper-binding constants of mb from both γ-proteobacterial MOB. In addition, the mb from M. trichosporium OB3b removed Cu(I) from the mb of both γ-proteobacterial MOB. Taken together the results suggest mb may be a factor in regulating methanotrophic community structure in copper-limited environments.

Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8.

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a sigma(54)-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

Methylobacter tundripaludum sp. nov., a methane-oxidizing bacterium from Arctic wetland soil on the Svalbard islands, Norway (78 degrees N).

A Gram-negative, rod-shaped, non-motile, non-spore forming bacterium (SV96T) was isolated from wetland soil near Ny-Alesund, Svalbard. On the basis of 16S rRNA gene sequence similarity, strain SV96T was shown to belong to the Gammaproteobacteria, related to Methylobacter psychrophilus Z-0021T (99.1 %), Methylobacter luteus ATCC 49878T (97.3 %), Methylobacter marinus A45T (97.0 %) and Methylobacter whittenburyi ATCC 51738T (95.8 %); the closest related species within the genus Methylomicrobium with a validly published name was Methylomicrobium album ATCC 33003T (95.0 %). Chemotaxonomic data (including the major fatty acids: 16 : 1omega8, 16 : 1omega7 and 16 : 1omega5t) supported the affiliation of strain SV96T to the genus Methylobacter. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV96T from the four Methylobacter species mentioned above. Strain SV96T therefore represents a novel species, for which the name Methylobacter tundripaludum sp. nov. is proposed (type strain SV96T = DSM 17260T = ATCC BAA-1195T).

Abundance, activity, and community structure of pelagic methane-oxidizing bacteria in temperate lakes.

The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a 14CH4 transformation method. The fatty acid analyses indicated that type I MOB most similar to species of Methylomonas, Methylomicrobium, and Methylosarcina made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (V) ranged from 0.001 to 2.77 mg CH4-C mg(-1) C day(-1) and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH4.

[New thermophilic methanotrophs of the genus Methylocaldum]. / Novye termofil'nye metanotrofy roda Methylocaldum.

Two pure cultures of obligate methanotrophs, strains H-11 and 0-12, growing in the temperature range from 30 to 61 degrees C with an optimum at 55 degrees C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes, membrane-bound methane monooxygenase, and phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulose phosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55 degrees C that in cells grown at 37 degrees C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH4+ assimilation occurred through reductive amination of alpha-ketoglutarate and via the glutamate cycle. The relationship between the physiological-biochemical peculiarities of the isolates and their thermophilic nature is discussed.

Estimation of methanotroph abundance in a freshwater lake sediment.

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.

Characterization of methanotrophic bacteria on the basis of intact phospholipid profiles.

The intact phospholipid profiles (IPPs) of seven species of methanotrophs from all three physiological groups, type I, II and X, were determined using liquid chromatography/electrospray ionization/mass spectrometry. In these methanotrophs, two major classes of phospholipids were found, phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) as well as its derivatives phosphatidylmethylethanolamine (PME) and phosphatidyldimethylethanolamine (PDME). Specifically, the type I methanotrophs, Methylomonas methanica, Methylomonas rubra and Methylomicrobium album BG8 were characterized by PE and PG phospholipids with predominantly C16:1 fatty acids. The type II methanotrophs, Methylosinus trichosporium OB3b and CSC1 were characterized by phospholipids of PG, PME and PDME with predominantly C18:1 fatty acids. Methylococcus capsulatus Bath, a representative of type X methanotrophs, contained mostly PE (89% of the total phospholipids). Finally, the IPPs of a recently isolated acidophilic methanotroph, Methylocella palustris, showed it had a preponderance of PME phospholipids with 18:1 fatty acids (94% of total). Principal component analysis showed these methanotrophs could be clearly distinguished based on phospholipid profiles. Results from this study suggest that IPP can be very useful in bacterial chemotaxonomy.

Sterols in a psychrophilic methanotroph, Methylosphaera hansonii.

A suite of six sterols, lanosterol, lanost-8(9)-en-3beta-ol, 4, 4-dimethylcholesta-8(14),24-dien-3beta-ol, 4, 4-dimethylcholest-8(14)-en-3beta-ol, 4-methylcholesta-8(14), 24-dien-3beta-ol and 4-methylcholest-8(14)-en-3beta-ol, were identified in the psychrophilic methanotrophic bacterium, Methylosphaera hansonii. Their presence suggests that the capacity for sterol biosynthesis in methanotrophic bacteria is limited to the family Methylococcaceae but which have widely different optimal growth temperatures.

Bacterial triterpenoids of the hopane series from the methanotrophic bacteria Methylocaldum spp.: phylogenetic implications and first evidence for an unsaturated aminobacteriohopanepolyol.

The hopanoid content of the two methanotrophic bacteria Methylocaldum szegediense and Methylocaldum tepidum was investigated. 35-Aminobacteriohopane-30R,31R,32R,33S, 34S-pentol and its 3beta-methyl homologue were present in both strains. In M. tepidum, they were accompanied by 35-aminobacteriohopane-31R,32R,33S, 34S-tetrol and its 3beta-methyl homologue. The side chain structure was identical to those previously reported from two other obligate methanotrophs, Methylococcus capsulatus and Methylomonas methanica. The two Methylocaldum species shared with the Methylococcus species the presence of 3beta-methylhopanoid as well as of a hopanoid releasing adiantol upon H(5)IO(6)/NaBH(4) treatment. A rare feature was in addition found in M. szegediense. The saturated hopanoids were accompanied by an unsaturated aminobacteriohopanepentol with a Delta(11) double bond. Comparison of the hopanoid fingerprints was in accordance with the close phylogenetic relationship of Methylococcus and Methylocaldum. The major difference was the absence of sterols in Methylocaldum which were always detected in the Methylococcus species.

Radioactive fingerprinting of microorganisms that oxidize atmospheric methane in different soils.

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with (14)CH(4) followed by analysis of radiolabelled phospholipid ester-linked fatty acids ((14)C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The (14)C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1omega8c (up to 9.0% of the total (14)C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1omega9, 18:1omega7, and 18:0). The (14)C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH(4). The (14)C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the (14)C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.

Crystallization and preliminary X-ray study of iso-2 azurin from the methylotrophic bacterium, Methylomonas J.

The obligate methylotroph Methylomonas J possesses two distinct azurins. The iso-2 azurin, which functions as an electron acceptor for methylamine dehydrogenase, has been crystallized using two kinds of precipitants: PEG 4000 and ammonium sulfate. The crystals precipitated with PEG belong to the monoclinic system, space group P21, with unit-cell parameters a = 32.96, b = 33.67, c = 47.34 A and beta = 101.35 degrees. The crystals precipitated with ammonium sulfate belong to the orthorhombic system, space group C2221, with unit-cell parameters a = 31.52, b = 62.49 and c = 135.41 A. The crystals diffract to 1.6 and 1.9 A resolution, respectively, and were suitable for X-ray crystallographic studies. A Patterson search is being conducted using the recently reported structure of Alcaligenes xylosoxidans NCIMB 11015 as a starting model.

Detection and partial characterization of bacteriocin in the methanotrophic bacterium Methylobacter bovis.

The strain Methylobacter bovis 98 was selected among methanotrophic bacteria as one of the most active producers of secretory bacteriocin-like compounds. In the above strain this compound was shown to be a protein with a molecular weight of about 70 kD, relatively thermostable, having a bactericidal effect on closely related organisms. Its properties as a whole are consistent with the accepted definition of bacteriocins, which so far have not been found in this group of microorganisms. A methodical approach that combines electrophoretic separation of secretory proteins and testing their antibacterial activity directly in polyacrylamide gel allowed us for the first time to identify bacteriocin in methanotrophic bacterial culture.

The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.

Concentration of Cu, EPR-detectable Cu, and formation of cupric-ferrocyanide in membranes with pMMO.

EPR spectra were obtained for the type 2 Cu(2+) site in particulate methane monooxygenase, pMMO, from membrane fractions of Methylomicrobium album BG8. In addition to the EPR signal with g parallel = 2.24 and A parallel = 185 G found in both cells and membrane fractions, a second EPR signal with g parallel = 2.29 and A parallel = 146 G was found in membrane fractions and attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by atomic absorption suggests that there are two or three EPR-silent coppers for every EPR-detectable copper and that there are approximately four coppers per enzyme composed of the 47, 27, and 25 kDa subunits. Treatment of membrane fractions loaded with pMMO with Fe(CN)6(3-) results in a new EPR signal that is attributed to CuFe(CN)6(2-), not to an intrinsic trimeric copper cluster as previously reported in studies with a related bacterium.

Outer membrane proteins of Methylococcus capsulatus (Bath).

Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.