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Background: Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. Results: The major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. Conclusions: In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off. [ABSTRACT FROM AUTHOR]

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities. [ABSTRACT FROM AUTHOR]

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the A lphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N-methylglutamate pathway, the two types of methanol dehydrogenase ( MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl- CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of A lphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions. [ABSTRACT FROM AUTHOR]

We describe five novel Methylophilaceae ecotypes from a single ecological niche in Lake Washington, USA, and compare them to three previously described ecotypes, in terms of their phenotype and genome sequence divergence. Two of the ecotypes appear to represent novel genera within the Methylophilaceae. Genome-based metabolic reconstruction highlights metabolic versatility of Methylophilaceae with respect to methylotrophy and nitrogen metabolism, different ecotypes possessing different combinations of primary substrate oxidation systems (MxaFI-type methanol dehydrogenase versus XoxF-type methanol dehydrogenase; methylamine dehydrogenase versus N-methylglutamate pathway) and different potentials for denitrification (assimilatory versus respiratory nitrate reduction). By comparing pairs of closely related genomes, we uncover that site-specific recombination is the main means of genomic evolution and strain divergence, including lateral transfers of genes from both closely- and distantly related taxa. The new ecotypes and the new genomes contribute significantly to our understanding of the extent of genomic and metabolic diversity among organisms of the same family inhabiting the same ecological niche. These organisms also provide novel experimental models for studying the complexity and the function of the microbial communities active in methylotrophy. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/ folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-mole-cule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agree-ment with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein-water hydrogen bonds were also replaced with protein-protein hydrogen bonds, addition-ally contributing to aggregation. Despite the increase in protein-pro-tein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as pro-teins aggregate at high temperature at an atomic level. [ABSTRACT FROM AUTHOR]

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process. [ABSTRACT FROM AUTHOR]

In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine- and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism's fitness, while GndB and FDH1 are auxiliary. [ABSTRACT FROM AUTHOR]

Methylotrophs, organisms able to gain energy and carbon from compounds containing no carbon–carbon bonds, such as methane, methanol and methylated amines, are widespread in nature. However, knowledge of their nutrient preference and their metabolism is mostly based on experiments with cultures grown in defined laboratory conditions. Here, we use transcriptomics to explore the activity of one methylotroph, Methyotenera mobilis in its natural environment, lake sediment from which it has been previously isolated. Cells encapsulated in incubation cassettes were exposed to sediment conditions, with or without supplementation with a carbon/energy source (methylamine), and gene-expression patterns were compared for those cells to patterns for cells incubated in a defined medium supplemented with methylamine. A few specific trends in gene expression were observed at in situ conditions that may be of environmental significance, as follows. Expression of genes for the linear formaldehyde oxidation pathway linked to tetrahydromethanopterin increased, suggesting an important role for this pathway in situ, in contrast to laboratory condition culture, in which the cyclic ribulose monophosphate pathway seemed to be the major route for formaldehyde oxidation. Along with the ribulose monophosphate cycle that is also a major pathway for assimilating C1 units, the methylcitric acid cycle seemd to be important in situ, suggesting that multicarbon compounds may be the natural carbon and/or energy substrates for M. mobilis, challenging the notion of an obligately methylotrophic lifestyle for this bacterium. We also detected a major switch in expression of genes responsible for the mode of motility between different conditions: from flagellum-enabled motility in defined medium to in situ expression of pili known to be involved in twitching motility and adherence. Overall, this study offers a novel approach for gaining insights into the lifestyle of individual microbes in their native environments. [ABSTRACT FROM AUTHOR]

Here, we compare the distributions of main chain (ΦΨ) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of Gly-Gly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (ΦΨ) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation. and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the "intrinsic propensities" of amino acids for particular (4,'I') conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the "random coil" state. [ABSTRACT FROM AUTHOR]

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 µs of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Φ values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated. [ABSTRACT FROM AUTHOR]

The correct treatment of van der Waals and electrostatic nonbonded interactions in molecular force fields is essential for performing realistic molecular dynamics (MD) simulations of solvated polypeptides. The most computationally tractable treatment of nonbonded interactions in MD utilizes a spherical distance cutoff (typically, 8-12 A) to reduce the number of pairwise interactions. In this work, we assess three spherical atom-based cutoff approaches for use with all-atom explicit solvent MD: abrupt truncation, a CHARMM-style electrostatic shift truncation, and our own force-shifted truncation. The chosen system for this study is an end-capped 17-residue alanine-based a-helical peptide, selected because of its use in previous computational and experimental studies. We compare the time-averaged helical content calculated from these MD trajectories with experiment. We also examine the effect of varying the cutoff treatment and distance on energy conservation. We find that the abrupt truncation approach is pathological in its inability to conserve energy. The CHARMM-style shift truncation performs quite well but suffers from energetic instability. On the other hand, the force-shifted spherical cutoff method conserves energy, correctly predicts the experimental helical content, and shows convergence in simulation statistics as the cutoff is increased. This work demonstrates that by using proper and rigorous techniques, it is possible to correctly model polypeptide dynamics in solution with a spherical cutoff. The inherent computational advantage of spherical cutoffs over Ewald summation (and related) techniques is essential in accessing longer MD time scales. [ABSTRACT FROM AUTHOR]

Mass-Suite (MSS) is a Python-based, open-source software package designed to analyze high-resolution mass spectrometry (HRMS)-based non-targeted analysis (NTA) data, particularly for water quality assessment and other environmental applications. MSS provides flexible, user-defined workflows for HRMS data processing and analysis, including both basic functions (e.g., feature extraction, data reduction, feature annotation, data visualization, and statistical analyses) and advanced exploratory data mining and predictive modeling capabilities that are not provided by currently available open-source software (e.g., unsupervised clustering analyses, a machine learning-based source tracking and apportionment tool). As a key advance, most core MSS functions are supported by machine learning algorithms (e.g., clustering algorithms and predictive modeling algorithms) to facilitate function accuracy and/or efficiency. MSS reliability was validated with mixed chemical standards of known composition, with 99.5% feature extraction accuracy and ~ 52% overlap of extracted features relative to other open-source software tools. Example user cases of laboratory data evaluation are provided to illustrate MSS functionalities and demonstrate reliability. MSS expands available HRMS data analysis workflows for water quality evaluation and environmental forensics, and is readily integrated with existing capabilities. As an open-source package, we anticipate further development of improved data analysis capabilities in collaboration with interested users. [ABSTRACT FROM AUTHOR]

The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO2 and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050. [ABSTRACT FROM AUTHOR]

Background: The toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels production. Methylobacterium extorquens AM1, a facultative methylotrophic a-proteobacterium, has been engineered to generate 1-butanol from cheap carbon feedstocks through a synthetic metabolic pathway. However, M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. Only a few studies have reported the general stress response of M. extorquens AM1 to solvent stress. Therefore, it is highly desirable to obtain a strain with ameliorated 1-butanol tolerance and elucidate the molecular mechanism of 1-butnaol tolerance in M. extorquens AM1 for future strain improvement. Results: In this work, adaptive laboratory evolution was used as a tool to isolate mutants with 1-butanol tolerance up to 0.5 %. The evolved strains, BHBT3 and BHBT5, demonstrated increased growth rates and higher survival rates with the existence of 1-butanol. Whole genome sequencing revealed a SNP mutation at kefB in BHBT5, which was confirmed to be responsible for increasing 1-butanol tolerance through an allelic exchange experiment. Global metabolomic analysis further discovered that the pools of multiple key metabolites, including fatty acids, amino acids, and disaccharides, were increased in BHBT5 in response to 1-butanol stress. Additionally, the carotenoid synthesis pathway was significantly down-regulated in BHBT5. Conclusions: We successfully screened mutants resistant to 1-butanol and provided insights into the molecular mechanism of 1-butanol tolerance in M. extorquens AM1. This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future. [ABSTRACT FROM AUTHOR]

The metabolism of one- and two-carbon compounds by the methylotrophic bacterium Methylobacterium extorquens AM1 involves high carbon flux through the ethylmalonyl coenzyme A (ethylmalonyl-CoA) pathway (EMC pathway). During growth on ethylamine, the EMC pathway operates as a linear pathway carrying the full assimilatory flux to produce glyoxylate, malate, and succinate. Assimilatory carbon enters the ethylmalonyl-CoA pathway directly as acetyl-CoA, bypassing pathways for formaldehyde oxidation/assimilation and the regulatory mechanisms controlling them, making ethylamine growth a useful condition to study the regulation of the EMC pathway. Wild-type M. extorquens cells were grown at steady state on a limiting concentration of succinate, and the growth substrate was then switched to ethylamine, a condition where the cell must make a sudden switch from utilizing the tricarboxylic acid (TCA) cycle to using the ethylmalonyl-CoA pathway for assimilation, which has been an effective strategy for identifying metabolic control points. A 9-h lag in growth was observed, during which butyryl-CoA, a degradation product of ethylmalonyl-CoA, accumulated, suggesting a metabolic imbalance. Ethylmalonyl-CoA mutase activity increased to a level sufficient for the observed growth rate at 9 h, which correlated with an upregulation of RNA transcripts for ecm and a decrease in the levels of ethylmalonyl-CoA. When the wild-type strain overexpressing ecm was tested with the same substrate switchover experiment, ethylmalonyl-CoA did not accumulate, growth resumed earlier, and, after a transient period of slow growth, the culture grew at a higher rate than that of the control. These findings demonstrate that ethylmalonyl-CoA mutase is a metabolic control point in the EMC pathway, expanding our understanding of its regulation. [ABSTRACT FROM AUTHOR]

The utilization of methane, a potent greenhouse gas, is an important component of local and global carbon cycles that is characterized by tight linkages between methane-utilizing (methanotrophic) and nonmethanotrophic bacteria. It has been suggested that the methanotroph sustains these nonmethanotrophs by cross-feeding, because subsequent products of the methane oxidation pathway, such as methanol, represent alternative carbon sources. We established cocultures in a microcosm model system to determine the mechanism and substrate that underlay the observed cross-feeding in the environment. Lanthanum, a rare earth element, was applied because of its increasing importance in methylotrophy. We used co-occurring strains isolated from Lake Washington sediment that are involved in methane utilization: a methanotroph and two nonmethanotrophic methylotrophs. Gene-expression profiles and mutant analyses suggest that methanol is the dominant carbon and energy source the methanotroph provides to support growth of the nonmethanotrophs. However, in the presence of the nonmethanotroph, gene expression of the dominant methanol dehydrogenase (MDH) shifts from the lanthanide-dependent MDH (XoxF)-type, to the calcium-dependent MDH (MxaF)-type. Correspondingly, methanol is released into the medium only when the methanotroph expresses the MxaF-type MDH. These results suggest a cross-feeding mechanism in which the nonmethanotrophic partner induces a change in expression of methanotrophMDHs, resulting in release of methanol for its growth. This partner-induced change in gene expression that benefits the partner is a paradigm for microbial interactions that cannot be observed in studies of pure cultures, underscoring the importance of synthetic microbial community approaches to understand environmental microbiomes. [ABSTRACT FROM AUTHOR]

Background: Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. Results: A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1-0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion efficiency of different pathways for C1-utilization, including different variants of the ribulose monophosphate pathway and the serine cycle. Conclusion: We demonstrate that the metabolic model can provide an effective tool for predicting metabolic parameters for different nutrients and genetic perturbations, and as such, should be valuable for metabolic engineering of the central metabolism of M. buryatense strains. [ABSTRACT FROM AUTHOR]

Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane- based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane. [ABSTRACT FROM AUTHOR]

Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum, a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N-3-hydroxydecanoyl-l-homoserine lactone (3-OH-C10-HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. [ABSTRACT FROM AUTHOR]

The genomes of three representatives of the family Methylophilaceae, Methylotenera mobilis JLW8, Methylotenera versatilis 301, and Methylovorus glucosetrophus SIP3-4, all isolated from a single study site, Lake Washington in Seattle, WA, were completely sequenced. These were compared to each other and to the previously published genomes of Methylobacillus flagellatus KT and an unclassified Methylophilales strain, HTCC2181. Comparative analysis revealed that the core genome of Methylophilaceae may be as small as approximately 600 genes, while the pangenome may be as large as approximately 6,000 genes. Significant divergence between the genomes in terms of both gene content and gene and protein conservation was uncovered, including the varied presence of certain genes involved in methylotrophy. Overall, our data demonstrate that metabolic potentials can vary significantly between different species of Methylophilaceae, including organisms inhabiting the very same environment. These data suggest that genetic divergence among the members of this family may be responsible for their specialized and nonredundant functions in C1 cycling, which in turn suggests means for their successful coexistence in their specific ecological niches. [ABSTRACT FROM AUTHOR]