Highly efficient methane biocatalysis revealed in a methanotrophic bacterium.

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

Respiration response imaging for real-time detection of microbial function at the single-cell level.

The ability to detect specific functions of uncultured microbial cells in complex natural communities remains one of the most difficult tasks of environmental microbiology. Here we present respiration response imaging (RRI) as a novel fluorescence microscopy-based approach for the identification of microbial function, such as the ability to use C(1) substrates, at a single-cell level. We demonstrate that RRI could be used for the investigation of heterogeneity of a single microbial population or for functional profiling of microbial cells from complex environmental communities, such as freshwater lake sediment.

Utility of environmental primers targeting ancient enzymes: methylotroph detection in Lake Washington.

Methods have been explored for detection of methylotrophs in natural samples, using environmental primers based on genes involved in the tetrahydromethanopterin (H4MPT)-linked C1 transfer pathway. The underlying hypotheses were that the H4MPT-linked pathway is an ancient methylotrophy pathway, based on gene divergence, and that primers targeting more divergent genes will detect a broader variety of methylotrophs compared to the variety uncovered using probes and primers targeting highly conserved genes. Three groups of novel primer sets were developed targeting mch, mtdB, and fae, key genes in the H4MPT-linked pathway, and these were used to assess the variety of microorganisms possessing these genes in sediments from Lake Washington in Seattle, WA. Environmental clone libraries were constructed for each of the genes and were analyzed by RFLP, and representatives of different RFLP groups were sequenced and subjected to phylogenetic analysis. A combination of all three sets of novel primers allowed detection of the two previously characterized groups of methylotrophs in the site: methanotrophs of the (alpha- and the gamma-proteobacterial groups, belonghg to genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, Methylomicrobium, and Methylococcus. In addition to the genes belonging to known methanotroph populations, novel genes were identified, suggesting existence of previously undetected microbial groups possessing C1 transfer functions in this site. These included sequences clustering with the well-characterized methylotrophic phyla, Methylobacterium, Hyphomicrobium, and Xanthobacter. In addition, sequences divergent from those known for any groups of methylotrophs or methanogens were obtained, suggesting the presence of a yet unidentified microbial group possessing this H4MPT-linked C1 transfer pathway.

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden.

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.