Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Haemerythrin proteins comprise a family of O 2 -carrrying proteins mainly found in a few phyla of marine invertebrates. Members of this family differ from haemoglobin and haemocyanin in that they contain a nonheme diiron site that reversibly binds one molecule of O 2 . This oxygen-binding binuclear iron complex is a characteristic feature of haemerythrins. The two iron ions are bound to the protein via seven conserved amino acid residues; five histidines, one glutamate and one aspartate [1]. All known haemerythrins also share a four-helix bundle fold which surrounds the diiron site. Furthermore, the haemerythrins are divided into two subfamilies; the haemerythrins (Hr) and myohaemerythrins (MHr). Hrs are found in coelomic cells and typically exist as homopolymers composed of subunits of 113-117 amino acid residues. MHrs are For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O 2 . We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O 2 may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV ⁄ vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.Abbreviations 2DE, two-dimensional gel electrophoresis; Hr, haemerythrin; ICP-MS, inductively coupled plasma atomic emission-mass spectrometry; IPG, immobilized pH gradient; IPTG, isopropyl thio-b-D-galactoside; McHr, Methylococcus capsulatus haemerythrin; MHr, myohaemerythrin; MMO, methane monooxygenase; NMS, nitrate mineral salt; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase.

Copper plays a very significant role in the physiology of the methanotrophic bacterium Methylococcus capsulatus (Bath). The availability of this metal ion regulates expression of the two forms of the methane-oxidizing enzyme methane monooxygenase (MMO) the bacterium possesses and formation of an extensive intracytoplasmic membrane network [1][2][3][4]. When copper is scarce, at a low copper-to-biomass ratio, a soluble cytoplasmic MMO (sMMO) is responsible for the oxidation of methane. At high copper-to-biomass ratios the particulate MMO (pMMO) is expressed and there is no detectable sMMO expression. Furthermore, copper also influences the expression of at least two of the four M. capsulatus formaldehyde dehydrogenases [5][6][7].

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