and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4 T , SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 6C (optimum 20-23 6C).The major cellular fatty acid was 18 : 1v7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4 T , SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and partial mmoX gene sequences of strains AR4 T , SOP9 and LAY are FR686343-FR686345 (16S rRNA gene) and FR686346-FR686348 (mmoX), respectively, and those for the partial mxaF, nifH and rbcL sequences of strain AR4T are FR686349, FR686351 and FR686352, respectively.

Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.

An aerobic, methanotrophic bacterium, designated KYG T , was isolated from a forest soil in Germany. Cells of strain KYG T were Gram-negative, non-motile, slightly curved rods that multiplied by binary fission and produced yellow colonies. The cells contained intracellular granules of poly-b-hydroxybutyrate at each cell pole, a particulate methane monooxygenase (pMMO) and stacks of intracytoplasmic membranes (ICMs) packed in parallel along one side of the cell envelope. Strain KYG T grew at pH 5.2-7.2 and 2-33 6C and could fix atmospheric nitrogen under reduced oxygen tension. The major cellular fatty acid was C 18 : 1 v7c (81.5 %) and the DNA G+C content was 61.4 mol%. Strain KYG T belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the obligate methanotroph Methylocapsa acidiphila B2 T (98.1 % 16S rRNA gene sequence similarity and 84.7 % pmoA sequence similarity). Unlike Methylocapsa acidiphila B2 T , which grows only on methane and methanol, strain KYG T was able to grow facultatively on acetate. Facultative acetate utilization is a characteristic of the methanotrophs of the genus Methylocella, but the genus Methylocella does not produce pMMO or ICMs. Strain KYG T differed from Methylocapsa acidiphila B2 T on the basis of substrate utilization pattern, pigmentation, pH range, cell ultrastructure and efficiency of dinitrogen fixation. Therefore, we propose a novel species, Methylocapsa aurea sp. nov., to accommodate this bacterium. The type strain is KYG T (5DSM 22158 T 5VKM B-2544 T ).All known aerobic methanotrophic bacteria belong to the phyla Proteobacteria or Verrucomicrobia (Op den Camp et al., 2009). The proteobacterial methanotrophs are affiliated to two families of the Alphaproteobacteria (Methylocystaceae and Beijerinckiaceae) and one family of the Gammaproteobacteria (Methylococcaceae). The family Beijerinckiaceae is particularly metabolically diverse and contains obligate and facultative methanotrophs and non-methanotrophic chemoheterotrophs. The two methanotrophic genera in this family, Methylocella and Methylocapsa, are abundant in acidic soils and peats (Dedysh et al., 2001(Dedysh et al., , 2003 and are physiologically distinct from each other. Methylocapsa acidiphila is an obligate methanotroph capable of growth only on one-carbon substrates. It has a particulate methane monooxygenase (pMMO) and an extensive intracytoplasmic membrane (ICM) system (Dedysh et al., 2002). In contrast, Methylocella species are the only known methanotrophs that lack pMMO and use only a soluble methane monooxygenase (sMMO) for methane oxidation. The genus Methylocella contains the first-described facultative methanotrophs and they are capable of growth on a few multicarbon substrates as well as methane (Dedysh et al., 2005). Here, we describe the isolation of a new methanotroph in the family Beijerinckiaceae.Abbreviations: ICM, intracytoplasmic membrane; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase.The GenBank/EMBL/DDBJ accession num...

Phylogenetic positions, and genotypic and phenotypic characteristics of three novel methylotrophic isolates, strains 301 T , 30S and SIP3-4, from sediment of Lake Washington, Seattle, USA, are described. The strains were restricted facultative methylotrophs capable of growth on single carbon compounds (methylamine and methanol) in addition to a limited range of multicarbon compounds. All strains used the N-methylglutamate pathway for methylamine oxidation. Strain SIP3-4 possessed the canonical (MxaFI) methanol dehydrogenase, but strains 301 T and 30S did not. All three strains used the ribulose monophosphate pathway for C1 assimilation. The major fatty acids in the three strains were C 16 : 0 and C 16 : 1 v7c. The DNA G+C contents of strains 301 T and SIP3-4 were 42.6 and 54.6 mol%, respectively. Based on 16S rRNA gene sequence phylogeny and the relevant phenotypic characteristics, strain SIP3-4 was assigned to the previously defined species Methylovorus glucosotrophus. Strains 301 T and 30S were closely related to each other (100 % 16S rRNA gene sequence similarity) and shared 96.6 % 16S rRNA gene sequence similarity with a previously described isolate, Methylotenera mobilis JLW8 T . Based on significant genomic and phenotypic divergence with the latter, strains 301 T and 30S represent a novel species within the genus Methylotenera, for which the name Methylotenera versatilis sp. nov. is proposed; the type strain is 301 T

Unlike biologically available nitrogen and phosphorus, which are often at limiting concentrations in surface seawater, sulfur in the form of sulfate is plentiful and not considered to constrain marine microbial activity. Nonetheless, in a model system in which a marine bacterium obtains all of its carbon from co-cultured phytoplankton, bacterial gene expression suggests that at least seven dissolved organic sulfur (DOS) metabolites support bacterial heterotrophy. These labile exometabolites of marine dinoflagellates and diatoms include taurine, N-acetyltaurine, isethionate, choline-O-sulfate, cysteate, 2,3-dihydroxypropane-1-sulfonate (DHPS), and dimethylsulfoniopropionate (DMSP). Leveraging from the compounds identified in this model system, we assessed the role of sulfur metabolites in the ocean carbon cycle by mining the Tara Oceans dataset for diagnostic genes. In the 1.4 million bacterial genome equivalents surveyed, estimates of the frequency of genomes harboring the capability for DOS metabolite utilization ranged broadly, from only 1 out of every 190 genomes (for the C2 sulfonate isethionate) to 1 out of every 5 (for the sulfonium compound DMSP). Bacteria able to participate in DOS transformations are dominated by Alphaproteobacteria in the surface ocean, but by SAR324, Acidimicrobiia, and Gammaproteobacteria at mesopelagic depths, where the capability for utilization occurs in higher frequency than in surface bacteria for more than half the sulfur metabolites. The discovery of an abundant and diverse suite of marine bacteria with the genetic capacity for DOS transformation argues for an important role for sulfur metabolites in the pelagic ocean carbon cycle.