Abstract:Mud volcanism is an important natural source of the greenhouse gas methane to hydrosphere and atmosphere 1,2 . Recent investigations show that the number of active submarine mud volcanoes may be much higher than anticipated (eg. 3-5), and that gas emitted from deep-sea seeps may reach the upper mixed ocean [6][7][8] . Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release is unknown 9 . Another uncertainty is the efficiency of methane oxidizing microorganisms in methane removal. Here we investigated the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72°N, 14°44'E; 1250 m water depth), to provide quantitative estimates of in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities; aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a novel clade of archaea (ANME-3) associated with bacterial mats. We found that upward flow of sulphate-and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to <40% of the total flux.The HMMV (Fig. 1), a circular structure of 1 km diameter and <10 m elevation above the adjacent seafloor, has been studied since the 1990s as a typical example of an active mud volcano 9 . Its formation may have coincided with a submarine landslide during the late Pleistocene, 330-200 ka before present 10 . Today, fluids, gas and muds rise from 2-3 km depth through a conduit below the HMMV 11,10 . The emitted gas is of a mixed microbial/thermogenic origin and consists of >99% CH4 with a δ 13 C-isotope signature of -60‰ 12,13 . The rising fluids are depleted in sulphate, chloride and magnesium caused by subsurface clay dewatering 11 . Investigation of the HMMV with RV POLARSTERN and ROV VICTOR 6000 in 2003 showed extensive outcroppings 1 2006-01-01028b_Boetius_MS 3 of fresh subsurface muds associated with steep thermal gradients 14 , gas and fluid vents, and a large gas plume reaching the mixed upper water column above the HMMV 12,8 .Seafloor videography in combination with geochemical measurements provided in situ estimates of gas flux 8 , fluid flow 15 and habitat distribution 16 . We focused on the three main concentric habitats above the gassy muds (Fig. 2): the centre of HMMV, which was devoid of epifauna; thiotrophic bacterial mats dominated by a Beggiatoa species; and surrounding fields of siboglinid tubeworms. Gas concentrations in sediments and bottom water were elevated in all three habitats (Tab. (Fig. 3a). Only minor amounts of methanotroph lipids (<0.1 µg gdw -1 ) and very low cell numbers (~10 7 cells cm -3 ) were found below 5 cm sediment depth ( Fig. 3a3-3a4). ANME cells were not microscopically detectable in the centre cores using all know...

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methaneconsuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56% ؎ 8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94% ؎ 2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.Large amounts of methane are stored in the subsurface ocean as crystalline gas hydrate, dissolved in porewater, and as free gas. Most of the methane which rises from subsurface reservoirs is consumed by anaerobic microorganisms inhabiting sulfate-penetrated sediment layers before it reaches the seafloor (65). This microbially mediated anaerobic oxidation of methane (AOM) controls the emission of the greenhouse gas methane from the ocean to the atmosphere (see reference 23 and references therein).In marine habitats, the metabolic process of AOM is proposed to be a reversed methanogenesis coupled to the reduction of sulfate involving methanotrophic archaea (ANME archaea) and sulfate-reducing bacteria (SRB). ANME archaea and SRB are assumed to interact syntrophically (26) and form microbial consortia which oxidize methane with equimolar amounts of sulfate, yielding bicarbonate and sulfide (26, 51, 52). Sulfide produced as a by-product of AOM at cold seeps often supports chemosynthetic communities which derive energy from sulfide oxidation. ...

BackgroundMethylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared.Methodology/Principal FindingsThe 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name “island integration determinant” (iid).Conclusion/SignificanceThese two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.