Microbial consortia mediating the anaerobic oxidation of methane with sulfate are composed of methanotrophic Archaea (ANME) and Bacteria related to sulfate-reducing Deltaproteobacteria. Cultured representatives are not available for any of the three ANME clades. Therefore, a metagenomic approach was applied to assess the genetic potential of ANME-1 archaea. In total, 3.4 Mbp sequence information was generated based on metagenomic fosmid libraries constructed directly from a methanotrophic microbial mat in the Black Sea. These sequence data represent, in 30 contigs, about 82-90% of a composite ANME-1 genome. The dataset supports the hypothesis of a reversal of the methanogenesis pathway. Indications for an assimilatory, but not for a dissimilatory sulfate reduction pathway in ANME-1, were found. Draft genome and expression analyses are consistent with acetate and formate as putative electron shuttles. Moreover, the dataset points towards downstream electron-accepting redox components different from the ones known from methanogenic archaea. Whereas catalytic subunits of [NiFe]-hydrogenases are lacking in the dataset, genes for an [FeFe]-hydrogenase homologue were identified, not yet described to be present in methanogenic archaea. Clustered genes annotated as secreted multiheme c-type cytochromes were identified, which have not yet been correlated with methanogenesis-related steps. The genes were shown to be expressed, suggesting direct electron transfer as an additional possible mode to shuttle electrons from ANME-1 to the bacterial sulfate-reducing partner.

A basic problem of the metagenomic approach in microbial ecology is the assignment of genomic fragments to a certain species or taxonomic group, when suitable marker genes are absent. Currently, the (G + C)-content together with phylogenetic information and codon adaptation for functional genes is mostly used to assess the relationship of different fragments. These methods, however, can produce ambiguous results. In order to evaluate sequence-based methods for fragment identification, we extensively compared (G + C)-contents and tetranucleotide usage patterns of 9054 fosmid-sized genomic fragments generated in silico from 118 completely sequenced bacterial genomes (40 982 931 fragment pairs were compared in total). The results of this systematic study show that the discriminatory power of correlations of tetranucleotide-derived z-scores is by far superior to that of differences in (G + C)-content and provides reasonable assignment probabilities when applied to metagenome libraries of small diversity. Using six fully sequenced fosmid inserts from a metagenomic analysis of microbial consortia mediating the anaerobic oxidation of methane (AOM), we demonstrate that discrimination based on tetranucleotide-derived z-score correlations was consistent with corresponding data from 16S ribosomal RNA sequence analysis and allowed us to discriminate between fosmid inserts that were indistinguishable with respect to their (G + C)-contents.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as *

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosomeassociated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.Magnetotactic bacteria are capable of forming magnetosomes, which are specific intracellular structures that enable the cells to orient along magnetic field lines (3, 4, 41). The superior crystalline and magnetic properties of magnetosomes make them potentially useful as a highly ordered biomaterial in a number of applications, e.g., in the immobilization of bioactive compounds, magnetic drug targeting, or as a contrast agent for magnetic resonance imaging (24,41). Recently, the characteristics of bacterial magnetosomes have even been considered for use as biosignatures to identify presumptive Martian magnetofossils (49). Moreover, understanding bacterial magnetosome formation is expected to provide insights into more complex biomineralization systems in higher organisms (19). The biomineralization of magnetosome particles is achieved by a complex mechanism with control over the uptake, accumulation, and precipitation of iron, which, however, is poorly understood at the molecular and biochemical level.The magnetotactic ␣-proteobacterium Magnetospirillum gryphiswaldense microaerobically produces up to 60 cubo-octahedral magnetosomes, which are approximately 45 nm in size and consist of membrane-bounded crystals of the iron mineral magnetite (Fe 3 O 4 ) (34,42). In contrast to most other magnetotactic bacteria, methods for mass culture and genetic manipulation of M. gryphiswaldense are available (17,38,44), which has facilitated its analysis in a number of studies (37,39,40,43).In Magnetospirillum species, the deposition of the mineral particle occurs within a specific compartment, which is provided by the magnetosome membrane ...

Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with 14 C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70-86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with 14 Cbicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up 450% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.

At deep-sea hydrothermal vents, primary production is carried out by chemolithoautotrophic microorganisms, with the oxidation of reduced sulfur compounds being a major driver for microbial carbon fixation. Dense and highly diverse assemblies of sulfur-oxidizing bacteria (SOB) are observed, yet the principles of niche differentiation between the different SOB across geochemical gradients remain poorly understood. In this study niche differentiation of the key SOB was addressed by extensive sampling of active sulfidic vents at six different hydrothermal venting sites in the Manus Basin, off Papua New Guinea. We subjected 33 diffuse fluid and water column samples and 23 samples from surfaces of chimneys, rocks and fauna to a combined analysis of 16S rRNA gene sequences, metagenomes and real-time in situ measured geochemical parameters. We found Sulfurovum Epsilonproteobacteria mainly attached to surfaces exposed to diffuse venting, while the SUP05-clade dominated the bacterioplankton in highly diluted mixtures of vent fluids and seawater. We propose that the high diversity within Sulfurimonas- and Sulfurovum-related Epsilonproteobacteria observed in this study derives from the high variation of environmental parameters such as oxygen and sulfide concentrations across small spatial and temporal scales.

SummaryThe anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor is mediated by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Whereas three clades of ANME have been repeatedly studied with respect to phylogeny, key genes and genomic capabilities, little is known about their sulfate-reducing partner. In order to identify the partner of anaerobic methanotrophs of the ANME-2 clade, bacterial 16S rRNA gene libraries were constructed from cultures highly enriched for ANME-2a and ANME-2c in consortia with Deltaproteobacteria of the Desulfosarcina/ Desulfococcus group (DSS). Phylogenetic analysis of those and publicly available sequences from AOM sites supported the hypothesis by Knittel and colleagues that the DSS partner belongs to the diverse SEEP-SRB1 cluster. Six subclusters of SEEP-SRB1, SEEP-SRB1a to SEEP-SRB1f, were proposed and specific oligonucleotide probes were designed. Using fluorescence in situ hybridization on samples from six different AOM sites, SEEP-SRB1a was identified as sulfate-reducing partner in up to 95% of total ANME-2 consortia. SEEP-SRB1a cells exhibited a rodshaped, vibrioid, or coccoid morphology and were found to be associated with subgroups ANME-2a and ANME-2c. Moreover, SEEP-SRB1a was also detected in 8% to 23% of ANME-3 consortia in Haakon Mosby Mud Volcano sediments, previously described to be predominantly associated with SRB of the Desulfobulbus group. SEEP-SRB1a contributed to only 0.3% to 0.7% of all single cells in almost all samples indicating that these bacteria are highly adapted to a symbiotic relationship with ANME-2.