We present data on the co-registered geochemistry (in situ mass spectrometry) and microbiology (pyrosequencing of 16S rRNA genes; V1, V2, V3 regions) in five fluid samples from Irina II in the Logatchev hydrothermal field. Two samples were collected over 24 min from the same spot and further three samples were from spatially distinct locations (20 cm, 3 m and the overlaying plume). Four low-temperature hydrothermal fluids from the Irina II are composed of the same core bacterial community, namely specific Gammaproteobacteria and Epsilonproteobacteria, which, however, differs in the relative abundance. The microbial composition of the fifth sample (plume) is considerably different. Although a significant correlation between sulfide enrichment and proportions of Sulfurovum (Epsilonproteobacteria) was found, no other significant linkages between abiotic factors, i.e. temperature, hydrogen, methane, sulfide and oxygen, and bacterial lineages were evident. Intriguingly, bacterial community compositions of some time series samples from the same spot were significantly more similar to a sample collected 20 cm away than to each other. Although this finding is based on three single samples only, it provides first hints that single hydrothermal fluid samples collected on a small spatial scale may also reflect unrecognized temporal variability. However, further studies are required to support this hypothesis.

Hydrothermal vent systems host microbial communities among which several microorganisms have been considered endemic to this type of habitat. It is still unclear how these organisms colonize geographically distant hydrothermal environments. Based on 16S rRNA gene sequences, we compare the bacterial communities of sixteen Atlantic hydrothermal vent samples with our own and publicly available global open ocean samples. Analysing sequences obtained from 63 million 16S rRNA genes, the genera we could identify in the open ocean waters contained 99.9% of the vent reads. This suggests that previously observed vent exclusiveness is, in most cases, probably an artefact of lower sequencing depth. These findings are a further step towards elucidating the role of the open ocean as a seed bank. They can explain the predicament of how species expected to be endemic to vent systems are able to colonize geographically distant hydrothermal habitats and contribute to our understanding of whether 'everything is really everywhere'.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. For more than a decade, metagenome research has demonstrated that it is a powerful tool for the discovery of novel biocatalysts and other valuable biomolecules by using either function-or sequence-based screening technologies (1-3). Sequencebased approaches allow the identification of candidate genes. In particular, the development of next-generation sequencing (NGS) technology and improved bioinformatic tools have significantly advanced this methodology (4). However, a major drawback of sequence-based screening technologies is that they do not allow direct conclusions about the functionality and biochemical parameters of the encoded enzymes. Furthermore, sequence-based searches are limited to the identification of homologs of already known motifs (5). Yet another problem associated with the sequence-based approach is that it often reveals only partial genes, which make subsequent expression and detailed biochemical analysis of the gene products difficult if not impossible. In contrast, the function-driven approach is usually much slower and more labor-intensive and costly but results in the detection of complete and active enzyme clones. It is of course well known that function-driven metagenomics is hampered due to the problems of expressi...

The microbial community composition and its functionality was assessed for hydrothermal fluids and volcanic ash sediments from Haungaroa and hydrothermal fluids from the Brothers volcano in the Kermadec island arc (New Zealand). The Haungaroa volcanic ash sediments were dominated by epsilonproteobacterial Sulfurovum sp. Ratios of electron donor consumption to CO2 fixation from respective sediment incubations indicated that sulfide oxidation appeared to fuel autotrophic CO2 fixation, coinciding with thermodynamic estimates predicting sulfide oxidation as the major energy source in the environment. Transcript analyses with the sulfide-supplemented sediment slurries demonstrated that Sulfurovum prevailed in the experiments as well. Hence, our sediment incubations appeared to simulate environmental conditions well suggesting that sulfide oxidation catalyzed by Sulfurovum members drive biomass synthesis in the volcanic ash sediments. For the Haungaroa fluids no inorganic electron donor and responsible microorganisms could be identified that clearly stimulated autotrophic CO2 fixation. In the Brothers hydrothermal fluids Sulfurimonas (49%) and Hydrogenovibrio/Thiomicrospira (15%) species prevailed. Respective fluid incubations exhibited highest autotrophic CO2 fixation if supplemented with iron(II) or hydrogen. Likewise catabolic energy calculations predicted primarily iron(II) but also hydrogen oxidation as major energy sources in the natural fluids. According to transcript analyses with material from the incubation experiments Thiomicrospira/Hydrogenovibrio species dominated, outcompeting Sulfurimonas. Given that experimental conditions likely only simulated environmental conditions that cause Thiomicrospira/Hydrogenovibrio but not Sulfurimonas to thrive, it remains unclear which environmental parameters determine Sulfurimonas’ dominance in the Brothers natural hydrothermal fluids.