In situ chemistry and microbial community compositions in five deep-sea hydrothermal fluid samples from Irina II in the Logatchev field.

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.

Current status of carbon monoxide dehydrogenases (CODH) and their potential for electrochemical applications.

Anthropogenic carbon dioxide (CO2) levels are rising to alarming concentrations in earth's atmosphere, causing adverse effects and global climate changes. In the last century, innovative research on CO2 reduction using chemical, photochemical, electrochemical and enzymatic approaches has been addressed. In particular, natural CO2 conversion serves as a model for many processes and extensive studies on microbes and enzymes regarding redox reactions involving CO2 have already been conducted. In this review we focus on the enzymatic conversion of CO2 to carbon monoxide (CO) as the chemical conversion downstream of CO production render CO particularly attractive as a key intermediate. We briefly discuss the different currently known natural autotrophic CO2 fixation pathways, focusing on the reversible reaction of CO2, two electrons and protons to CO and water, catalyzed by carbon monoxide dehydrogenases (CODHs). We then move on to classify the different type of CODHs, involved catalyzed chemical reactions and coupled metabolisms. Finally, we discuss applications of CODH enzymes in photochemical and electrochemical cells to harness CO2 from the environment transforming it into commodity chemicals.

Approaches to Unmask Functioning of the Uncultured Microbial Majority From Extreme Habitats on the Seafloor.

Researchers have recognized the potential of enzymes and metabolic pathways hidden among the unseen majority of Earth's microorganisms for decades now. Most of the microbes expected to colonize the seafloor and its subsurface are currently uncultured. Thus, their ability and contribution to element cycling remain enigmatic. Given that the seafloor covers ∼70% of our planet, this amounts to an uncalled potential of unrecognized metabolic properties and interconnections catalyzed by this microbial dark matter. Consequently, a tremendous black box awaits discovery of novel enzymes, catalytic abilities, and metabolic properties in one of the largest habitats on Earth. This mini review summarizes the current knowledge of cultivation-dependent and -independent techniques applied to seafloor habitats to unravel the role of the microbial dark matter. It highlights the great potential that combining microbiological and biogeochemical data from in situ experiments with molecular tools has for providing a holistic understanding of bio-geo-coupling in seafloor habitats and uses hydrothermal vent systems as a case example.

Environmental changes affect the microbial release of hydrogen sulfide and methane from sediments at Boknis Eck (SW Baltic Sea).

Anthropogenic activities are modifying the oceanic environment rapidly and are causing ocean warming and deoxygenation, affecting biodiversity, productivity, and biogeochemical cycling. In coastal sediments, anaerobic organic matter degradation essentially fuels the production of hydrogen sulfide and methane. The release of these compounds from sediments is detrimental for the (local) environment and entails socio-economic consequences. Therefore, it is vital to understand which microbes catalyze the re-oxidation of these compounds under environmental dynamics, thereby mitigating their release to the water column. Here we use the seasonally dynamic Boknis Eck study site (SW Baltic Sea), where bottom waters annually fall hypoxic or anoxic after the summer months, to extrapolate how the microbial community and its activity reflects rising temperatures and deoxygenation. During October 2018, hallmarked by warmer bottom water and following a hypoxic event, modeled sulfide and methane production and consumption rates are higher than in March at lower temperatures and under fully oxic bottom water conditions. The microbial populations catalyzing sulfide and methane metabolisms are found in shallower sediment zones in October 2018 than in March 2019. DNA-and RNA profiling of sediments indicate a shift from primarily organotrophic to (autotrophic) sulfide oxidizing Bacteria, respectively. Previous studies using data collected over decades demonstrate rising temperatures, decreasing eutrophication, lower primary production and thus less fresh organic matter transported to the Boknis Eck sediments. Elevated temperatures are known to stimulate methanogenesis, anaerobic oxidation of methane, sulfate reduction and essentially microbial sulfide consumption, likely explaining the shift to a phylogenetically more diverse sulfide oxidizing community based on RNA.

Impact of high Fe-concentrations on microbial community structure and dissolved organics in hydrothermal plumes: an experimental study.

Iron (Fe) is an essential trace element for life. In the ocean, Fe can be exceptionally scarce and thus biolimiting or extremely enriched causing microbial stress. The ability of hydrothermal plume microbes to counteract unfavorable Fe-concentrations up to 10 mM is investigated through experiments. While Campylobacterota (Sulfurimonas) are prominent in a diverse community at low to intermediate Fe-concentrations, the highest 10 mM Fe-level is phylogenetically less diverse and dominated by the SUP05 clade (Gammaproteobacteria), a species known to be genetically well equipped to strive in high-Fe environments. In all incubations, Fe-binding ligands were produced in excess of the corresponding Fe-concentration level, possibly facilitating biological Fe-uptake in low-Fe incubations and detoxification in high-Fe incubations. The diversity of Fe-containing formulae among dissolved organics (SPE-DOM) decreased with increasing Fe-concentration, which may reflect toxic conditions of the high-Fe treatments. A DOM-derived degradation index (IDEG) points to a degradation magnitude (microbial activity) that decreases with Fe and/or selective Fe-DOM coagulation. Our results show that some hydrothermal microbes (especially Gammaproteobacteria) have the capacity to thrive even at unfavorably high Fe-concentrations. These ligand-producing microbes could hence play a key role in keeping Fe in solution, particularly in environments, where Fe precipitation dominates and toxic conditions prevail.

Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments.

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

Parameters Governing the Community Structure and Element Turnover in Kermadec Volcanic Ash and Hydrothermal Fluids as Monitored by Inorganic Electron Donor Consumption, Autotrophic CO2 Fixation and 16S Tags of the Transcriptome in Incubation Experiments.

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.

Unraveling RubisCO Form I and Form II Regulation in an Uncultured Organism from a Deep-Sea Hydrothermal Vent via Metagenomic and Mutagenesis Studies.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first major step of carbon fixation in the Calvin-Benson-Bassham (CBB) cycle. This autotrophic CO2 fixation cycle accounts for almost all the assimilated carbon on Earth. Due to the primary role that RubisCO plays in autotrophic carbon fixation, it is important to understand how its gene expression is regulated and the enzyme is activated. Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18' S along the Mid-Atlantic Ridge. It is 13,046 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. In silico analyses uncovered several putative LysR binding sites and promoter structures. Functions of some of these DNA motifs were experimentally confirmed. For example, according to mobility shift assays LysR1's binding ability to the intergenic region of lysR1 and cbbL appears to be intensified when CbbL or LysR2 are present. Binding of LysR2 upstream of cbbM appears to be intensified if CbbM is present. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR's binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression. A model summarizes the findings and predicts putative interactions of the different proteins influencing RubisCO gene regulation and expression.

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model.

Previously, Planted Fixed-Bed Reactors (PFRs) have been used to investigate microbial toluene removal in the rhizosphere of constructed wetlands. Aerobic toluene degradation was predominant in these model systems although bulk redox conditions were hypoxic to anoxic. However, culture-independent approaches indicated also that microbes capable of anaerobic toluene degradation were abundant. Therefore, we aimed at isolating anaerobic-toluene degraders from one of these PFRs. From the obtained colonies which consisted of spirilli-shaped bacteria, a strain designated 15-1 was selected for further investigations. Analysis of its 16S rRNA gene revealed greatest similarity (99%) with toluene-degrading Magnetospirillum sp. TS-6. Isolate 15-1 grew with up to 0.5 mM of toluene under nitrate-reducing conditions. Cells reacted to higher concentrations of toluene by an increase in the degree of saturation of their membrane fatty acids. Strain 15-1 contained key genes for the anaerobic degradation of toluene via benzylsuccinate and subsequently the benzoyl-CoA pathway, namely bssA, encoding for the alpha subunit of benzylsuccinate synthase, bcrC for subunit C of benzoyl-CoA reductase and bamA for 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase. Finally, most members of a clone library of bssA generated from the PFR had highest similarity to bssA from strain 15-1. Our study provides insights about the physiological capacities of a strain of Magnetospirillum isolated from a planted system where active rhizoremediation of toluene is taking place.

Endemic hydrothermal vent species identified in the open ocean seed bank.

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'.

A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

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.