Description: In 2014 we described a new activity-based screen suited to seek active recombinant RubisCOs from the environment - independent of the native host's culturability. Within this study we aimed at seeking novel RubisCO active enzymes from the environment. For this purpose we screened six metagenomic fosmid libraries from hydrothermal vent environments, differing with respect to their (i) geographic origin (north or south Mid Atlantic Ridge) and their (ii) environmental characteristics (basalt vs. ultramafic hosted, temperature, and pH). Overall we screened roughly 12500 metagenomic fosmid clones 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.

Description: Here we used the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. Hydrothermal vent fluids derived from the interface zone between hot fluids emanating from Drachenschlund and ambient cold seawater at 8°18'S/13°30'W served as initial sample for library construction. Among 1056 screened fosmid clones 12 exhibited RubisCO activity. The metagenomic fragments of all twelve clones resembled genes from Hydrogenovibrio crunogenus. 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 12 genes and 2 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.

Description: 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 13,023 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. To date, only one study has ever investigated regulatory mechanisms in metagenome-derived RubisCO gene clusters (Böhnke and Perner 2015). Here, total RubisCO activity was significantly influenced when cbbL and cbbM neighboring genes were knocked out (Böhnke and Perner 2015), but it remained unclear which of the two RubisCOs was primarily affected by these mutations. 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.

Description: Metal cycling in the ocean is likely more affected by hydrothermal emissions of volcanic arcs than by those of mid- ocean ridges. This can be explained by generally higher metal fluxes and the release of biogeochemically essential metals at shallower water depths. Hydrothermal fluids are typically enriched in hydrogen, methane, sulfide and other reduced inorganic compounds, which can be oxidized by microorganisms to gain energy. To study this microbially mediated element cycling in hydrothermal fluids, vents at the Kermadec Arc offer an eminent potential. Fluids of this location can have very low pH as well as low sulfide and hydrogen contents, but they are often enriched in trace metals, especially in iron. Based on the local environmental conditions the aim of this study was to determine which of the available inorganic compounds primarily fuel autotrophic CO2 fixation and which organisms are responsible for catalyzing these processes. Here we report on incubation experiments with hydrothermal fluids from four actively venting sites on the Kermadec arc. Hydrothermal emissions were supplemented either with iron(II), sulfide or hydrogen and radioactively labeled bicarbonate followed by incubation for eight hours. Microbial consumption of inorganic electron donors as well as the amount of autotrophically fixed CO2 into biomass was measured. At the end of the experiments, the active microorganisms responsible for element cycling were identified through transcriptome analyses. Interestingly, the highest autotrophic CO2 fixation rates were observed in iron(II) supplemented fluids with Epsilonproteobacteria and Thiomicrospira spp. as the likely responsible organisms.

Description: Carbon monoxide dehydrogenases (CODHs) are a promising resource for biotechnological and industrial applications as these enzymes catalyze the reversible reaction of carbon monoxide (CO) with water to carbon dioxide (CO2) protons and two electrons. Therefore, these enzymes are helping to convert the greenhouse gas CO2 into valuable commodities. CODHs are used by a variety of phylogenetically diverse aerobic and anaerobic microbial organisms in autotrophic carbon fixation and energy conservation such as in the reductive acetyl-CoA pathway, even though CO has a toxic nature. However, the identification of environmental CODH enzymes is limited as the vast microbial majority is currently not cultured. Thus, the exploration of a large biochemical potential remains inaccessible using culture-dependent and sequence-based methods. Therefore, we have developed a cultivation-independent method using an activity-based colorimetric screening approach that enables us to recover novel CODH enzymes from the environment by detecting the oxidation of CO to CO2. To investigate which CODHs from different microbial phyla can be targeted by this method, the screen was successfully applied to fosmid clones prepared with genomic material from Rhodospirillum rubrum, Desulfovibrio vulgaris, Moorella thermoacetica and Methanosarcina mazei. To discover highly energetic and novel CODHs from the environment, our screen is currently being applied to a metagenomic fosmid library constructed with anoxic marine sediments from the Eckernförde Bight (Baltic Sea, Germany).

Description: Ni-dependent carbon monoxide dehydrogenases (CODHs) are a promising resource for biotechnological and industrial applications as these enzymes catalyze the reversible reaction of carbon monoxide to carbon dioxide and therefore, are helping to convert the greenhouse gas CO2 into valuable commodities. Even though, carbon monoxide is toxic to most organisms, it is used by CODHs of a variety of phylogenetically diverse aerobic and anaerobic microbial organisms in autotrophic carbon fixation and energy conservation such as in the reductive acetyl-CoA pathway (Wood-Ljungdahl pathway). However, the ability to identify environmental CODH enzymes is limited as the vast microbial majority is currently not culturable and thus the exploration of a large biochemical potential remains inaccessible using culture-dependent and sequence-based methods. Therefore, we have developed a cultivation-independent method using an activity-based colorimetric screening approach that enables us to recover novel CODH enzymes from the environment by detecting the oxidation of CO to CO2. To investigate which CODHs from different microbial phyla can be targeted by this method, the screen was successfully applied to fosmid clones prepared with genomic material from Rhodospirillum rubrum, Desulfovibrio vulgaris, Moorella thermoacetica and Methanosarcina mazei. Our screen is currently being applied to a metagenomic fosmid library constructed using anoxic marine sediments from the Eckernförde Bight (Baltic Sea, Germany) to discover highly energetic and novel CODHs from the environment.

Description: Carbon monoxide dehydrogenases (CODHs) catalyze the reversible conversion between carbon monoxide and carbon dioxide. These enzymes are additionally involved in autotrophic carbon fixation and energy conservation by the reductive acetyl-CoA pathway (Wood-Ljungdahl pathway), which enables acetogenic bacteria, autotrophic sulfate-reducing bacteria, planctomycetes as well as methanogenic archaea to grow on inorganic carbon sources. Since the vast majority of microbes are still unculturable, the exploration of a large biochemical potential remains limited using culture-dependent or sequence-based methods. Applying metagenomic approaches for the investigation of genes and enzymes associated with the CODH expression and activation, may reveal an enormous potential among autotrophic unculturable organisms. Therefore, we here present the development of a novel colorimetric function-based screening approach to seek oxygen sensitive CODHs directly from metagenomes by detecting the oxidation of CO to CO2. For this purpose, three genomic fosmid libraries of Moorella thermoacetica, Desulfovibrio vulgaris and Rhodospirillum rubrum were constructed as references for CODH activity within different phyla. The CODH activity assay was performed in 96 Deep-Well plates using oxidized methyl viologen as an electron acceptor. Active CODH clones are then indicated by a blue color change upon the reduction of methyl viologen. The activity of recombinant CODHs from D. vulgaris clones has already been measured, suggesting that the developed screen suits Deltaproteobacteria. For the discovery of highly energetic and novel CODHs from the environment, the screening of a metagenomic fosmid library, derived from marine sediment of the Eckernförde Bay (Baltic Sea, Germany), was recently started.

Description: Carbon monoxide dehydrogenases catalyze the reversible reaction of carbon monoxide (CO) and water to carbon dioxide (CO2), protons, and electrons. These enzymes are used by a variety of phylogenetically diverse aerobic and especially anaerobic microbes for autotrophic carbon fixation and energy conservation such as in the reductive acetyl-CoA pathway. Therefore, the enzymatic inventory involved in this CO metabolism is a promising resource for industrial as well as biotechnological applications as CODHs drive the conversion of the greenhouse gas CO2 into valuable commodities. However, given that the vast majority of microbes on earth are currently not cultured, identification of truly novel environmental CODH enzymes is limited and thus the exploration of a large biochemical potential remains inaccessible using present culture-dependent and sequence-based approaches. Facing this problem, we use a culture-independent method and apply an activity-based colorimetric screen that enables us to detect (novel) CODH enzymes from the environment. To investigate which CODHs from different microbial phyla can be targeted by this method, the screen was successfully applied to fosmid clones prepared with genomic material from Rhodospirillum rubrum, Desulfovibrio vulgaris, Moorella thermoacetica and Methanosarcina mazei. Our screen was then applied to two different metagenomic fosmid libraries constructed with both anoxic marine sediments from Eckernförde Bight (Baltic Sea, Germany) and material from hydrothermal vents (Sisters Peak, Mid-Atlantic-Ridge). Screening latter metagenomic fosmid library recently resulted in the detection of two putative CODH active fosmid clones, which will be further investigated, characterized, and optimized for application in electrochemical cells to potentially reduce anthropogenically emitted CO2.