110 research outputs found
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 degrees 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
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 CalvinâBensonâBassham (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 environmentâindependent of the native hostâs 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
Characterization of Two Hydrogen-Oxidizing Hydrogenovibrio Strains From Kermadec Volcanic Island Arc Hydrothermal Vents
The genus Hydrogenovibrio consists of chemolithotrophic sulfur- and hydrogen-oxidizing bacteria that are found in diverse marine environments including hydrothermal vents where they can reach high cell numbers. Although several vent Hydrogenovibrio genomes encode for [NiFe]-hydrogenases (enzymes catalyzing the reversible reaction of hydrogen into protons and electrons), different attempts to grow these strains on hydrogen failed for a long time. Not long ago it was shown that some Hydrogenovibrio strains from hydrothermal vents are indeed able to oxidize hydrogen, which broadens their physiological spectrum in a competitive environment for energy sources. We here identify two active hydrogen consuming bacteria of the Hydrogenovibrio genus with different hydrogenase genes from vents in the South Pacific Ocean. Based on our results, hydrogen consuming Hydrogenovibrio species seem to be much more widespread in the oceans than expected
Comparison of Microbial Communities Inhabiting Ultramafic- and Basalt-Hosted Hydrothermal Systems: Genetic Characterization of 16S rRNA Genes, Hydrogen Oxidation and CO<sub>2</sub> Fixation Pathways
The host rock of a hydrothermal system defines that system's specific fluid chemical composition. Therefore the type of host rock (ultramafic rocks versus basalts) plays a major role in determining the most abundant energy sources which are available for microorganisms. This study was conducted to investigate the influence the host rock and resulting fluid chemistry have on the distribution of the indigenous microorganisms. For this purpose hydrothermal fluids were collected from the ultramafic-hosted Logatchev hydrothermal field (LHF) and the basalt-hosted Lilliput hydrothermal field. The microbial community analysis was based on 16S rRNA genes and fluorescence in situ hybridization. The functionality of chemoautotrophic communities was assessed in terms of putative CO2 fixation pathways and energy sources used. Ultramafic host rocks at the LHF result in fluids with extremely high hydrogen contents. Possibly as a consequence, phylogenetically diverse hydrogen-oxidizing chemolithotrophs were detected. High abundance in hydrogen concentrations seem to coincide with the diversification of putative hydrogen-oxidizing prokaryotes at the three sites investigated. By contrast, the fluids from the Lilliput field are very low in hydrogen but high in sulfide. Significantly less diverse hydrogen-oxidizing microorganisms were detected at the basalt-hosted Lilliput hydrothermal field. Nevertheless, diverse bacteria were related to organisms involved in sulfur cycling. This suggests that the host rock indirectly determines the presence of microorganisms with specific physiologies. However, in addition to the importance of the host rock it can be shown that mixing processes of hydrothermal fluids with ambient seawater have a significant impact on the distribution of microorganisms. Contrary, for CO2 fixation pathways the type of host rock is not relevant. Key enzymes of the two CO2 fixation pathways investigated tolerate different levels of oxygen. The presence of multiple environmental niches with different oxygen contents is reflected in the phylogenetic diversity of Calvin Benson-Bassham and reverse tricarboxylic acid cycle genes detected at the LHF. At the Lilliput hydrothermal field the diversity of these different genes suggests that the chemoautotrophic community is better adapted to low oxygen contents. The generally high diversity of CO2 fixation pathway genes demonstrates the ability of metabolically versatile prokaryotes to exhaust the potential of every available habitat
Handling Temperature Bursts Reaching 464 C: Different Microbial Strategies in the Sisters Peak Hydrothermal Chimney
The active venting Sisters Peak (SP) chimney on the Mid-Atlantic Ridge holds the current temperature record for the hottest ever measured hydrothermal fluids (400 degrees C, accompanied by sudden temperature bursts reaching 464 degrees C). Given the unprecedented temperature regime, we investigated the biome of this chimney with a focus on special microbial adaptations for thermal tolerance. The SP metagenome reveals considerable differences in the taxonomic composition from those of other hydrothermal vent and subsurface samples; these could be better explained by temperature than by other available abiotic parameters. The most common species to which SP genes were assigned were thermophilic Aciduliprofundum sp. strain MAR08-339 (11.8%), Hippea maritima (3.8%), Caldisericum exile (1.5%), and Caminibacter mediatlanticus (1.4%) as well as to the mesophilic Niastella koreensis (2.8%). A statistical analysis of associations between taxonomic and functional gene assignments revealed specific overrepresented functional categories: for Aciduliprofundum, protein biosynthesis, nucleotide metabolism, and energy metabolism genes; for Hippea and Caminibacter, cell motility and/or DNA replication and repair system genes; and for Niastella, cell wall and membrane biogenesis genes. Cultured representatives of these organisms inhabit different thermal niches; i.e., Aciduliprofundum has an optimal growth temperature of 70 degrees C, Hippea and Caminibacter have optimal growth temperatures around 55 degrees C, and Niastella grows between 10 and 37 degrees C. Therefore, we posit that the different enrichment profiles of functional categories reflect distinct microbial strategies to deal with the different impacts of the local sudden temperature bursts in disparate regions of the chimney
Hydrogenase Gene Distribution and H2 Consumption Ability within the Thiomicrospira Lineage
Thiomicrospira were originally characterized as sulfur-oxidizing chemolithoautotrophs. Attempts to grow them on hydrogen failed for many years. Only recently we demonstrated hydrogen consumption among two of three tested Thiomicrospira and posited that hydrogen consumption may be more widespread among Thiomicrospira than previously assumed. Here, we investigate and compare the hydrogen consumption ability and the presence of group 1 [NiFe]-hydrogenase genes (enzyme catalyzes H2â2H+ + 2e-) for sixteen different Thiomicrospira species. Seven of these Thiomicrospira species encoded group 1 [NiFe]-hydrogenase genes and five of these species could also consume hydrogen. All Thiomicrospira species exhibiting hydrogen consumption were from hydrothermal vents along the Mid-Atlantic ridge or Eastern Pacific ridges. The tested Thiomicrospira from Mediterranean and Western Pacific vents could not consume hydrogen. The [NiFe]-hydrogenase genes were categorized into two clusters: those resembling the hydrogenase from Hydrogenovibrio are in cluster I and are related to those from Alpha- and other Gammaproteobacteria. In cluster II, hydrogenases found exclusively in Thiomicrospira crunogena strains are combined and form a monophyletic group with those from Epsilonproteobacteria suggesting they were acquired through horizontal gene transfer. Hydrogen consumption appears to be common among some Thiomicrospira, given that five of the tested sixteen strains carried this trait. The hydrogen consumption ability expands their competitiveness within an environment
The globally widespread genus Sulfurimonas: versatile energy metabolisms and adaptations to redox clines
Sulfurimonas species are commonly isolated from sulfidic habitats and numerous 16S rRNA sequences related to Sulfurimonas species have been identified in chemically distinct environments, such as hydrothermal deep-sea vents, marine sediments, the oceanâs water column, and terrestrial habitats. In some of these habitats, Sulfurimonas have been demonstrated to play an important role in chemoautotrophic processes. Sulfurimonas species can grow with a variety of electron donors and acceptors, which may contribute to their widespread distribution. Multiple copies of one type of enzyme (e.g., sulfide:quinone reductases and hydrogenases) may play a pivotal role in Sulfurimonasâ flexibility to colonize disparate environments. Many of these genes appear to have been acquired through horizontal gene transfer which has promoted adaptations to the distinct habitats. Here we summarize Sulfurimonasâ versatile energy metabolisms and link their physiological properties to their global distribution
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 Earthâs 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 âŒ70% 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
Microbially Mediated Hydrogen Cycling in Deep-Sea Hydrothermal Vents
Deep-sea hydrothermal vents may provide one of the largest reservoirs on Earth for hydrogen-oxidizing microorganisms. Depending on the type of geological setting, hydrothermal environments can be considerably enriched in hydrogen (up to millimolar concentrations). As hot, reduced hydrothermal fluids ascend to the seafloor they mix with entrained cold, oxygenated seawater, forming thermal and chemical gradients along their fluid pathways. Consequently, in these thermally and chemically dynamic habitats biochemically distinct hydrogenases (adapted to various temperature regimes, oxygen and hydrogen concentrations) from physiologically and phylogenetically diverse Bacteria and Archaea can be expected. Hydrogen oxidation is one of the important inorganic energy sources in these habitats, capable of providing relatively large amounts of energy (237 kJ/mol H-2) for driving ATP synthesis and autotrophic CO2 fixation. Therefore, hydrogen-oxidizing organisms play a key role in deep-sea hydrothermal vent ecosystems as they can be considerably involved in light-independent primary biomass production. So far, the specific role of hydrogen-utilizing microorganisms in deep-sea hydrothermal ecosystems has been investigated by isolating hydrogen-oxidizers, measuring hydrogen consumption (ex situ), studying hydrogenase gene distribution and more recently by analyzing metatranscriptomic and metaproteomic data. Here we summarize this available knowledge and discuss the advent of new techniques for the identification of novel hydrogen-uptake and - evolving enzymes from hydrothermal vent microorganisms
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