Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. Both the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex catalyze this reaction. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic conditions while the PDH complex is inactivated under the same conditions. We found that cells rely on a general shift from utilizing NAD(H)-dependent to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

Multiple integrated metabolic strategies allow foraminiferan protists to thrive in anoxic marine sediments

Oceanic deoxygenation is increasingly affecting marine ecosystems; many taxa will be severely challenged, yet certain nominally aerobic foraminifera (rhizarian protists) thrive in oxygen-depleted to anoxic, sometimes sulfidic, sediments uninhabitable to most eukaryotes. Gene expression analyses of foraminifera common to severely hypoxic or anoxic sediments identified metabolic strategies used by this abundant taxon. In field-collected and laboratory-incubated samples, foraminifera expressed denitrification genes regardless of oxygen regime with a putative nitric oxide dismutase, a characteristic enzyme of oxygenic denitrification. A pyruvate:ferredoxin oxidoreductase was highly expressed, indicating the capability for anaerobic energy generation during exposure to hypoxia and anoxia. Near-complete expression of a diatom’s plastid genome in one foraminiferal species suggests kleptoplasty or sequestration of functional plastids, conferring a metabolic advantage despite the host living far below the euphotic zone. Through a unique integration of functions largely unrecognized among “typical” eukaryotes, benthic foraminifera represent winning microeukaryotes in the face of ongoing oceanic deoxygenation.

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

The genus Nitrospira is the most widespread group of chemolithoautotrophic nitrite-oxidizing bacteria that thrive in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T represents the type genus and was isolated from the oceanic water column over 30 years ago, however, its genome has not yet been analyzed. Here, we analyzed the complete genome sequence of N. marina and performed select physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to combat oxidative, osmotic and UV-light induced stress and low dissolved pCO2, and is able to grow chemoorganotrophically on formate. We further investigated the metabolic response of N. marina to low (∼5.6 µM) O2 concentrations commonly encountered in marine environments with high nitrite concentrations. In response to O2-limited conditions, the abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high affinity cbb3-type terminal oxidase increased, suggesting a role in sustaining nitrite oxidation-driven autotrophy under O2 limitation. Additionally, a Cu/Zn-binding superoxide dismutase increased in abundance potentially protecting this putatively more O2-sensitive POR complex from oxidative damage. An increase in abundance of proteins involved in alternative energy metabolisms, including type 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides important insights into the metabolic diversity and niche differentiation of NOB in marine environments.

An ectosymbiosis-based mechanism of eukaryogenesis

The findings of a deep branching Microsporidia clade on the SSU rRNA tree, and diversity of sequence motifs in eukaryotic Hsp70s rendered invalid the endosymbiosis-first theory that mitosome- and hydrogenosome-containing amitochondriate eukaryotes (AMIs) arose from mitochondriate eukaryotes (MTEs) via reductive evolution. Instead, evidence of widespread ectosymbioses indicated that eukaryogenesis was started by an archaeal parent via its acquisition of archaeal proteins through ‘accelerated gene adoption’, and bacterial proteins from ectosymbionts including a clostridial ectosymbiont that supplied its [Fe] hydrogenase and pyruvate:ferredoxin oxidoreductase genes to the AMIs. Subsequent endosymbiosis with Tistrella gave rise to mitochondria with the participation of other alphaproteobacteria. The high frequencies of top similarity bitscores displayed by Giardia, Edhazardia and Trichomonas toward Aciduliprofundum boonei (Abo) pertaining to the enzymes of DNA biology, far surpassing the frequencies toward any Asgard or TACK archaeon, established Abo as the source of these enzymes in eukaryotes, and the archaeal parent of Eukarya.

Genomic analysis of the mesophilic Thermotogae genusMesotogareveals phylogeographic structure and genomic determinants of its distinct metabolism

SummaryThe genusMesotoga, the only described mesophilicThermotogaelineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily,Mesotogadisplays lineage-specific phenotypes, such as no or little H2production and dependence on sulfur-compound reduction, which may influence its ecological role. We used comparative genomics of 18Mesotogastrains (pairwise 16S rRNA identity > 99%) and a transcriptome ofM. primato investigate how life at moderate temperatures affects phylogeography and to interrogate the genomic features of its lineage-specific metabolism. We propose thatMesotogaaccomplish H2oxidation and thiosulfate reduction using a sulfide dehydrogenase and a hydrogenase-complex and that a pyruvate:ferredoxin oxidoreductase acquired fromClostridiais responsible for oxidizing acetate. Phylogenetic analysis revealed three distinctMesotogalineages (89.6-99.9% average nucleotide identity [ANI] within lineages, 79.3-87.6% ANI between lineages) having different geographic distribution patterns and high levels of intra-lineage recombination but little geneflow between lineages. Including data from metagenomes, phylogeographic patterns suggest that geographical separation historically has been more important forMesotogathan hyperthermophilicThermotogaand we hypothesize that distribution ofMesotogais constrained by their anaerobic lifestyle. Our data also suggest that recent anthropogenic activities and environments (e.g., wastewater treatment, oil exploration) have expandedMesotogahabitats and dispersal capabilities.Originality-Significance StatementThis study comprises one of the first whole-genome-based phylogeographic analyses of anaerobic mesophiles, and our data suggest that such microbes are more restricted by geography than are thermophiles (and mesophilic aerobes). This is likely to be a general trait for similar anaerobic organisms – and therefore broadly relevant to and testable in other environments. Moreover,Mesotogabacteria are part of the largely understudied subsurface ecosystem that has relatively recently been recognized as a new and important biosphere. Understanding the forces responsible for the distribution of organisms in the subsurface, as well as the identification of genes responsible forMesotoga’s distinct metabolism, will contribute to the understanding of these communities.

Binding site for coenzyme A revealed in the structure of pyruvate:ferredoxin oxidoreductase from Moorella thermoacetica

Pyruvate:ferredoxin oxidoreductase (PFOR) is a microbial enzyme that uses thiamine pyrophosphate (TPP), three [4Fe-4S] clusters, and coenzyme A (CoA) in the reversible oxidation of pyruvate to generate acetyl-CoA and carbon dioxide. The two electrons that are generated as a result of pyruvate decarboxylation are used in the reduction of low potential ferredoxins, which provide reducing equivalents for central metabolism, including the Wood–Ljungdahl pathway. PFOR is a member of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily, which plays major roles in both microbial redox reactions and carbon dioxide fixation. Here, we present a set of crystallographic snapshots of the best-studied member of this superfamily, the PFOR from Moorella thermoacetica (MtPFOR). These snapshots include the native structure, those of lactyl-TPP and acetyl-TPP reaction intermediates, and the first of an OFOR with CoA bound. These structural data reveal the binding site of CoA as domain III, the function of which in OFORs was previously unknown, and establish sequence motifs for CoA binding in the OFOR superfamily. MtPFOR structures further show that domain III undergoes a conformational change upon CoA binding that seals off the active site and positions the thiolate of CoA directly adjacent to the TPP cofactor. These structural findings provide a molecular basis for the experimental observation that CoA binding accelerates catalysis by 105-fold.

Hydrogen production bySulfurospirillumspp. enables syntrophic interactions of Epsilonproteobacteria

Hydrogen-producing bacteria are of environmental and biotechnological importance in anoxic environments, since hydrogen is an important electron donor for prokaryotes and of interest as an alternative energy source. Epsilonproteobacteria, inhabiting ecologically, clinically or biotechnologically relevant environments, are currently considered to be hydrogen-oxidizing bacteria exclusively. Here, we report hydrogen production for a genus of free-living Epsilonproteobacteria,Sulfurospirillumspp. inhabiting sediments, wastewater plants, bioelectrodes, oil reservoirs, contaminated areas, or marine habitats. The amount of hydrogen production was largely different in two subgroups ofSulfurospirillumspp., represented byS. cavoleiandS. multivorans. The former is shown to be the more potent hydrogen producer and excretes acetate as sole organic acid, while the latter exhibited a more flexible fermentation, producing additionally lactate and succinate. The observed hydrogen production could be assigned to a group 4 hydrogenase similar to Hydrogenase 4 (Hyf) inE. coli. We propose thatSulfurospirillumspp. produce molecular hydrogen with electrons derived from pyruvate oxidation by pyruvate:ferredoxin oxidoreductase and reduced ferredoxin. This hypothesis is supported by comparative proteome data, in which both PFOR and ferredoxin as well as hydrogenase 4 are up-regulated. A co-culture experiment withS. multivoransandMethanococcus voltaecultivated with lactate as sole substrate shows a syntrophic interaction between both organisms, since the former cannot grow fermentatively on lactate alone and the latter relies on hydrogen as electron donor. This opens up new perspectives on microbial communities, since Epsilonproteobacteria could play a yet unrecognized role as hydrogen producers in anoxic microbial communities.

CO2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO2 to formate serves as a CO2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO2 via two biochemical reactions: the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO2 uptake, and provided physical evidence of a distinct in vivo “rPFOR-PFL shunt” to reduce CO2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO2 fixation via the reductive C1 metabolic pathway in C. thermocellum. These findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO2 as well.