Kinetics of the ancestral carbon metabolism pathways in deep-branching bacteria and archaea

The origin of life is believed to be chemoautotrophic, deriving all biomass components from carbon dioxide, and all energy from inorganic redox couples in the environment. The reductive tricarboxylic acid cycle (rTCA) and the Wood–Ljungdahl pathway (WL) have been recognized as the most ancient carbon fixation pathways. The rTCA of the chemolithotrophic Thermosulfidibacter takaii, which was recently demonstrated to take place via an unexpected reverse reaction of citrate synthase, was reproduced using a kinetic network model, and a competition between reductive and oxidative fluxes on rTCA due to an acetyl coenzyme A (ACOA) influx upon acetate uptake was revealed. Avoiding ACOA direct influx into rTCA from WL is, therefore, raised as a kinetically necessary condition to maintain a complete rTCA. This hypothesis was confirmed for deep-branching bacteria and archaea, and explains the kinetic factors governing elementary processes in carbon metabolism evolution from the last universal common ancestor.

Co-occurring activity of two autotrophic pathways in symbionts of hydrothermal vent tubeworm Riftia pachyptila

Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and Calvin-Benson-Bassham cycle (CBB) in “ Candidatus Endoriftia persephonae”, the autotrophic sulfur-oxidizing bacterial endosymbiont from giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than the CBB. R. pachyptila were incubated under in situ conditions in high-pressure aquaria under low (28-40 μmol hr −1 ) or high (180 - 276 μmol hr −1 ) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under either condition were capable of similar rates of CO 2 fixation. Activities of rCAC enzyme ATP-dependent citrate lyase and CBB enzyme RubisCO did not differ between the two conditions, though transcript abundances for ATP-dependent citrate lyase were 4 to 5-fold higher under low sulfide conditions. δ 13 C values of internal dissolved inorganic carbon pools were variable, and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ 13 C values of lipids fell between those collected for organisms using either the rCAC or CBB cycles exclusively. These observations are consistent with co-occurring activities of rCAC and CBB cycles in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from –omics studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila were maintained and monitored in high-pressure aquaria prior to measuring their CO 2 -fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO 2 -fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.

Pangenomics reveals alternative environmental lifestyles among chlamydiae

Chlamydiae are highly successful strictly intracellular bacteria associated with diverse eukaryotic hosts. Here we analyzed metagenome-assembled genomes of the “Genomes from Earth’s Microbiomes” initiative from diverse environmental samples, which almost double the known phylogenetic diversity of the phylum and facilitate a highly resolved view at the chlamydial pangenome. Chlamydiae are defined by a relatively large core genome indicative of an intracellular lifestyle, and a highly dynamic accessory genome of environmental lineages. We observe chlamydial lineages that encode enzymes of the reductive tricarboxylic acid cycle and for light-driven ATP synthesis. We show a widespread potential for anaerobic energy generation through pyruvate fermentation or the arginine deiminase pathway, and we add lineages capable of molecular hydrogen production. Genome-informed analysis of environmental distribution revealed lineage-specific niches and a high abundance of chlamydiae in some habitats. Together, our data provide an extended perspective of the variability of chlamydial biology and the ecology of this phylum of intracellular microbes.

Growth Enhancement Facilitated by Gaseous CO2 through Heterologous Expression of Reductive Tricarboxylic acid Cycle Genes in Escherichia coli

The enzymatic mechanisms of carbon fixation by autotrophs, such as the reductive tricarboxylic acid cycle (rTCA), have inspired biotechnological approaches to producing bio-based chemicals directly through CO2. To explore the possibility of constructing an rTCA cycle in Escherichia coli and to investigate their potential for CO2 assimilation, a total of ten genes encoding the key rTCA cycle enzymes, including α-ketoglutarate:ferredoxin oxidoreductase, ATP-dependent citrate lyase, and fumarate reductase/succinate dehydrogenase, were cloned into E. coli. The transgenic E. coli strain exhibited enhanced growth and the ability to assimilate external inorganic carbon with a gaseous CO2 supply. Further experiments conducted in sugar-free medium containing hydrogen as the electron donor and dimethyl sulfoxide (DMSO) as the electron acceptor proved that the strain is able to undergo anaerobic respiration, using CO2 as the major carbon source. The transgenic stain demonstrated CO2-enhanced growth, whereas the genes involved in chemotaxis, flagellar assembly, and acid-resistance were upregulated under the anaerobic respiration. Furthermore, metabolomic analysis demonstrated that the total concentrations of ATP, ADP, and AMP in the transgenic strain were higher than those in the vector control strain and these results coincided with the enhanced growth. Our approach offers a novel strategy to engineer E. coli for assimilating external gaseous CO2.

Systems-informed genome mining for electroautotrophic microbial production

Microbial electrosynthesis (MES) systems can store renewable energy and CO2 in many-carbon molecules inaccessible to abiotic electrochemistry. Here, we develop a multiphysics model to investigate the fundamental and practical limits of MES enabled by direct electron uptake and we identify organisms in which this biotechnological CO2-fixation strategy can be realized. Systematic model comparisons of microbial respiration and carbon fixation strategies revealed that, under aerobic conditions, the CO2 fixation rate is limited to <6 μmol/cm2/hr by O2 mass transport despite efficient electron utilization. In contrast, anaerobic nitrate respiration enables CO2 fixation rates >50 μmol/cm2/hr for microbes using the reductive tricarboxylic acid cycle. Phylogenetic analysis, validated by recapitulating experimental demonstrations of electroautotrophy, uncovered multiple probable electroautotrophic organisms and a significant number of genetically tractable strains that require heterologous expression of <5 proteins to gain electroautotrophic function. The model and analysis presented here will guide microbial engineering and reactor design for practical MES systems.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria)

Most autotrophs use the Calvin–Benson–Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts (“Candidatus Thiobarba”) of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that “Ca. Thiobarba” switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated “Ca. Thiobarba”. Direct stable isotope fingerprinting showed that “Ca. Thiobarba” has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.

Metals likely promoted protometabolism in early ocean alkaline hydrothermal systems

One of the most plausible scenarios of the origin of life assumes the preceding prebiotic autotrophic metabolism in sulfide-rich hydrothermal vent environments. However, geochemical mechanisms to harness the reductive power provided by hydrothermal systems remain to be elucidated. Here, we show that, under a geoelectrochemical condition realizable in the early ocean hydrothermal systems, several metal sulfides (FeS, Ag2S, CuS, and PbS) undergo hour- to day-scale conversion to the corresponding metals at ≤−0.7 V (versus the standard hydrogen electrode). The electrochemically produced FeS-Fe0 assemblage promoted various reactions including certain steps in the reductive tricarboxylic acid cycle with efficiencies far superior to those due to pure FeS. The threshold potential is readily generated in the H2-rich alkaline hydrothermal systems that were probably ubiquitous on the Hadean seafloor. Thus, widespread metal production and metal-sustained primordial metabolism were likely to occur as a natural consequence of the active hydrothermal processes on the Hadean Earth.

Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria)

Although the majority of known autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts (“Candidatus Thiobarba”) of deep-sea mussels that have acquired a complete CBB cycle and lost key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that “Ca. Thiobarba” switched from the rTCA to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated “Ca. Thiobarba”. Direct stable isotope fingerprinting showed that “Ca. Thiobarba” has typical CBB signatures, additional evidence that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways across the tree of life, and the interpretation of stable isotope measurements in the environment.