Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments

Abstract

In nature, concentrations of dissolved inorganic carbon (DIC; CO₂ + HCO₃^- + CO₃^2-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO₂ fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations, were chosen for study. A high-CO₂-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) ΔyadF ΔcynT to grow under low-CO₂ conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO₂ was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO₂, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with various subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO₂.

Keywords: autotroph; carbon dioxide; carbon dioxide-concentrating mechanism; carbon fixation; dissolved inorganic carbon.

FIG 1

Models for structure and function of DACs. Possible configurations of 1-, 2-, and 3-subunit forms of DACs are depicted on the left. Both T and M subunits are predicted by TMHMM (32) to have multiple membrane-spanning alpha helices and are accordingly shown spanning the membrane. On the right, two possible models for DAC function are shown, one in which DACs couple proton translocation to the conversion of intracellular CO₂ to HCO₃⁻ and one in which DACs carry out symport of CO₂ and protons and this CO₂ is converted to HCO₃⁻ during or upon translocation to the cytoplasm. In autotrophic cells, this HCO₃⁻ is consumed by the Calvin-Benson-Bassham cycle (CBB) via carboxysomes (c’some) or the reductive citric acid cycle (rCAC). This figure was created with BioRender.com.

FIG 2

Growth curves of E. coli expressing complete DACs under low-CO₂ conditions. Each plot represents growth of a construct expressing a DAC from a different species: (A) Am. ferrooxidans DAC genes in pET28; (B) At. ferrooxidans in pET28; (C) At. thiooxidans in pBAD202; (D) H. crunogenus in pBAD202; (E) H. neapolitanus in pBAD202; (F) Sulfurovum sp. strain AR in pBAD202; (G) T. ruber in pET28. Circles, no inducer or repressor; triangles, inducer (IPTG for pET28 constructs; arabinose for pBAD202 constructs); squares, repressor (rhamnose for pET28 constructs; glucose for pBAD202).

FIG 3

Growth curves of E. coli expressing DACs with missing subunits under high-CO₂ (red) and low-CO₂ (blue) conditions. Each plot represents growth of E. coli expressing a DAC from a different species: (A) At. ferrooxidans DAC genes in pET28, M subunit only; (B) At. thiooxidans DAC genes in pBAD202, M subunit only; (C) At. thiooxidans DAC genes in pBAD202, C and M subunits only (missing T subunit gene); (D) H. crunogenus DAC genes in pBAD202, M subunit only; (E) H. neapolitanus DAC genes in pBAD202, M subunit only; (F) H. neapolitanus DAC genes in pBAD, C and M subunits only (missing T subunit gene); (G) Sulfurovum sp. strain AR DAC genes in pBAD202, M subunit only; (H) Sulfurovum sp. strain AR DAC genes in pBAD202, C and M subunits only (missing T subunit); (I) T. ruber DA genes in in pET28, M subunit only. Circles, no inducer or repressor; triangles, inducer (IPTG for pET28 constructs; arabinose for pBAD202 constructs); squares, repressor (rhamnose for pET28 constructs; glucose for pBAD202).

FIG 4

Intracellular DIC concentrations measured in E. coli expressing DACs from seven species (Am. fer., Acidimicrobium ferrooxidans; At. fer., Acidithiobacillus ferrooxidans; At. thio., Acidithiobacillus thiooxidans; H. cru., Hydrogenovibrio crunogenus; H. nea., Halothiobacillus neapolitanus; S. AR, Sulfurovum sp. AR; T. rub., Thermocrinis ruber). Results are shown for cells grown under a 5% CO₂ headspace in the presence of inducer (I) or repressor (R). When indicated, cells were missing either cytoplasmic (C) or tiny (T) subunits. Black bars indicate median values (n = 8).

FIG 5

Intracellular DIC concentrations measured for E. coli expressing DACs when provided with either CO₂ or HCO₃⁻ out of chemical equilibrium. Am. fer., Acidimicrobium ferrooxidans; H. cru., Hydrogenovibrio crunogenus; S. AR, Sulfurovum sp. strain AR. Results are shown for cells grown in the presence of inducer (I) or repressor (R). Black bars indicate median values (n = 4).

FIG 6

Intracellular DIC concentrations measured in E. coli expressing DACs when incubated in the presence of either DMSO (solvent control) or CCCP plus DMSO. Am. fer., Acidimicrobium ferrooxidans; H. cru., Hydrogenovibrio crunogenus; S. AR, Sulfurovum sp. strain AR. Black bars indicate median values (n = 8).

FIG 7

Chromosomal colocation of genes encoding DACs with those encoding enzymes from the CBB and rCAC pathways for autotrophic CO₂ fixation.