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ABSTRACT Autotrophic bacteria are able to fix CO₂in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO₂fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO₂-concentrating mechanisms (CCMs). CCMs consist of CO₂and HCO₃⁻transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophicBacteriathat use the Calvin-Benson-Basham cycle. Members of the genusThiomicrospiralack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO₂fixation by carboxysomes from members ofThiomicrospiraand potentially otherBacteria. Carboxysome enrichments fromThiomicrospira pelophilaandThiomicrospira aerophilawere found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted inT. pelophila, cells could no longer grow under low-CO₂conditions, and CA activity was no longer detectable in their carboxysomes. WhenT. pelophilaιCA was expressed in a strain ofEscherichia colilacking native CA activity, this strain recovered an ability to grow under low CO₂conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCEHere, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO₂fixation by some organisms with CO₂-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions. 

In nature, concentrations of dissolved inorganic carbon (DIC; = CO 2 + HCO 3 - + CO 3 2- ) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO 2 fixation. Such mechanisms have been well-studied in Cyanobacteria , but much remains to be learned about their activity in other phyla. Novel multi-subunit 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-CO 2 requiring, carbonic anhydrase-deficient ( yadF - cynT - ) strain of E. 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) yadF - cynT - to grow under low CO 2 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 CO 2 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 CO 2 , autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multi-subunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, begging 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 varied 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 CO 2 .