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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. 

Two sulphur-oxidizing, chemolithoautotrophic aerobes were isolated from the chemocline of an anchialine sinkhole located within the Weeki Wachee River of Florida. Gram-stain-negative cells of both strains were motile, chemotactic rods. Phylogenetic analysis of the 16S rRNA gene and predicted amino acid sequences of ribosomal proteins, average nucleotide identities, and alignment fractions suggest the strains HH1 T and HH3 T represent novel species belonging to the genus Thiomicrorhabdus . The genome G+C fraction of HH1 T is 47.8 mol% with a genome length of 2.61 Mb, whereas HH3 T has a G+C fraction of 52.4 mol% and 2.49 Mb genome length. Major fatty acids of the two strains included C 16 : 1 , C 18 : 1 and C 16 : 0 , with the addition of C 10:0 3-OH in HH1 T and C 12 : 0 in HH3 T . Chemolithoautotrophic growth of both strains was supported by elemental sulphur, sulphide, tetrathionate, and thiosulphate, and HH1 T was also able to use molecular hydrogen. Neither strain was capable of heterotrophic growth or use of nitrate as a terminal electron acceptor. Strain HH1 T grew from pH 6.5 to 8.5, with an optimum of pH 7.4, whereas strain HH3 T grew from pH 6 to 8 with an optimum of pH 7.5. Growth was observed between 15–35 °C with optima of 32.8 °C for HH1 T and 32 °C for HH3 T . HH1 T grew in media with [NaCl] 80–689 mM, with an optimum of 400 mM, while HH3 T grew at 80–517 mM, with an optimum of 80 mM. The name Thiomicrorhabdus heinhorstiae sp. nov. is proposed, and the type strain is HH1 T (=DSM 111584 T =ATCC TSD-240 T ). The name Thiomicrorhabdus cannonii sp. nov is proposed, and the type strain is HH3 T (=DSM 111593 T =ATCC TSD-241 T ).