Expression and Function of Four Carbonic Anhydrase Homologs in the Deep-Sea Chemolithoautotroph Thiomicrospira crunogena

Abstract

The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena grows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO2 + HCO3 − + CO3 −2). Its genome encodes α-carbonic anhydrase (α-CA), β-CA, carboxysomal β-like CA (CsoSCA), and a protein distantly related to γ-CA. The purposes of this work were to characterize the gene products, determine whether they were differentially expressed, and identify those that are necessary for DIC uptake and fixation. When expressed in Escherichia coli, CA activity was detectable for α-CA, β-CA, and CsoSCA but not for the γ-CA-like protein. α-CA and CsoSCA but not β-CA were inhibited by sulfonamide inhibitors. CsoSCA was also inhibited by dithiothreitol. When grown under DIC limitation in chemostats, T. crunogena transcribed csoSCA more frequently than when ammonia limited, while genes encoding α-CA and β-CA were not differentially transcribed under these conditions. Cell extracts from T. crunogena grown under both DIC- and ammonia-limited conditions had CA activity that was strongly inhibited by sulfonamides, though extracts from nitrogen-limited cells had some CA activity that was resistant, perhaps due to a higher level of β-CA activity. Based on predictions from the SignalP software program, subcellular location when expressed in E. coli, and carbonic anhydrase assays conducted on intact T. crunogena cells, α-CA is located in the periplasm. However, inhibition of α-CA by acetazolamide had only a minor impact on rates of DIC uptake or fixation. Conversely, inhibition of CsoSCA with ethoxyzolamide inhibited carbon fixation but not DIC uptake, consistent with this enzyme functioning to facilitate DIC interconversion and fixation within carboxysomes.

A novel hydrogen oxidizer amidst the sulfur-oxidizing Thiomicrospira lineage

“…On the basis of the stoichiometry, the oxygen availability (30 000 nmol) in these incubations would support oxidation of at most 15 000 nmol thiosulfate. In addition to growth stagnation related to oxygen limitation in the T-ASW medium, the higher cell densities in the MJ-T medium may be related to considerably higher carbonate concentrations in the MJ-T medium, which have been shown to stimulate autotrophic CO 2 fixation in Thiomicrospira (Dobrinski et al, 2010). TH-55 also exhibited uptake hydrogenase activity (Supplementary Figure S1).…”

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

“…Cells were cultivated under either DIC or NH 3 limitation, as previous work had demonstrated that DIC limitation induced the expression of a CCM in this organism while NH 3 limitation did not (15,20,22). To cultivate cells under DIC limitation, TASW was prepared with 2 mM NaHCO 3 and the growth chamber was pulsed with O 2 to maintain oxygen tensions at 5 to 20% atmospheric saturation using the BioFlo dO 2 controller.…”

“…RT-PCRs with primers directed to Tcr_0841, encoding carboxysomal carbonic anhydrase, were used to verify that cells were responding to low-DIC conditions (22), and reactions with primers targeting Tcr_0350 (Table S1), encoding citrate synthase, were used as a positive control for RNA integrity, as this gene has been found to be transcribed similarly under low-and high-DIC conditions (20). RT-PCR amplicons were examined after subjecting the reaction products to gel electrophoresis (Tris-borate-EDTA [TBE], 1% agarose) and staining with ethidium bromide (48).…”

“…The T. crunogena genome encodes a periplasmic carbonic anhydrase, which does not appear to play a role in DIC uptake under low-DIC conditions. This carbonic anhydrase is not upregulated by low-DIC conditions, and inhibiting it with acetazolamide does not hinder cells' ability to create high intracellular DIC concentrations (22). Intracellular DIC accumulation by T. crunogena cells is sensitive to CCCP (carbonyl cyanide-m-chlorophenylhydrazone) and DCCD (N,N-dicyclohexylcarbodiimide) and therefore likely driven by either proton potential, ATP, or both (21).…”

Transcriptional Response of the Sulfur Chemolithoautotroph Thiomicrospira crunogena to Dissolved Inorganic Carbon Limitation

“…Elsewhere in the genome, two other RubisCOs are encoded (one form I and one form II), as well as an alpha carbonic anhydrase (␣-CA) and two ␤-CA genes (including csoSCA). However, CsoSCA is the only carbonic anhydrase that appears to play a role in DIC uptake and fixation (9), and no orthologs to the bicarbonate transporters found in cyanobacteria are apparent in the T. crunogena genome (28). The objectives of this study were to determine whether CCM induction in T. crunogena occurs at the level of transcription and to compare the transcriptomes of T. crunogena cultivated under low-and high-DIC conditions to identify the genes whose expression is stimulated by growth under low-DIC conditions as a step in resolving all of the components of this proteobacterial CCM.…”