Dissolved Inorganic Carbon Uptake in \u3ci\u3eThiomicrospira crunogena\u3c/i\u3e XCL–2 is ATP–sensitive and Enhances RubisCO–mediated Carbon Fixation

Many autotrophic organisms living in environments with episodically low dissolved inorganic carbon (DIC) concentrations compensate for these fluctuations by employing a carbon concentrating mechanism (CCM). By utilizing a CCM, these organisms can generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. Carbon concentrating mechanisms have been well-studied in cyanobacteria but studies are lacking in other autotrophs. The gammaproteobacterium Thiomicrospira crunogena XCL-2 is a hydrothermal vent chemolithoautotroph that has a CCM, which is functionally similar to that of cyanobacteria. At hydrothermal vents, DIC concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 µM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low. The CCM in T. crunogena includes -carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 - transporter to generate the elevated intracellular concentrations of DIC previously measured in this organism. In this study, to determine whether RubisCO itself might be adapted to low CO2 concentrations, the affinity (KCO2) for purified carboxysomal RubisCO was measured, and found to be 250 M (SD ± 40) which was much greater than that of whole cells (1.03 µm). This finding suggests that the primary adaptation by T. crunogena to low-DIC conditions has been to enhance DIC uptake, presumably by energy-dependent membrane transport systems that are either ATP- v dependent and/or dependent on membrane potential (). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis and . After separate incubations with the ATP synthase inhibitor N, N’- dicyclohexylcarbodiimide (DCCD) and the protonophore carbonyl cyanide 3- chlorophenylhydrazone (CCCP), intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite an absence of an inhibitory effect on in the DCCD-incubated cells. In some organisms, DCCD inhibits the NADH dehydrogenase (NDH-1) and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 mol/s for NDH-1 and 3.4 mol/s for bc1) and uninhibited treatments (271.7 mol/s for NDH-1 and 3.6 mol/s for bc1). These data support the hypothesis that an ATP-dependent transporter is responsible for HCO3 - transport in T. crunogena; however, the activity of a secondary transporter could not be ruled out. The gene encoding the solute-binding protein (cmpA), of the ATPdependent bicarbonate transporter in Synechococcus elongatus PCC 7942, was used to perform a BLAST query and Tcr_1153 was the closest match in the T. crunogena genome. Examination of the Tcr_1153 gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Bacterial DIC uptake has been very well-studied only in cyanobacteria. Finding the genes that encode the transporter(s) responsible for DIC uptake in T. crunogena will aid in metagenomic studies of environmental samples to help determine how widely distributed these genes are, and will help to characterize CCMs in other cultivated organisms