The carbon-concentrating mechanism of the hydrothermal vent chemolithoautotroph Thiomicrospira crunogena.

Chemolithoautotrophic bacteria grow in habitats with a variety of dissolved inorganic carbon (DIC) concentrations and are likely to have transport-related adaptations to DIC scarcity. Carbon-concentrating mechanisms (CCMs) are present in many species of cyanobacteria, enabling them to grow in the presence of low concentrations of CO2 by utilizing bicarbonate transporters and CO2 traps to generate high intracellular concentrations of DIC. Similar CCMs may also be present in many other autotrophic bacteria. The sulfur-oxidizing -proteobacterial chemolithoautotroph Thiomicrospira crunogena experiences broad fluctuations in DIC availability at its hydrothermal vent habitat and may use a CCM to facilitate growth during periods of CO2 scarcity. T. crunogena was cultivated in chemostats under DIC limitation to determine whether it has a CCM. Its KDIC for growth was 0.22 mM, with a maximum growth rate of 0.44 h . In short-term incubations with [C]DIC, DIClimited cells had higher affinities for DIC (0.026 mM) than DIC-sufficient cells (0.66 mM). DIC-limited cells demonstrated an ability to use both extracellular CO2 and HCO3 , as assayed by isotopic disequilibrium incubations. These cells also accumulated intracellular DIC to concentrations 100 higher than extracellular, as determined using the silicone oil centrifugation technique. Cells that were not provided with an electron donor did not have elevated intracellular DIC concentrations. The inducible changes in whole-cell affinity for DIC, the ability to use both extracellular CO2 and HCO3 , and the energy-dependent generation of elevated intracellular concentrations of DIC are all consistent with the presence of a CCM in T. crunogena. Some 2.7 billion years ago, this planet was geochemically, ecologically, and biologically distinct from what it is today. Atmospheric carbon dioxide concentrations were 1 to 3 orders of magnitude higher (16, 30). Representatives from all three domains of life (Archaea, Bacteria, and Eukarya) were already present and had begun to diversify (3). Confronted with the precipitous fall of atmospheric and oceanic CO2 concentrations in the late Proterozoic, many autotrophic lineages likely responded with adaptations to maintain an adequate supply of CO2 for growth (27, 35). Carbon-concentrating mechanisms (CCMs) can facilitate rapid autotrophic growth in environments where the CO2 and/or HCO3 concentrations are chronically or episodically low. CCMs are present in many species of cyanobacteria and generate an elevated intracellular concentration of HCO3 by using active HCO3 transport (22, 32) and CO2 traps (33). Carboxysomal carbonic anhydrase (EC 4.2.1.1) converts intracellular HCO3 to CO2, which is fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) (2, 15, 26). The elevated intracellular concentrations of dissolved inorganic carbon (DIC) resulting from active transport expedite carbon fixation by Rubisco by enhancing substrate availability and mitigating the Rubisco oxygenase reaction (15). CCMs have not been rigorously studied for any other prokaryotic autotrophs (e.g., autotrophic Proteobacteria, planktomycetes, green sulfur bacteria, Aquificales, and Archaea). This is surprising, as CCMs are likely to be quite relevant to primary productivity in the diverse habitats where autotrophic microorganisms are found. CCMs with similarities to those present in cyanobacteria may be widespread. For example, the recently sequenced genomes of the photosynthetic -proteobacterium Rhodopseudomonas palustris and the ammonia-oxidizing -proteobacterium Nitrosomonas europaea contain genes for carbonic anhydrase and potential HCO3 transporters (4, 18). Furthermore, it has recently been demonstrated that carboxysomes from the chemolithoautotroph Halothiobacillus neapolitanus contain carbonic anhydrase and are believed to function similarly to those present in cyanobacteria (34). A CCM could facilitate the growth of chemolithoautotrophs at hydrothermal vents, where there is an enormous degree of spatial and temporal variability in the concentration of CO2 (9). The hydrothermal vent -proteobacterium Thiomicrospira crunogena is an obligate sulfur-oxidizing chemoautotroph that was originally isolated from a deep-sea hydrothermal vent habitat where the CO2 concentration oscillates between 20 M and 1 mM, and HCO3 is always the most abundant form of DIC (equal to the sum of CO2, HCO3 , and CO3 2 ) (9, 11). This is one of the fastest-growing chemoautotrophs, with a doubling time as low as 1 h (11). It continues to grow rapidly in batch culture even after drawing the concentration of DIC down to less than 20 M, and it has carboxysomes (31). Both characteristics are consistent with the presence of a CCM. Detailed physiological experiments were undertaken to determine whether this organism has a carbon-concentrating mechanism. Understanding how T. crunogena and other autotrophic microorganisms adapt to low concentrations of DIC during growth is relevant to understanding the physiology of these unique organisms and will provide insights into the response of autotrophic carbon fixation to changes in global geochemistry over Earth history.