Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

Expression and function of four carbonic anhydrase homologs in the deep-sea chemolithoautotroph Thiomicrospira crunogena.

The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena grows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO(2) + HCO(3)(-) + CO(3)(-2)). Its genome encodes alpha-carbonic anhydrase (alpha-CA), beta-CA, carboxysomal beta-like CA (CsoSCA), and a protein distantly related to gamma-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 alpha-CA, beta-CA, and CsoSCA but not for the gamma-CA-like protein. alpha-CA and CsoSCA but not beta-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 alpha-CA and beta-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 beta-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, alpha-CA is located in the periplasm. However, inhibition of alpha-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.

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.