Influence of form IA RubisCO and environmental dissolved inorganic carbon on the δ¹³C of the clam‐chemoautotroph symbiosis Solemya velum

Abstract

Many nutritive symbioses between chemoautotrophic bacteria and invertebrates, such as Solemya velum, have delta(13)C values of approximately -30 to -35%, considerably more depleted than phytoplankton. Most of the chemoautotrophic symbionts fix carbon with a form IA ribulose 1,5-bisphosphate carboxylase (RubisCO). We hypothesized that this form of RubisCO discriminates against (13)CO(2) to a greater extent than other forms. Solemya velum symbiont RubisCO was cloned and expressed in Escherichia coli, purified and characterized. Enzyme from this recombinant system fixed carbon most rapidly at pH 7.5 and 20-25 degrees C. Surprisingly, this RubisCO had an epsilon-value (proportional to the degree to which the enzyme discriminates against (13)CO(2)) of 24.4 per thousand, similar to form IB RubisCOs, and higher than form II RubisCOs. Samples of interstitial water from S. velum's habitat were collected to determine whether the dissolved inorganic carbon (DIC) could contribute to the negative delta(13)C values. Solemya velum habitat DIC was present at high concentrations (up to approximately 5 mM) and isotopically depleted, with delta(13)C values as low as approximately -6%. Thus environmental DIC, coupled with a high degree of isotopic fractionation by symbiont RubisCO likely contribute to the isotopically depleted delta(13)C values of S. velum biomass, highlighting the necessity of considering factors at all levels (from environmental to enzymatic) in interpreting stable isotope ratios.

CO 2 Uptake and Fixation by Endosymbiotic Chemoautotrophs from the Bivalve Solemya velum

“…The apparent K CO2 for S. velum symbionts (28 M) is lower than CO 2 concentrations measured from sediments where S. velum was collected (up to 200 M [39]). Provided boundary layers and other diffusive distances between the symbionts and their environmental source of CO 2 are small enough to prevent the intracellular CO 2 concentration from being substantially lower than the sediment CO 2 concentration, the rate of symbiont carbon fixation should not be limited by CO 2 availability.…”

“…A carbon flux model, derived to estimate the sizes of the CO 2 concentration gradients between the environment and the symbiont cytosol for R. pachyptila tubeworms (35) 2(s) ] is the concentration of CO 2 within the symbiont cells, ␣ R is the kinetic isotope effect for carbon fixation by S. velum symbiont RubisCO, and ␣ D is the kinetic isotope effect for CO 2 diffusion. Using published values for the isotope ratios of S. velum biomass (0.01088 to 0.01085 [10]) and environmental CO 2 (0.01113 to 0.01108 [39]), as well as for the kinetic isotope effects for S. velum symbiont RubisCO (1.0245 [39]) and diffusion (1.0007 [32]), the calculated symbiont intracellular CO 2 concentration is at least 70% of that present in the bivalve's environment. CO 2 gradients between the environment and the symbiont cells are likely to be mitigated by rapid flushing of the mantle cavity and gill carbonic anhydrase.…”

“…This may reflect differences in the ribulose 1,5-bisphosphate (RuBP) supply. The RubisCO assays (39) were conducted in the presence of a large excess of RuBP (0.4 mM). For the carbon fixation assays reported here, the symbionts were generating RuBP via the Calvin-Benson cycle.…”

Kinetic isotope effect and biochemical characterization of form IA RubisCO from the marine cyanobacterium Prochlorococcus marinus MIT9313

“…RubisCO characterization-The responses of enzyme activity to pH and temperature were determined radiometrically by an isotopic disequilibrium technique (Scott et al 2004b). Preliminary experiments indicated that the K CO2 values for these enzymes were quite large, requiring incubations in the presence of very high concentrations of CO 2 .…”

“…Subsequent work with high precision methods has indicated that form I enzymes discriminate against 13 C to a greater extent than form II. Form IA RubisCO from the gamma proteobacterial chemolithoautotrophic endosymbionts of the protobranch bivalve Solemya velum has an e value of 24.5% (Scott et al 2004b), whereas form IB RubisCOs from spinach and from the freshwater cyanobacterium Synechococcus PCC6301 have e values of 29% and 22%, respectively (Roeske and O'Leary 1984;Guy et al 1993;McNevin et al 2006). Fractionation by form II RubisCOs from the photosynthetic alpha proteobacterium Rhodospirillum rubrum and from the symbionts of the hydrothermal vent tubeworm Riftia pachyptila is less than that demonstrated by the form I RubisCOs.…”