Biochemistry and molecular biology of rubisco expression and stable carbon isotope fractionation in chemoautotrophic symbioses

The discovery of deep-sea hydrothermal vents and associated biota precipitated the discovery of a new form of symbiotic relationship between sulfur-oxidizing chemoautotrophic bacteria and marine invertebrates. The symbionts reduce dissolved inorganic carbon (DIC) via the Calvin-Benson cycle and oxidize sulfur compounds as an energy source, providing the host animal with a source of autotrophically fixed carbon. Evidence of carbon sources utilized by these organisms has been provided by stable carbon isotope analyses, which have shown that essentially all organic carbon at the vents is of local origin. Two classes of symbioses exist based on their {dollar}deltasp{lcub}13{rcub}{dollar}C values, those with depleted values of ca. {dollar}-{dollar}30{dollar}perthous{dollar} and those with enriched values of ca. {dollar}-{dollar}11{dollar}perthous.{dollar} The depleted group resembles free-living chemoautotrophic bacteria while the enriched group has escaped satisfactory explanation. It is proposed that the difference in {dollar}deltasp{lcub}13{rcub}{dollar}C values between the two groups is due to fixation of CO{dollar}sb2{dollar} by different forms of the enzyme ribulose-1,5-bisphosphate caroxylase/oxygenase (RubisCO), called form I and form II, which catalyze the same reaction but discriminate against the isotope {dollar}sp{lcub}13{rcub}{dollar}C to different degrees.; A survey of RubisCO expression has been conducted, with form I found in the great majority of depleted species and the form II in all enriched symbioses. The hydrothermal vent tubeworm Riftia pachyptila, which has an enriched {dollar}deltasp{lcub}13{rcub}{dollar}C value and expresses a form II RubisCO, has been used as a model for further studies. The gene encoding RubisCO has been cloned, characterized at a nucleic acid level, and expressed in Escherichia coli. The native protein was purified from the bacterial symbionts and its kinetic isotope effect was measured by high precision methods. The obtained fractionation factor of 18.5{dollar}perthous{dollar} is consistent with the enriched isotopic values of the symbiosis. These studies provide important new information about the role of form I and form II RubisCO in determining an organism's {dollar}deltasp{lcub}13{rcub}{dollar}C value, and provide a biochemical mechanism for the differences in {dollar}deltasp{lcub}13{rcub}{dollar}C values measured for chemoautotrophic symbioses.