Journal of General Microbiology (1991), 137, 1491-1496. Printed in Great Britain 1491 Kinetic properties of ribulose bisphosphate carboxylase/oxygenase from Thiobacillus thyasiris, the putative symbiont of Thyasirajlexuosa (Montagu), a bivalve mussel C. M. COOK,'* T. LANARAS,' A. P. WOOD,^ G. A. CODD~ and D. P. KELLY~ Department of Botany, University of Thessaloniki, P.O. Box 109, GR-540 06 Thessaloniki, Greece Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 3Department of Biological Sciences, University of Dundee, Dundee, D D l 4HN, UK (Received 2 November 1990; revised 5 March 1991 ; accepted 26 March 1991) Some kinetic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenasefrom Thiobaciffus thyasiris, a marine, facultatively heterotrophic, sulphur-oxidizingbacterium and putative symbiont of Thyasira fixuosa (Montagu), a bivalve mussel, have been determined. The kinetic parameters for the COz/Mg2+-activated enzyme were: K,,,(RuBP) 24.3 pM, K,,,(CO,) 125.5 pM, Km(02) 900 pM and Km(Mg2+) 1.53 mM. The low C o t affinity suggeststhat T. thyusiris may possess a Cot-concentrating mechanism. RuBP oxygenase activity was inhibited by increasing C o t concentration. Divalent metal ions were essential for RuBP carboxylase activity; activity of the Mg2+-free enzyme could be restored by the addition of Mg2+, Mn2+ or Cat+. The pH optimum was 7.8. The temperature optimum for RuBP carboxylase activity was 55 "C, although the enzyme rapidly lost activity at this temperature. An Arrhenius plot was biphasic, with a break at 40 "C. The activation energies were 55.5 x lo3 J mol-I and 32-9 x lo3 J mol-I over the temperature ranges 10-40 "C and 40-55 "C, respectively. Qlo was 2.12 for any 10 "C increment between 1 0 4 0 "C, and 1.47 between 40-55 "C. RuBP carboxylase activity was stable at 35 "C, the optimum growth temperature of T. fhyasiris and at 7.5 "C, the temperature of the habitat of TiryaSira ~~XUOSU, but the activity was 40% and 3.5%, respectively, of the potential activity at 55 "C. RuBP carboxylase activity was stimulated by NaCl concentrations of up to 0.3 M, with a maximum (33 %), occurring between 0.1 and 0.2 M-Naa. At higher concentrations of NaCl ( > 0.3 M) RuBP carboxylase activity was inhibited. Introduction Symbioses between marine invertebrates and endosym- biotic chemoautotrophic sulphur bacteria are now well documented in several phyla of worms and in five families of bivalve molluscs, found in a variety of habitats where reduced chemical species are present (Cavanaugh, 1985; Southward, 1987). In members of the Pogonophora, gutless marine worms, the symbionts are internally localized in the trophosome, a highly vascular- ised organ, where they are supplied with oxygen and other metabolites (Southward & Southward, 1988). In most molluscs, with the exception of the thyasirids, the bacteria are located in the epidermal cells of the gills (Southward, 1987). In thyasirids and some oligochaetous annelids, the bacteria are located under the cuticle of the epidermal cells (Southward, 1986). Epidermally located I Abbreviations : RuBisCO, ribulose 1,5-bisphosphate carboxylase/ oxygenase; RuBP, ribulose 1,5-bisphosphate. bacteria are in close proximity to the external environ- ment and the necessary metabolites C02, O2 and sulphide. It has been estimated that chemoautotrophic sym- bionts may contribute from 50400% of the host carbon, either through the provision of extracellular products or by the digestion of the symbionts themselves (South- ward, 1986, 1987). However, further investigation of the nutritional basis of the symbioses requires the identifica- tion and characterization of the symbionts. Their isolation in pure culture has proved difficult; however, the putative symbiont from ThyasiraJIexuosa (Montagu), a thyasirid bivalve mussel from a low-sulphide environ- ment, has recently been isolated and characterized (Wood & Kelly, 1989). The symbiont, Thiobacillus thyasiris, is a novel, marine, facultatively heterotrophic Thiobacillus unlike any previously described. Auto- trophicallygrown culturescontained ri bulose 1,5-bisp hos- phate carboxylase/oxygenase(RuBisCO; EC 4.1.1.39) and excreted up to 20% of their fixed carbon into the 0001-6613 0 1991 SGM