Arch Microbiol (1991) 156:338- 343 030289339100141J Archwes of Hicrnbiology 9 Springer-Verlag 1991 Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasiraflexuosa (Montagu) Tom Lanaras J, Catherine M. Cook 1, Ann P. Wood 2, Don P. Kelly 2, and Geoffrey A. Codd 3 1 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 3 Department of Biological Sciences,University of Dundee, Dundee, DD1 4HN, UK ReceivedJune 14, 1991/AcceptedJuly 5, 1991 Abstract. The bacterial symbionts of many marine invert- ebrates contain ribulose 1,5-bisphosphate (RuBP) car- boxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50- 90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small sub- units, with molecular weights of 53,110 and 11,100 respec- tively. Particulate RuBP carboxylase activity was associ- ated with polyhedral bodies 50-100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were in- ferred to be carboxysomes. Native electrophoresis of iso- lated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl- sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 2I % of the total carboxysomal protein. The most abun- dant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function. Key words: Thiobacillus thyasiris - Bivalve symbiont - Ribulose 1,5-bisphosphate carboxylase/oxygenase - Polyhedral bodies - Carboxysomes - Protein compo- sition -- Facultative chemolithoautotroph Offprint requests to: T. Lanaras Abbreviations: FPLC, fast performanceliquid chromatography; IB, isolation buffer; PAGE, polyacrylamidegel electrophoresis; RuBP carboxylase, ribulose 1,5-bisphosphate carboxylase/oxygenase; SDS, sodium dodecyl-sulphate Symbioses between many marine invertebrates and chemoautotrophic sulphur bacteria are well documented (Cavanaugh 1985; Southward 1987) and the chemoautotrophic symbionts may contribute from 50% to 100% of the host carbon, depending on species and circumstances (Southward 1987). Thyasira flexuosa (Montagu) a marine mollusc and inhabitant of low-sul- phide environments is partially provided with carbon by autotrophic symbiotic sulphur bacteria, either by the excretion of extracellular products, or by the endocytosis and digestion of bacterial cells (Dando and Southward 1986; Southward 1986). The putative symbiont of Thyasira flexuosa was isolated from the gill tissue and named Thiobacillus thyasiris (Wood and Kelly 1989). When grown chemoautotrophically on thiosulphate it contained ribulose 1,5-bisphosphate (RuBP) carboxy- lase/oxygenase (EC 4A.1.39), a primary CO2-fixing en- zyme of autotrophic organisms, and excreted up to 20% of the fixed carbon into the medium. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of RuBP carboxylase. Some kinetic properties of T. thyasiris RuBP carboxylase have been determined (Cook et al. 1991). RuBP carboxylases from autotrophic prokaryotes, with a few exceptions among the photosynthetic purple non-sulphur bacteria, are of high molecular weight (500,000-600,000) and consist of large (mol. wt. 50,000- 56,000) and small (tool. wt. 11,000-15,000) sub- units with an LsSs quaternary structure (Codd 1988). In many autotrophic prokaryotes, including several mem- bers of the thiobacilli, RuBP carboxylase is localised in the cell in polyhedral bodies known as carboxysomes (Shively 1974; Allen 1984; Katayama-Fujimura 1984; Codd 1988). However, in the symbiotic chemoauto- trophic bacteria polyhedral bodies appear to be absent in thin cell sections examined by electron microscopy and immuno-electron microscopy (Southward 1987; Cavanaugh et al. 1988). On a microscopical level the polyhedral bodies identified in the Lucinoma annulata symbiont (Vetter 1985) have been questioned to be poly- phosphate granules (Southward 1987), while polyhedral