Thomas E. Hanson and F. Robert Tabita

Department of Microbiology, The Ohio State University

Rm. 376 Biological Sciences, 484 W. 12^th Ave

Columbus, OH 43210

Chlorobium tepidum is a moderately thermophilic green sulfur photosynthetic bacterium that fixes carbon dioxide (CO₂) primarily by the reverse tricarboxylic acid cycle. Similarity searches, however, of preliminary C. tepidum genomic sequence via the NCBI Unfinished Microbial Genomes BLAST server identified a single gene encoding a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) homolog, the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway of CO₂ fixation. The deduced amino acid sequence of the C. tepidum homolog was shown to be most closely related to RubisCO homolog genes found in Archaeoglobus fulgidus and Bacillus subtilis, however several key residues important for catalysis are altered. Molecular phylogenetic analyses indicated these deduced sequences form a group separate from the well described and phylogenetically distinct form I and form II RubisCO proteins and bona fide archaeal RubisCO genes. Sequence analysis indicated that the RubisCO homolog is the second of a group of four open reading frames (ORF’s) in the same transcriptional orientation. A 2.8 kb transcript was found when C. tepidum RNA was probed with an internal fragment of the RubisCO homolog which can accommodate the putative RubisCO gene and the two downstream ORF’s. The RubisCO homolog was cloned and expressed in E. coli and recombinant protein purified. Thus far, we have detected no RubisCO activity in the purified recombinant protein. Only slight, perhaps insignificant, RubisCO activity was found in C. tepidum cell extracts. However, when the RubisCO homolog gene was disrupted in the C. tepidum chromosome there was a reduction in the photoautotrophic growth rate and the level of photosynthetic pigments. Most importantly, the whole cell CO₂ fixation rate was diminished. Physiological characterization of the mutant strain is underway to further elucidate the RubisCO homolog’s role in the physiology of C. tepidum and its potential role in CO₂ fixation.