AMPLIFICATION OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT (RUBISCO LSU) GENE FRAGMENTS FROM THIOBACILLUS-FERROOXIDANS AND A MODERATE THERMOPHILE USING POLYMERASE CHAIN-REACTION

Southern blot analysis of DNA from an iron-oxidising moderate thermoph ile NMW-6 and from Thiobacillus ferrooxidans strain TFI-35 demonstrate d sequences homologous to the RuBisCO LSU gene of Synechococcus. DNA f ragments (457 bp) encoding part of the RuBisCO LSU gene (amino acids 7 3-200) were amplified from the genomic DNA of Thiobacillus ferrooxidan s and the moderate thermophile NMW-6 using the polymerase chain reacti on (PCR) technique (Saiki et al. (1985) Science 233, 1350-1354). A com parison with the LSU sequences from T ferrooxidans, Alcaligenes eutrop hus, Chromatium vinosum, Synechococcus and Spinacea oleracea, which al l have RuBisCOs with a hexadecameric structure, showed that the RuBisC O LSU gene sequence from NMW-6 appeared to be most closely related to that of the hydrogen bacterium A. eutrophus which showed 71.9% homolog y at the amino acid level. Despite its physiological similarity, T fer rooxidans showed only 64.1% homology to the amino acid sequence from N MW-6 and had the lowest DNA homology (60.9%) of the hexadecameric type RuBisCOs. In the region sequenced, T ferrooxidans and the RuBisCOs of the phototrophs C vinosum, Synechococcus and S. oleracea, had 17 resi dues that were completely conserved which were substituted in both NMW -6 and A. eutrophus, 11 of these being identical substitutions. Compar ison of the nucleotide and derived amino acid sequences of the RuBisCO LSU fragment from T ferrooxidans with other RuBisCO sequences indicat ed a closer relationship to the hexadecameric type LSU genes of photos ynthetic origin than to that of A. eutrophus. The T ferrooxidans amino acid sequence showed 93.8%, 78.9% and 77.3% homology, respectively, t o the C vinosum, Synechococcus and S. oleracea (spinach) sequences but only 56.2% to A. eutrophus. The DNA sequence from Rhodospirillum rubr um, which has the atypical large subunit dimer RuBisCO structure with no small subunit, showed 39.2% and 42.7% homology, respectively, with the sequences of NMW-6 and T ferrooxidans, and 25.0% and 29.7% amino a cid homology, indicating that the DNA homology was substantially rando m in nature. PCR fragments (126 bp) that overlaped the last 15 codons of the fragments above were also amplified and sequenced. They showed incomplete homology with the larger fragments, supporting evidence obt ained from Southern hybridizations that T ferrooxidans and the moderat e thermophile NMW-6 have multiple copies of RuBisCO LSU genes.