| Thiobacillus neapolitanus, an obligate autotrophic bacterium, contains two Forms of ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the key enzyme of the Calvin cycle. Form I is a hexadecamer composed of 8 large (CbbL) and 8 small subunits (CbbS). Form II is comprised of only large subunits (CbbM). Many autotrophic bacteria have been shown to contain one or more cbbR genes encoding a regulatory protein belonging to the Lys-R family of transcriptional activators. CbbR is involved in the regulation of cbb genes by binding upstream of the target genes/operons. The gene for the Form II RuBisCO was isolated from a lambda library of T. neapolitanus genomic DNA, subcloned and sequenced. Sequencing data revealed that a cbbR is located upstream of cbbM and is transcribed in the opposite direction. Binding of CbbR to the cbbR/cbbM intergenic region was determined by gel-shift analysis.; In order to investigate the role of cbbR gene in the regulation of cbbM and cbbL genes of T. neapolitanus, a cbbR knock-out mutant and a cbbR/cbbL knock-out mutant were created. Interruption of cbbR by insertion of a kanamycin resistance gene results in a mutant, which requires an elevated CO2 level for growth. The cbbR/cbbL double knock-out mutant has an elevated CO2 requirement for growth.; Thiobacillus denitrificans ATCC 25259, an obligate chemolithotroph, contains two Forms of RuBisCO. Most Thiobacillus species contain polyhedral inclusion bodies called carboxysomes that enhance CO 2 fixation by RuBisCO and are surrounded by a protein shell. Unlike other thiobacilli, T. denitrificans doesn't possess carboxysomes. Previously, both Form I and Form II genes of T. denitrificans were isolated from a cosmid library. By southern blot analysis a gene for carboxysome shell peptide (csoS1A) was detected on the cosmid clone containing Form II RuBisCO. The Form II clone was subcloned and sequenced. Sequence analysis revealed that, unlike the other Thiobacillus species, the T. denitrificans genes are located upstream of the Form II gene which is located 17 kbp upstream of Form I gene and are transcribed in the same direction. Interestingly, an open reading frame upsteram of csoS2 is found to be similar to LysR type regulatory gene sequences. |
