| It has been established that Thiobacillus neapolitanus fixes CO{dollar}sb2{dollar} using a form I RuBisCO and that much of the enzyme is sequestered into carboxysomes. In the present study, the genes encoding the form I RuBisCO, cbbL and cbbS, were cloned and sequenced. Analysis of RNA showed a transcript of approximately 2000 nucleotides containing both cbbL and cbbS. The insertion of a kanamycin resistance cartridge into cbbL resulted in a polar mutant in which cbbS is no longer transcribed. The mutant is able to fix CO{dollar}sb2,{dollar} but requires a CO{dollar}sb2{dollar} supplement for growth. The expression of form II RuBisCO by the mutant was demonstrated by immunoblot analysis as well as by analysis of RuBisCO activity upon separation of cellular proteins on sucrose gradients. The mutant does not possess carboxysomes; however, Northern analysis showed that some of the shell components of the carboxysome are produced.; The carboxysomes from T. neapolitanus have been purified and partially characterized. When analyzed by SDS-PAGE, 7-8 major peptides are observed, two of which are the large and small subunits of RuBisCO. The remaining peptides are thought to be components of the shell. Three shell genes have been identified by "reverse genetics" and were named csoS for CarboxySOme Shell. The product of the csoS3 gene was identified by expressing the gene in E. coli and raising antibodies against the recombinant protein. When the antibodies were used to probe an immunoblot containing carboxysomes, a 60 kDa protein was recognized. The csoS genes, along with cbbL and cbbS and two unidentified open reading frames, appear to constitute an operon.; Thiobacillus denitrificans expresses both form I and form II RuBisCO, which are encoded by cbbLS and CbbM, respectively. Sequencing upstream of cbbL and cbbM revealed two open reading frames subsequently designated cbbRI and cbbRII. Both cbbRI and cbbRII are transcribed in the opposite direction of cbbL or cbbM, respectively. CbbRI and CbbRII are 40% identical at the amino acid level and share significant homology to members of the LysR family of transcriptional activators. Heterologous expression of cbbRI and cbbRII resulted in insoluble proteins of approximately 31 kDa which is in agreement with their predicted sizes. |
