Shively 9513481 During carbon limitation, many bacteria are able to enhance CO2 fixation by sequestering ribulose bisphosphate carboxylase/oxygenase (RuBisCO) into carboxysomes. This research addresses the mechanisms by which the carboxvsome aids C02 fixation. The research is divided into three areas. The first area involves the isolation and analysis of genes encoding s1 three additional carboxysome peptides. Presumably, these genes will be located in the putative carboxysome operon of Thiobacillus neapolitanus and/or Thiobacillus intermedius. The putative operon of T. neapolitanus has been totally sequenced (9.75 kbp). Already identified genes include four shell genes and the genes for the large and small subunits of RuBisCO. Three other open reading frames are present in the operon, but are yet to be identified. Also, some peptides appear to be coded for by over-lapping genes. The additional new genes will be isolated by the technique of reverse genetics, sequenced, and expressed in Escherichia coli. Their identity will be confirmed by comparing the amino acid and DNA sequences. Insertion mutants of the new genes will be created and their phenotypes (growth, cell structure, RuBisCO activity, proteins, transcription, etc.) carefully analyzed in comparison to the wild type. The second area will be the assay for the presence of carbonic anhydrase and the determination of the specificity factor for RuBisCO in highly purified carboxysomes. Carbonic anhydrase has been hypothesized to be the component in the carboxysome which is responsible for the enhancement of CO2 fixation by RuBisCO, however all of the assays thus far have been accomplished with grossly contaminated cyanobacterial carboxyxomes. Highly purified carboxysomes of T. neapolitanus will be analyzed by membrane inlet-mass spectrometry for carbonic anhydrase. The specificity factor is a measure of the ability of RuBisCO to discriminate between CO2 and O2 It is hypothesized that the carboxysome via some unknown mechanism is able to i ncrease the specificity factor. The factor will be determined for purified RuBisCO and for the RuBisCO in the purified carboxysome by simultaneous assay of both carboxylase and oxygenase activities according to standard procedures. The third area will be to begin the examination of what role, if any, cbbR, a transcriptional regulatory gene of the LysR family, might play in the expression of the putative carboxysome operon. CbbR, a positive transcriptional regulator has been shown to play a significant role in the expression of Calvin cycle operons in a variety of bacteria. The organization of the cbbL and cbbS of RuBisCO with carboxysome genes and the coordinated transcriptional regulation of RuBisCO and the carboxysome in T. neapolitanus and T. intennedius suggest that they might be controlled by the same regulatory protein, perhaps CbbR. A cbbR gene has been detected in both of these thiobacilli by heterologous hybridization using a cbbR from Thiobacillus denitrificans as the probe. The gene will be isolated, sequenced, expressed, mutagenized, and the mutants analyzed phenotypically as accomplished with the carboxysome genes. It is anticipated that the information gained from this project will contribute substantially to our knowledge of the carboxysome, the overall process of bacterial CO2 fixation, and how bacteria can adapt to changing environmental conditions. ***
