Polyhedral organelles involved in the B(12)-dependent metabolism of 1,2-propanediol in Salmonella enterica Serovar Typhimurium LT2.

摘要

In an effort to better understand salmonella physiology and coenzyme B₁₂-dependent processes, Salmonella enterica has been used as a model to study coenzyme B₁₂-dependent degradation of 1,2-propanediol. One of the most surprising findings was the observation of polyhedral organelles in S. enterica cells grown on propanediol. Although these organelles appeared similar to carboxysomes, which are involved in CO₂-fixation in autotrophic bacteria, a similar function in S. enterica was unlikely since this organism is not an autotroph and does not fix CO₂. This dissertation investigates the structure and function of these unusual bacterial organelles.; The conditions required for organelle formation were defined, and a time course of organelle formation revealed that their synthesis correlated to a metabolic shift to growth on propanediol. In addition, two genes, pduA and pduB, were shown to be involved in organelle formation. To further investigate the role of the PduA protein in polyhedral organelle formation, the pduA gene was cloned and overexpressed. The protein product was then used to generate polyclonal antiserum. Immunolabeling with this antiserum demonstrated that the PduA protein localized to the organelle periphery, suggesting it was a component of the shell. Additional studies demonstrated that S. enterica pduA null mutants did not make polyhedral organelles, and when grown on propanediol minimal medium they exhibited a period of arrested growth. Subsequent physiological tests suggested that the organelles might function as a B₁₂ barrier, and the arrested growth observed in organelle mutants may result from increased production of a toxic intermediate.; In order to determine the constituent proteins of the polyhedral organelles, a purification scheme was developed to obtain stable, homogenous preparations. After purification, preparations were separated using one and two-dimensional gel electrophoresis. The major proteins of the organelle were identified using peptide mass fingerprinting, N-terminal sequencing, and immunoblotting. A total of 14 proteins was identified, including four enzymes: coenzyme B ₁₂-dependent-diol dehydratase, CoA-dependent propionaldehyde dehydrogenase, adenosyltransferase, and a putative two-component diol dehydratase-reactivating factor. Based on these findings, a model is proposed wherein the polyhedral organelles serve to prevent cellular toxicity by channeling and sequestering propionaldehyde as well as by moderating the rate of its production.