Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2 - PubMed (original) (raw)

Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2

Gregory D Havemann et al. J Bacteriol. 2003 Sep.

Abstract

Salmonella enterica forms polyhedral organelles during coenzyme B(12)-dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B(12)-dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B(12)-dependent 1,2-PD degradation by S. enterica were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B(12)-dependent diol dehydratase. Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB'CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB'JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO(2) fixation), suggesting that the S. enterica organelles and carboxysomes have a related multiprotein shell. In addition, S. enterica organelles contained four enzymes: B(12)-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the S. enterica organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the S. enterica organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.

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Figures

FIG. 1.

Electron micrographs of polyhedral organelles purified from S. enterica. (A) Magnification, ×53,000. (B) Magnification, ×122,000. The marker bars are 100 nm. Prior to electron microscopy, organelles were fixed with Trump's reagent and then negatively stained with uranyl-acetate.

FIG. 2.

Analysis of the organelle purification protocol by SDS-PAGE. Lane 1, molecular mass markers; lane 2, crude lysate; lane 3, detergent-treated crude lysate; lane 4, supernatant from centrifugation at 12,000 × g; lane 5, pellet from centrifugation at 48,000 × g; lane 6, clarified supernatant from centrifugation at 12,000 × g; lane 7, density gradient-purified polyhedral organelles. Molecular masses in kilodaltons are shown at the left. Lanes 2 to 7 each contained 20 μg of protein.

FIG. 3.

Western analysis of purified polyhedral organelles. Lane 1, molecular mass markers; lanes 2 to 7 each contained 20 μg of purified polyhedral organelles. Lane 2 was stained with Coomassie brilliant blue and lanes 3 to 7 were probed with the following antisera: lane 3, anti-PduA; lane 4, anti-PduAJ; lane 5, anti-diol dehydratase; lane 6, anti-PduO; lane 7, anti-PduP.

FIG. 4.

Two-dimensional electrophoresis of purified polyhedral organelles of S. enterica. A purified polyhedral organelle preparation (115 μg of protein) was subjected to 2D-IEF-SDS-PAGE followed by colloidal blue staining. Molecular mass is indicated to the left and IEF pH at the top of the gel.

FIG. 5.

A model for the role of the pdu organelles in 1,2-PD degradation. The dashed line indicates the multiprotein shell of the organelle. The enzymes of the organelle are indicated graphically and in the legend. The pathway of 1,2-PD degradation is also shown. The proposed function of the organelles is to protect cytoplasmic components from the cytotoxic effects of propionaldehyde.

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Protein content of polyhedral organelles involved in coenzyme B12-dependent degradation of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2 - PubMed (original) (raw)