Protein Content of Polyhedral Organelles Involved in Coenzyme B ₁₂ -Dependent Degradation of 1,2-Propanediol in Salmonella enterica Serovar Typhimurium LT2

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.The vitamin B 12 coenzymes adenosyl-B 12 (Ado-B 12 ) and methyl-B 12 (CH 3 -B 12 ) are required cofactors for at least 15 different enzymes (5, 27, 30). These enzymes have a broad but uneven distribution among living forms and are vital to human health, are essential to the carbon cycle, and have important industrial applications (5, 27, 30). Historically, bacteria have provided excellent model systems for the study of vitamins, and recent investigations with several bacterial systems have found the molecular biology of B 12 -dependent processes to be unexpectedly complex (9,27,29,34,35). One of the most surprising findings in this area has been the identification of a polyhedral organelle involved in coenzyme B 12 -dependent 1,2-propanediol (1,2-PD) degradation by Salmonella enterica (9).Salmonella enterica utilizes 1,2-PD as a carbon and energy source in an Ado-B 12 -dependent fashion (19). Degradation occurs aerobically, or anaerobically if tetrathionate is added as a terminal electron acceptor (26). Based on biochemical studies, a pathway for 1,2-PD degradation has been ...

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase

“…Although the pathway of 1,2-PD degradation appears quite straightforward, the catabolism of this small molecule is in fact a very elaborate process (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003).…”

“…Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). These polyhedra are 100-150 nm across, (Havemann et al, 2002;Havemann & Bobik, 2003;Leal et al, 2003a).…”

“…Included are the pocR and pduF genes (which encode a positive transcriptional regulator and a 1,2-PD diffusion facilitator, respectively) (Bobik et al, 1992;Chen et al, 1995) as well as the adjacent, but divergently transcribed, pdu operon, which includes 21 additional genes (Bobik et al, 1999;Jeter, 1990). Of these 21 genes, six encode enzymes of the 1,2-PD degradative pathway (pduCDEPQW) (Bobik et al, 1997(Bobik et al, , 1999Leal et al, 2003a) and seven are thought to encode structural proteins required for polyhedral body formation (pduABJKNTU) (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). Based on high sequence similarity, the pduGH genes are likely to encode a protein required for reactivation of diol dehydratase following the breakdown of AdoCbl (Bobik et al, 1999).…”

“…Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). These polyhedra are 100-150 nm across, (Havemann et al, 2002;Havemann & Bobik, 2003;Leal et al, 2003a).Adding to the complexity of 1,2-PD degradation is the fact that this process requires systems for the acquisition and maintenance of AdoCbl. S. enterica can obtain AdoCbl by de novo synthesis and by uptake from the environment (Roth et al, 1996).…”

“…Subsequently, propionaldehyde is converted to propanol and propionate by alcohol dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacylase and propionate kinase (Obradors et al, 1988;Toraya et al, 1979). This pathway generates one ATP, an electron sink for the regeneration of NAD and an intermediate (propionyl-CoA) that can serve as a carbon and energy source via the methylcitrate pathway (Horswill & Escalante-Semerena, 1997).Although the pathway of 1,2-PD degradation appears quite straightforward, the catabolism of this small molecule is in fact a very elaborate process (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003).…”