Protein Content of Polyhedral Organelles Involved in Coenzyme B12-Dependent Degradation of 1,2-Propanediol in Salmonella enterica Serovar Typhimurium LT2

The vitamin B12 coenzymes adenosyl-B12 (Ado-B12) and methyl-B12 (CH3-B12) 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 B12-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 B12-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-B12-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 proposed (24, 37). Breakdown initiates with the conversion of 1,2-PD to propionaldehyde by Ado-B12-dependent diol dehydratase (1). The propionaldehyde is then reduced to propanol or oxidized to propionic acid via propionyl coenzyme A (propionyl-CoA) and propionyl-phosphate. Reduction of propionaldehyde serves to regenerate NAD from NADH, while its oxidation provides a source of ATP and cell carbon. Because the pathway of 1,2-PD degradation appeared relatively straightforward, it was somewhat surprising when DNA sequence analyses indicated that the 1,2-PD utilization (pdu) locus included 23 genes (9). Of these, six pdu genes are thought to encode enzymes needed for the 1,2-PD degradative pathway (pduCDEPQW); two are involved in transport and regulation (pduF and pocR); two are probably used for diol dehydratase reactivation (pduGH); one is needed for the conversion of CN-B12 to Ado-B12 (pduO); five are of unknown function (pduLMSVX); and seven (pduABJKNTU) share similarity to genes needed for the formation of carboxysomes, polyhedral organelles involved in autotrophic CO2 fixation (8-10, 13, 15, 20, 31, 32). The finding that the pdu locus included several homologues of carboxysome genes led to recent studies which showed that S. enterica forms polyhedral organelles during Ado-B12-dependent growth on 1,2-PD (9). Like carboxysomes, the pdu organelles are 100 to 150 nm in diameter and are composed of a proteinaceous interior surrounded by a 3- to 4-nm protein shell (9, 17). However, carboxysomes and the S. enterica organelles differ in a number of ways. Carboxysomes function to enhance autotrophic growth at low CO2 concentrations (2, 21, 25, 33), and the carboxysomes of Halothiobacillus neapolitanus (which are the best studied) consist of a protein shell composed of at least six different proteins which encases most of the cell's ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) (3, 4, 14, 16, 18). In contrast, the organelles of S. enterica do not contain RuBisCO but instead consist of Ado-B12-dependent diol dehydratase and a protein shell composed of the PduA protein as well as other unidentified proteins (9, 17). It has been proposed that the S. enterica organelles function to minimize aldehyde toxicity by moderating propionaldehyde production through control of Ado-B12 availability (17). However, this function has not been established, and a great deal remains to be learned about their structure. Here we report the purification and structural characterization of the unusual organelles involved in 1,2-PD degradation by S. enterica. The analyses performed include one and two-dimensional electrophoresis, immunoblotting, N-terminal sequencing, and protein mass fingerprinting via matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). By these methods, 15 proteins of the organelles were identified. These included Ado-B12-dependent diol dehydratase (PduCDE), CoA-dependent propionaldehyde dehydrogenase (PduP), adenosyltransferase (PduO), the large (PduG) and small (PduH) subunits of the putative diol dehydratase-reactivating factor, the PduA shell protein and six additional probable structural proteins (pduBB′JKTU), and one unidentified protein. Densitometry results indicated that of the seven possible structural proteins, PduABB′J are the more abundant structural proteins while PduKTU appear to be more minor structural elements. These findings are consistent with a role for the pdu organelles in aldehyde detoxification and also show that these organelles represent a complex mode of subcellular organization.