Macromolecular metabolosome formation associated with 12-propanediol utilisation

Propanediol is produced as a result of the fermentation of several plant sugars and it is found in quite high concentrations in the intestine. A number of enteric bacteria, such as Salmonella enterica, have the ability to utlise propanediol as a carbon source. However, what is remarkable about this metabolism is that these bacteria actually make their own proteinaceous organelle, or microcompartment, to house this metabolic activity. The proteinaceous organelle is called a metabolsome and its formation is associated with about twenty proteins, some of which form the shell of the capsid, some of which are associated with the metabolic enzymes and some of which are associated with transport, scaffolding and regulation. The genes for propanediol utilisation are contiguous and coregulated with the cobalamin biosynthetic genes, indicating that propanediol catabolism is the primary reason for de novo B12 synthesis in many bacteria. In this application we wish to study the form and function of the propanediol metabolosome by initiating research into the organelle at the molecular level. The aim of this application is to investigate the propanediol metabolosome. We plan to study the role played by the twenty or so proteins in the metabolic process, including their function and localisation. Moreover, we will investigate the metabolic advantage of the metabolosome over the production of individual enzymes. Finally, we will explore structural aspects of the metabolosome including the ability to transport elements into and out of the macromolecular complex

Although compartmentalisation is often quoted as one of the major differences between prokaryotes and eukaryotes, it is a little appreciated fact that many bacteria do actually have the ability to form proteinaceous microcompartments. These bacterial microcompartments are associated with a specific metabolic activity. Initially, these 'organelles' were observed in cyanobacteria and were termed carboxysomes. The carboxysome consists of a metabolic enzyme encased within a multiprotein shell, similar though quite distinct to a viral capsid. However, more recently 'carboxysome'-like structures have been observed with the metabolism of both ethanolamine and 1,2-propanediol, which are two separate vitamin B12-dependent processes that allow bacteria to use these substrates as carbon sources. Since these organelles are associated with distinct metabolic activities and in order to differentiate them from the CO2-concentrating carboxysomes, it has been suggested that they be referred to as metabolosomes. We have isolated the twenty three propanediol utilisation genes from Citrobacter freundii and, for the first time, have overproduced the encoded gene products in E. coli, giving rise a to metabolically engineered strain of E. coli that is able to metabolise propanediol. We aim to make a major contribution to the understanding of the physiology and biochemistry of propanediol metabolism, by investigating the metabolic activity of the pdu metabolosome and determining all the metabolic steps that take place within the organelle. We aim to find a function for all the proteins associated with the metabolosome and delineate how the complex is pieced to together. A major objective is to obtain molecular detail on the components of propanediol metabolism and investigate how they interact to generate this remarkable assembly, which can act as a selective barrier.