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While our long-term goal is to identify environmental reservoirs of Salmonella, our immediate focus is to elucidate the nature of the metabolic processes that should allow this reservoir to persist. Previous results suggested that all Salmonellae share a constellation of functions that support anaerobic B12-dependent growth on ethanolamine and propanediol in the presence of thiosulfate as electron acceptor. Growth on ethanolamine involves a protein-bounded organelle that is thought to contain the needed metabolites, cofactors and enzymes. This organelle is homologous to the carboxysome of photosynthetic cyanobacteria, which are responsible for about 30% of all global CO2 fixation. In neither organism is the role of the organelle understood. <P>We hope that by learning about the compartment in Salmonella, where genetic approaches can be taken, we will gain an understanding of the more general functions of the compartment in both CO2 fixation and maintenance of Salmonella reservoirs.<P> Our immediate objectives are the following:<ol> <LI>Make and characterize mutants of Salmonella that lack one or more (or the five) compartment shell proteins. <LI>Determine the physiological defects in these mutants under a variety of growth conditions. <LI> Isolate mutants in which the compartment is essential for growth on ethanolamine. <LI>Devise ways to fluorescently label the organelle so that mutants can be screened quickly for possession of an intact structure. <LI>Construct subtle mutations in genes for shell proteins that leave the organelle structurally intact but interfere with specific aspects of compartment function (identified above).</ol> Thus far we have found that the compartment is needed for growth at high pH (8.5), conditions under which the volatile intermediate acetaldehyde is most subject to loss as a gas and acetate lost from the cell is difficult to recapture. The labels we have attached to shell proteins thus far have all prevented assembly of the organelle.<P> We are now pursuing a genetic approach of looking for mutant backgrounds in which micro-compartments are essential. This may reveal how this compartment contributes to metabolism. <P>We are also studying mutants in which lack of carboxysomes does not prevent growth at high pH. This has revealed several classes of mutations in NAD synthesis and may suggest reducing conditions inside the cell have a big effect on how acetaldehyde is dealt with. We suspect that under oxidizing conditions it is converted to acetate and excreted. <P>We hope to be able to show the reason for this compartment and how it contributes not only to Salmonella metabolism but to the more global problem of CO2 fixation.
Non-Technical Summary: All isolates of the bacterium Salmonella share ability to make B12 and use it to degrade the 2-carbon compounds, enthanolamine and propanediol under anaerobic conditions using tetrathionate as electron acceptor. This constellation of functions is used in identification of Salmonella, testimony to the importance in the life history of Salmonella. We think it suggests a life in soil environments that we ought to be able to characterize by understanding the nature of the process. The process requires micro-compartments which contain the required enzymes metabolite and cofactors. These compartments are very similar to the carboxysomes of photosynthetic bacteria. Roughly one third of all CO2 fixation on earth is done by photosynthetic bacteria, mostly in marine environments. Much is known about how they do this. It is important to understand this process better since burning of fossil fuels contributes to increasing atmospheric CO2. By fixing CO2, living things harvest solar energy, create fuels, and remove CO2 from the atmosphere that contributes to global warming. Bacteria are likely to contribute to solutions since they can be genetically manipulated and engineered to enhance their performance. One open question is why these bacteria make little compartments that contain the enzymes that capture CO2. We suspect that the compartments are doing something other than concentrating CO2 and that we may be able to learn more about this better in Salmonella because this bacterium makes genetic approaches feasible. Our previous work has suggested that the Salmonella compartments may retain a volatile intermediate acetaldehyde, which otherwise escapes into the air. Several reasons lead us to seek a different purpose for the compartments. The compartments are made of protein, which makes them rather open lattices or cages that seem unlikely to be able to restrict movement of CO2 (or acetaldehyde). Another curiosity is that Salmonella makes compartments even though it does not do photosynthesis. Yet another puzzle is that CO2 corrects the problems of Salmonella mutants lacking little compartments, even though no CO2 is involved in degradation of ethanolamine. Genetic analysis is perhaps the best known way of approaching problems for which one has no hints as to the underlying mechanisms. By isolating random mutants that fail to do a process, one can learn what proteins are involved and can thereby approach the mechanisms. This requires no prior knowledge and the experimental process suggests which proteins are responsible. This can only be done in organisms for which a body of genetic methods and materials have been worked out. Salmonella is one of these. We believe we can figure out the importance of the cages to ethanolamine and photosynthesis and that it will reveal a common feature of the two processes. <P> Approach: The genetic methods to be used are standard ones in this lab and involve transposable genetic elements with drug resistance determinants that can be used to make random insertion mutants that can be screened for those with interesting phenotypes. The inserted resistance element makes it possible to quickly determine the affected gene. Knowing the genes affected should suggest conditions under which the compartments are essential (and therefore a testable functional reason for their existence). We need to identify more robust conditions under which the compartment is essential and would like to have multiple such conditions so that we can screen mutants to identify those that have one function (and thus must have an intact compartment) but lack another (suggesting that their compartments lack some property that can be pursued structurally. Other labs are working on three-dimensional structures of carboxysome proteins and how they fit together in a finished compartment. While their work is progressing well, it does not give any hints as to the function and seems unlike to do so in the future without some in vitro tests. We are hoping to bring the genetic approach up to speed so as to build upon the body of structural information.