Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Microbiology
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. 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 where it 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. Although Salmonella does not fix CO2, its mutants defective in making compartments have growth defects that are corrected by CO2, just as were those in photosynthetic bacteria. 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.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
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. 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. In the past period we have made and characterized mutants of Salmonella that lack one or more (or the five) compartment shell proteins. Isolate mutants in which use of Eut enzyme are needed for other pathways and show that the compartment does in fact retain enzymes and intermediates in the ethanolamine pathway. These tests provide evidence that the compartment restricts diffusion of acetaldehyde. We are now pursuing a genetic approach to elucidate how the compartment might enhance ability of Salmonella to grow on ethanolamine. The compartement is not essential for growth on ethanolamine under standard lab conditions which employ 40mM ethanolamine. We suspect that this is a far higher concentration than is normally available in the gut, but the high concentration is needed in the lab because the growth yield on ethanolamine is very small. Our collaborator, Michael Savageau has modeled that pathway and shown that in principle the compartment should accelerate growth on low ethanolamine. We will test this model by serial passage and perhaps chemostat growth on ethanolamine and we will measure the kinetic parameters (Km, Vmax) of the ethanolamine degradative enzymes thought to operate within the compartment. We will also pursue the idea that acetaldehyde exists in solution as hydride clathrates that increase the effective size of acetaldehyde molecules and allows the compartment to restrict diffusion. Previous chemistry has demonstrated that these clathrates can form under some conditions.
Project Methods
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. 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.