UW-Madison Dept of Bacteriology

Research Description

Our laboratory is interested in bacterial metabolism and physiology. Our group takes a comprehensive approach to our research problems. We do in vivo and in vitro genetics, enzymology, small-molecule biochemistry, metabolic pathway integration, and general physiology. Structural work is performed in collaboration with Ivan Rayment (UW-Madison, Biochemistry Department), and spectroscopy work is performed in collaboration with Thomas Brunold (UW-Madison, Chemistry Department). Much of the work we do is performed in Salmonella enterica because we can do sophisticated in vivo and in vitro genetic analyses of strains. Our work also includes other bacteria (some of them pathogenic) and archaea.

Research areas of interest are:

1. Metabolic Pathway Integration. In 1998, we identified the cobB gene of Salmonella enterica as a new member of the SIR2 family regulatory proteins in eukaryotes whose activities are needed for gene silencing and cell aging. Our report was an important contribution to this field of research and led to the identification of two enzymatic activities associated with these proteins. Knowledge of these activities is essential to better understand the exciting process of cell aging. We also established a link between sirtuins and central metabolism. We showed that the acetyl-CoA synthetase enzyme (Acs, AMP-forming) is under the control of the sirtuin-dependent acylation/deacylation system (SDPADS). The first half of the reaction, the conversion of acetate to acetyladenylate requires residue Lys609 for substrate positioning. Acetylation of Lys609 inactivates Acs. The SDPADS controls the activity of many other proteins in the cell. Recently, we discovered that propionyl-CoA synthetase (PrpE) is controlled by the SDPADS but not by acetylation, but by propionylation. We are using a proteomics approach to identify SDPADS substrates in S. enterica and other prokaryotes of interest to us, including archaea. We are currently studying in detail the function of the protein acetyltyransferase (Pat) enzyme, one of the enzymes of the SDPADS. We are using protein chips to identify putative substrates of Pat and other putative acetyltransferases present in enteric bacteria and the Gram-positive bacterium Bacillus subtilis. We are paying a great deal of attention to CoA homeostasis to help us explain the phenotypes we observe.

2. Metabolic Stress. A second focus of our work centers on the catabolism of poor carbon sources such as propionate and acetate, two short-chain fatty acids that are very abundant in soil and the gut. These compounds are used by many prokaryotes of relevance to the environment and health. Catabolism of these short-chain fatty acids has profound physiological implications for the cell. Both acetate and propionate are toxic to the cell as evidenced by the fact that the breakdown of propionate requires functional editing DNA polymerase, glutathione, and mismatch repair functions. We are investigating why these functions are need during growth on propionate. We would like to know why is propionate such a metabolic risk to the cell. Propionate catabolism is also a great model system to investigate strategies used by the cell to integrate its metabolic pathways.

3. Complex Metabolic Pathway Analysis. We are studying multiple aspects of adenosylcobalamin (AdoCbl, coenzyme B12) biosynthesis. This coenzyme is the largest non-polymeric molecule with biological activity. At least 24 genes of the Salmonella enterica genome are dedicated to the synthesis of coenzyme B12. Twenty of the twenty-four known cobalamin biosynthetic (cob) genes comprise a 17-kb superoperon. In Salmonella enterica, de novo synthesis of AdoCbl occurs only under anoxic growth conditions. This fact raises very interesting questions regarding gene regulation and oxygen lability of intermediates/enzymes. Transcriptional regulation of this superoperon is complex and requires the interplay of local as well as global regulatory proteins. We have discovered new enzymes and pathways in archaea for the assimilation of iincomplete corrinoids from the environment.