The Escherichia coli YfiD protein: an oxygen and acidity responsive regulator of carbon flux

Bacteria are the most biochemically versatile organisms on the planet. They adapt by altering patterns of gene expression in response to environmental and metabolic cues. The bacterium Escherichia coli is one of the best-characterised life forms. It is a model organism and much of our understanding of the fundamental processes of life has been revealed by intensive study of this bacterium. Yet there is still much to learn. For example, we do not know the function of approximately one third of all E. coli genes. This project aims to better understand the central metabolic processes of bacteria by using molecular biology and biochemistry to investigate the function of a protein (YfiD) that is important in adaptation to a number of different environmental conditions. Specifically, we would like to determine the function and extent of the influence of YfiD on bacterial central metabolism, how this contributes to the ability of the bacterium to adapt to different environments, and the molecular mechanism by which YfiD exerts its influence. A deeper understanding of how bacteria adapt and survive in a range of environmental conditions, some of which relate directly to pathogenesis, will help to underpin the search for new therapeutics. In addition, the project findings will be of interest to biotechnologists using E. coli as a factory for the production of recombinant proteins and chemicals by improving the efficiency of such processes.

Escherichia coli is able to adopt one of three basic metabolic modes in response to the availability of electron donors and acceptors. The fate of the key metabolite pyruvate is crucial for this adaptation. Under aerobic conditions pyruvate is oxidised the pyruvate dehydrogenase complex, whereas under anaerobic conditions pyruvate is metabolised non-oxidatively by pyruvate formate-lyase (PFL). When the active, glycyl radical containing, form of PFL is exposed to O2 it is cleaved in the vicinity of the free radical and is inactivated. The YfiD protein is similar to the glycyl radical containing region of PFL, and acts to repair O2-damaged PFL. Under microaerobic conditions, in acidic environments, and during phagocytosis by neutrophils, expression of the yfiD gene and synthesis of the YfiD protein is greatly enhanced, such that YfiD is one of the most abundant cellular proteins. Previously we have investigated the regulation of yfiD expression, demonstrated that YfiD is a glycyl radical protein, that the radical is introduced by the PFL activase, and that yfiD mutant has altered patterns of over-metabolite production in glucose-limited chemostat cultures. These observations led to the hypothesis that YfiD is an important component of bacterial adaptation to changes in O2 availability and acidity. The proposal considers the role of YfiD in the context of the whole organism by addressing the following questions: 1.What is the breadth of YfiD influence over central metabolism? 2.What is the role of YfiD in bacterial adaptation to changes in oxygen availability and pH? 3.What are the molecular mechanisms by which YfiD exerts its effects on metabolism? Thus, will obtain new data sets under strictly defined conditions that will allow integration of phenotypic, transcriptomic, proteomic, metabolomic, structural and biochemical data to determine how YfiD contributes to E.coli adaptation in response to two important environmental parameters, i.e. O2 availability and pH.