Carbon is an essential element for all known forms of life. Nearly all important cellular constituents contain carbon compounds and in many organisms these carbon-containing biomolecules are simultaneously used to obtain energy for growth. Carbon-containing biomolecules are converted into smaller molecules (precursor metabolites) that are key components for the major chemical reactions (metabolism) of living organisms. These precursor metabolites can then serve either as the building blocks for other cellular constituents or can be broken down in chemical reactions that generate energy. These different uses of carbon-containing biomolecules occur regardless of the carbon sources that are used by microbes for their growth and energy requirements. Consequently, it is essential to understand the processes by which the chemical reactions of precursor metabolites are controlled so that carbon can flow towards the molecules that will be used to build cells. This knowledge will enable the more efficient use of microorganisms to generate useful products, including those (such as biofuels) of biotechnological importance. This project will generate fundamental new information on how carbon is used in living systems, and through classroom and research experiences will provide interdisciplinary training to the next generation of scientists, including undergraduate and graduate students, in microbial physiology and genetics.
For every carbon substrate, the levels of the precursor metabolites pyruvate (C3), phosphoenolpyruvate (C3), and oxaloacetate (C4) have to be controlled: this is referred to as the C4/C3 node. Importantly, the pathways leading to these metabolites, as well as their fate, differ depending on where a given substrate has entered central carbon metabolism. In this project the regulation and activity of enzymes involved in the partition of carbon at the C4/C3 node will be examined and the question of why several enzymes apparently catalyzing the same reaction are active at the same time will be addressed. Rhodobacter sphaeroides will be used to expand our knowledge of assimilation of carbon since it is possible to disconnect energy metabolism from carbon assimilation during anoxygenic photoheterotrophic growth of this organism. R. sphaeroides is ideally suited for this study given its versatility in utilizing a wide spectrum of carbon substrates. This research will be used to determine the reactions required for 1) the conversion of C4- to C3-precursor metabolites under different growth conditions and 2) the inter-conversion of pyruvate and phosphoenolpyruvate. By capitalizing on the genetic tractability of R. sphaeroides mutants will be constructed for comparative experiments analyzing which specific enzymes are required under particular growth conditions. In addition, biochemical analyses of recombinant proteins will determine kinetic parameters, potential heteromeric composition, cofactor specificity, and posttranslational regulation of enzymes at the C4/C3 node.