Determination of fatty acid synthesis intermediates in escherichia coli and bacillus subtilis

Lipids play crucial roles in maintaining cellular structure and energy storage. Structural lipids in the form of phospholipids constitute almost 10% of the dry cell weight in bacteria with their synthesis requiring 32 moles of ATP per mole of lipid. This significant investment ensures that the flux through the fatty acid biosynthesis (FAS) and related metabolic pathways is very precisely coordinated. A key feature of the FAS pathway is the acyl carrier protein (ACP), which is a small acidic protein that tethers acyl intermediates via a high-energy thioester bond and shuttles them between enzymes. In Escherichia coli, ACP is one of the most abundant soluble proteins with about 60000 copies per cell. Despite being the subject of extensive biochemical and structural studies for several decades, a reliable snapshot of ACP-bound species in any organism under different conditions is unavailable. Previously used methods are severely limited in their capacity to differentiate fatty acid intermediates, suffer from poor reproducibility, require elaborate instrumentation and cannot be used in an ideal setting for determining intracellular fluxes. This dissertation describes a sensitive and facile method to identify and quantify the physiological level of acyl-ACP species in E. coli and Bacillus subtilis under varying conditions of growth. The method primarily relies on an inert strep-tagged ACP which is purified using a two-step purification strategy utilizing volatile buffers of low pH in order to preserve the thioester bond between the acyl group and ACP. Samples obtained via this method were extremely pure and free of unwanted salts. The full length purified strep-tagged ACP were subjected to ESI mass spectrometry. Short and medium chain saturated fatty acyl-ACPs as well as long chain saturated and unsaturated fatty acyl-ACP intermediates were observed. Apart from validating widely held ideas about acyl-ACP flux in basic metabolism, acetyl-ACP, a novel intermediate, was also observed in abundance along with much lower levels of holo-ACP than previously thought. Current efforts are focusing on developing and validating a new model for the initiation of fatty acid biosynthesis using a combination of FAS mutants and stable-isotope labelling studies. During our efforts to construct one such mutant, we identified and characterized the function of a FabG temperature-sensitive mutant missing eight residues at its dimerization interface. Surprisingly, this mutant behaved like a point mutant. We identified interactions within the dimerization/tetramerization interface that compensate for each other and showed that the wildtype FabG predominantly exists as a dimer but functions as a tetramer. The mutation also rendered the enzyme extremely sensitive to calcium in vitro and conferred resistance to a calmodulin inhibitor in vivo. In B. subtilis, we have determined that the cellular levels of ACP are ten times lower than those in E. coli. when normalized to the total protein content per cell. Even under our stringent purification conditions, ACP co-purified with several other proteins, most noticeably FabF. We are currently trying to identify if Bacillus ACP is part of a loose complex. Nevertheless, this method has also successfully resolved all expected fatty acid intermediates associated with ACP including the presence of acetyl-ACP as observed in E. coli. In addition, I also discuss the development of a new set of vectors with IPTG-dependent origins of replication and a robust CRISPR-Cas9 toolkit for E. coli based on this vector set.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste