Our research program centers on prokaryotic lipid metabolism. Lipids are an essential component of cell membranes and a source of regulators of metabolism and energy. Their production is complex and tightly regulated so that each membrane can be tailored to suit the needs of the cell, tissue, and organism. We have been studying how bacteria exert exquisite control over the fatty acid biosynthetic pathway and membrane phospholipid formation. The techniques of genetics, molecular biology, biochemistry, and structural biology are being applied to understand intra- and extracellular signaling mechanisms that regulate the expression of crucial genes encoding enzymes of lipid metabolism. Our current research focuses on defining the regulatory signals and proteins that control lipid synthesis, understanding the detailed mechanism of sensors and transcription factors, and determining the high-resolution structure of two key regulatory proteins. This research is needed to develop new targets for antibacterial compounds, and we are applying our findings on roles and structures of regulatory proteins to design effective inhibitors.
Bacteria stringently regulate the synthesis of their membrane phospholipids in response to nutritional and environmental conditions, but the regulatory mechanisms are incompletely understood. In contrast to the detailed information on the synthesis and regulation of lipid metabolism in the Gram-negative bacterium Escherichia coli, our knowledge of the regulatory mechanisms controlling lipid synthesis in Gram-positive bacteria remains fragmentary. Since 1990, our group has focused on the biosynthesis and function of membrane lipids in the model Gram-positive bacterium Bacillus subtilis, a harmless bacterium that lives in the soil. Our efforts have been devoted to understanding the control of fatty acid and phospholipid synthesis and the regulation of synthesis of unsaturated fatty acids by growth temperature.
Fatty acid biosynthesis is a vital facet of bacterial physiology and is carried out by a series of enzymatic steps, each enzyme encoded by a different gene, known as the type II fatty acid synthase (FASII) pathway. Given that this biosynthetic pathway is essential and expends considerable energy, organisms have developed homeostatic mechanisms that maintain the concentration of lipids at defined levels. Although significant progress has been made in recent years, the regulatory mechanisms elucidated so far in bacteria are those that act mainly at the level of lipid biosynthetic enzymes, rather than at the level of gene expression. Major advances in our understanding of the transcriptional control of bacterial lipid synthesis have emerged from our studies, leading to the isolation and characterization of FapR, a transcription factor that negatively controls the expression of several genes of the FASII and phospholipid biosynthesis (the fap regulon) pathways in B. subtilis. FapR is the first global regulator of lipid synthesis discovered in bacteria and is largely conserved in Gram-positive organisms, including virulent human pathogens such as Bacillus anthracis, Listeria monocytogenes, and Staphylococcus aureus.
Our studies have provided evidence that decreasing the cellular concentrations of malonyl-CoA, an essential building block for fatty acid elongation, inhibits expression of FapR-regulated genes and that this inhibition strictly depends on FapR. Using a series of biochemical and biophysical studies, we recently demonstrated that FapR is able to bind directly to malonyl-CoA, leading to inhibition, or prevention, of FapR-DNA binding and to relief of fap repression. In addition, we recently solved the structure of the C-terminal domain of FapR complexed with malonyl-CoA. Structure-based mutations that disrupt the FapR–malonyl-CoA interaction prevent regulation of DNA binding and result in a lethal phenotype in B. subtilis, suggesting that this homeostatic signaling pathway is a promising target for novel chemotherapeutic agents against Gram-positive pathogens.
The cis-unsaturated fatty acids (UFAs) play crucial roles in membrane biology and signaling processes in organisms ranging from bacteria to humans. The relative UFA content of cellular phospholipids exerts a major influence on the physical properties of most biological membranes. UFAs have a much lower transition temperature than saturated fatty acids because the steric hindrance imparted by the rigid kink of the cisdouble bond results in much poorer packing of the acyl chains. When organisms that are unable to regulate cell temperature—such as bacteria, plants, and fish—are exposed to suboptimal growth temperatures, their membrane lipids become more rigid, leading to subnormal functioning of cellular activities. Adaptation to such new conditions involves an increase in the proportion of UFAs in their membranes. The resulting increase in UFA content causes membrane lipid fluidity to return to its original state, or close to it, with concurrent restoration of normal cellular activity at the lower temperature.
Many attempts have been made to elucidate the regulatory cascades that sense and transduce low-temperature signals to adjust the membrane UFA synthesis. However, the only case in which the mechanism of cold induction of the entire pathway is well established and for which a functional connection between an induced protein and cold adaptation has been demonstrated is the DesK/DesR pathway of B. subtilis. This pathway, elucidated entirely in our laboratory, is designed to adjust the membrane lipid composition in response to growth temperature by regulating the expression of the acyl lipid desaturase Δ5-Des. The DesK/DesR pathway responds to a decrease in growth temperature by enhancing the expression of the des gene, which encodes Δ5-Des. The regulatory pathway is uniquely and stringently controlled by the two-component system DesK/DesR. DesK is a histidine kinase located in the membrane; DesR is a cytoplasmic response regulator that binds specifically to the promoter region it controls. Induction of the DesK/DesR pathway is brought about by the ability of DesK to assume different signaling states in response to changes in membrane fluidity. This is accomplished by regulating the ratio of kinase to phosphatase activities. An increase in the proportion of ordered membrane lipids favors a kinase-dominant state of DesK, so that the enzyme undergoes autophosphorylation on the conserved residue His188. The phosphorylated kinase then transfers the phosphate to the Asp54 of the dimeric effector DesR, leading to the stabilization of a DesR-P tetramer. The tetramer binds to two adjacent, nonidentical DesR-P binding sites within the des promoter, leading to recruitment of RNA polymerase and activation of des transcription. DesK-mediated phosphorylation of DesR promotes activation of des, resulting in the synthesis of Δ5-Des, which introduces double bonds into the acyl chains of membrane lipids. The newly synthesized UFAs decrease the phase transition temperature of the phospholipids, favoring the phosphatase activity of DesK, resulting in hydrolysis of DesR-P. The unphosphorylated regulator is unable to bind to the des promoter and, as consequence, des transcription is turned off.
Our studies are also supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica.
Last updated September 2008
