Cold-Adaptation of bacterial memebrane lipids.

ALTABE, SILVIA GRACIELA; DE MENDOZA, D

Rosario-Argentina

Workshop; Workshop in Cryobiology Applied to Medical Sciencies; 2009

Centro Binacional de Criobiología Clínica y Aplicada UNESCO

Variability and adaptability are crucial characteristics of organisms possessing the ability to survive and prosper in a wide variety of environmental conditions. A universally conserved adaptation-response observed among bacteria and most, if not all, poikilothermicorganisms is the adjustment of membrane lipid composition at low temperatures, referred to as homeoviscous adaptation. Temperature markedly affects membrane physical state, and changes in lipid composition occur to assure optimal membrane structure and function.In bacteria, membrane fluidity can be optimized by modifying the types of fatty acids that are attached to glycerol backbones since the structure of these fatty acids determine the biophysical properties of the membrane bilayer. The major way by which bacteria, lacking cholesterol, maintain this functional membrane physical state, is by increasing the proportion of low melting point fatty acids in the membrane lipids as the growth temperature decreases. Membrane fluidity can be optimized by modifying its fatty acids composition, that is: changing the proportion of branched-chain fatty acids, the acyl chain length or introducing double bonds. cis-Unsaturated fatty acids (UFAs) introduce a pronounced kink that disrupt the order of the bilayer and results in lower transition temperature than of one composed of straight –chain saturated fatty acids. Also, anteiso-branched chain fatty acids promote more fluid membranes than the iso-fatty acids.Our group has focused on the study of the biosynthesis and the regulation of UFAs by growth temperature in B. subtilis. Bacilli cells respond to a decrease in ambient growth temperature by introducing double bonds into fatty acids. These double bonds are inserted by specific fatty acid desaturase enzymes. B. subtilis contains a unique desaturase, which is encoded by the des gene which is tightly regulated by temperature at the transcriptional level. Desaturases are present in all groups of organisms and play a key role in the maintenance of the proper structures and function of biological membranes. They introduce a double bond at specific positions in fatty acids of defined chain lengths and utilize molecular oxygen and reducing equivalents from an electron transport chain. The importance of the desaturase enzymes in UFAs biosynthesis and the unique chemistry of the reaction that they catalyze have focused our interest in understanding the biochemistry of the desaturation reaction. It is known that the reaction requires oxygen and utilizes NAD(P)H and flavoproteins as electron donors, however the electron transport system of the Δ5-acyl lipid desaturase from B. subtilis has not been yet characterized. The “in silico” analysis of the genome of B. subtilis reveals the existence of two flavodoxins YkuN and YkuP, and a fer gene and we propose that they could act as putative electron donors for the Δ5-desaturase. We have demonstrated that mutants in any of these genes are not impaired in UFAs synthesis. Interestingly we found that in B. subtilis the function of de Δ5-desaturase is impeded when both, ferredoxin and flavodoxins are absent indicating that both flavoproteins can act as electron donors for UFAs synthesis in these bacteria. In a previous work we reported that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position,regardless of the growth temperature and the length chain of the fatty acids. However, the determinants of the enzymatic specificity of membrane desaturases are not understood. Genes with significant similarity to fatty acid desaturases have been found in the genomes ofmany bacteria. The existence of structurally related desaturases, with different substrate recognition and double bond-positioning properties offers the opportunity to compare the active site structures of members of this family of enzymes. In silico analysis on Bacillusgenomes allowed us the identification of highly similar desaturases. We have characterized two desaturases from B. licheniformis and two from B. cereus. Site-directed mutagenesis and domain swapping techniques will help us to elucidate which residues or regions in these enzymes are important for the substrate recognition and double bond-positioning activities of desaturases. The dissection of this molecular mechanism in these bacteria will provide important insights into the basic question on the desaturation reaction. Information gained from this research could potentially lead to the design of desaturases capable of producing new industrially useful isomers of monounsaturated fatty acids or specific inhibitors potentially useful as antibiotics.