“Structural and functional characterization of DesK, the membrane fluidity sensor of Bacillus subtilis”

ALBANESI, D; BUSCHIAZZO, A; MANSILLA MC; MARTÍN, M; AHMED HAOUZ; DE MENDOZA D; ALZARI, P

Tirrenia, Pisa. Italia

Congreso; The 14th International Conference on Bacilli; 2007

Structural and functional characterization of DesK, the membrane fluidity sensor of Bacillus subtilis   Daniela Albanesi1, Alejandro Buschiazzo2, María Cecilia Mansilla3, Mariana Martín3, Ahmed Haouz1, Diego de Mendoza3 and Pedro Alzari1.   1Unit of Structural Biology, Pasteur Institute, 25 rue du Dr. Roux, 75015 Paris, France. 2Laboratory of Structural Biology, Pasteur Institute of Montevideo, Mataojo 2020, Montevideo 11400, Uruguay. 3Department of Microbiology, National University of Rosario. IBR- CONICET. Suipacha 531, 2000 Rosario, Argentina. Email: albanesi@pasteur.fr               Bacillus subtilis, a gram-positive bacterium, frequently encounters stress conditions in its natural environment, the soil. In order to detect and respond to these stressful and variable conditions, it employs two-component signal transduction systems. The B. subtilis Des pathway is composed of the membrane Δ5-acyl lipid desaturase, Δ5-Des, and the two component system DesK/DesR (1, 2, 4). DesK is a histidine kinase located in the membrane (2) and DesR is a cytoplasmatic response regulator that binds specifically to the promoter region it controls (6). The induction of the Des pathway is brought about by the ability of DesK to assume different signalling states in response to changes in membrane fluidity (2, 5). This could be accomplished by regulating the ratio of kinase to phosphatase activities (3). Upon an increase in the proportion of ordered membrane lipids, a kinase-dominant state of DesK is favored, so that it undergoes autophosphorylation and subsequently the phosphoryl group is transferred to the cytoplasmic response regulator DesR. The DesK-mediated phosphorylation of DesR promotes binding of the DesR-P dimers to Pdes and this favors DesR-P tetramerization. Tetrameric DesR-P interacts specifically with RNA polymerase to turn on des transcription (6). Activation of des results in the synthesis of Δ5-Des, which introduces double bonds in the acyl chains of membrane lipids (1, 4). These newly synthesized unsaturated fatty acids 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 Pdes and, as a consequence, des transcription is turned off . Thus, the transcription of des gene is directly modulated by the levels of DesR-P in the cell. In order to characterize the signaling states corresponding to the autokinase, phosphotransfer and phosphatase activities associated with the cytoplasmic region of DesK and to gain insight into the mechanism by which this sensor protein, which is at the top of the Des pathway, adjust its signaling state in response to changes in membrane lipid fluidity we decided to reconstitute an in vitro phosphorylation system using whole DesK and to perform structural studies on this protein. Here, we show and analyze the crystallographic structure, at a 2.1Å resolution, of the complete soluble domain of DesK. This is the first crystallographic structure of the complete cytosolic domain of a histidine kinase with known biological relevance. This structure allows us to propose and discuss molecular aspects concerning different states of the sensor.   References: 1. Aguilar, P, Cronan, J.E., Jr. and de Mendoza, D. (1998). J. Bacteriol. 180, 2194-2200 2. Aguilar PS, Hernandez-Arriaga AM, Cybulski LE, Erazo A, and de Mendoza D The EMBO Journal 2001. 20: 1681-1691. 3. Albanesi, D., Mansilla, M.C and de Mendoza, D. (2004). J. Bacteriol. 186, 2655-2663 4. Altabe, S. G., Aguilar, P. S., Caballero G. M and de Mendoza, D. (2003). J. Bacteriol. 185, 3228-3231 5. Cybulski LE, Albanesi D, Mansilla MC, Altabe S, Aguilar P, and de Mendoza D Mol. Microbiol. 2002. 45: 1379-1388. 6. Cybulski LE, del Solar G, Craig P, Espinosa M, and de Mendoza D J. Biol. Chem . 2004, 279: 39340-39347.