CHARACTERIZATION OF THE INTERACTION BETWEEN BACILLUS SUBTILIS DESK AND ITS COGNATE RESPONSE REGULATOR DESR

MARTÍN, MARIANA; MANSILLA, M. C.; ALBANESI, D.; HAIMOVICH, A.; CYBULSKI, L. E.; DE MEMDOZA, D.

Pinamar, Buenos Aires, Argentina

Congreso; XLI Reunión Anual de la SAIB; 2005

The des gene of Bacillus subtilis codes for an acyl-lipid D5-desaturase, D5-Des, which introduces double bounds in membrane phospholipids. Its expression is strictly regulated in response to membrane fluidity changes, by the two-component system DesK/R, composed of a membrane associated kinase, DesK, and a soluble transcriptional regulator, DesR. To give additional biochemical and biophysical support to this model, we studied the interaction between DesK and DesR by Far Western Blots. We found that purified DesR is capable of interact with the cytoplasmatic domain of DesK (DesKC) fixed to a nitrocellulose membrane. We also found a 66 kDa subunit protein that being recognized by immunopurified anti-DesR antibodies, interacts with DesKC in vitro and its level is increased in the double mutant YvfT/U. The in vivo analysis of the DesK/R and DesK/D5-Des interactions will be performed by double hybrid systems assays. In addition, in order to structurally characterize this interaction we are performing crystallization assays. Until now, we had obtained crystals of DesKCV188, a punctual mutant derived from DesKC. Moreover, we performed in vivo studies of the localization of DesK/R and D5-Des by fluorescence microscopy. The des gene of Bacillus subtilis codes for an acyl-lipid D5-desaturase, D5-Des, which introduces double bounds in membrane phospholipids. Its expression is strictly regulated in response to membrane fluidity changes, by the two-component system DesK/R, composed of a membrane associated kinase, DesK, and a soluble transcriptional regulator, DesR. To give additional biochemical and biophysical support to this model, we studied the interaction between DesK and DesR by Far Western Blots. We found that purified DesR is capable of interact with the cytoplasmatic domain of DesK (DesKC) fixed to a nitrocellulose membrane. We also found a 66 kDa subunit protein that being recognized by immunopurified anti-DesR antibodies, interacts with DesKC in vitro and its level is increased in the double mutant YvfT/U. The in vivo analysis of the DesK/R and DesK/D5-Des interactions will be performed by double hybrid systems assays. In addition, in order to structurally characterize this interaction we are performing crystallization assays. Until now, we had obtained crystals of DesKCV188, a punctual mutant derived from DesKC. Moreover, we performed in vivo studies of the localization of DesK/R and D5-Des by fluorescence microscopy.