Development of a Reporter Gene Assay for Screening of DesK Mutants in Bacillus subtilis

DIAZ, ALEJANDRA R; MANSILLA, C. .; RE, F. ; DE MENDOZA, D

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2008

DEVELOPMENT OF A REPORTER GENE ASSAY FOR SCREENING OF DesK MUTANTS IN Bacillus subtilis Alejandra R. Díaz1, María Cecilia Mansilla2, Florencia Ré2 y Diego de Mendoza2   1Dpto. Biol., Bioq. y Farmacia, UNS, B. Blanca, 2IBR (CONICET) y Fac. CBF, UNR, Rosario. Argentina.   The B. subtilis Des pathway is composed by the membrane delta5-acyl lipid desaturase and the two component system DesK-DesR. DesK is a histidine kinase located in the membrane and DesR is a cytoplasmatic response regulator that binds specifically to the promoter region of the des gene. Induction of the Des pathway is brought about by the ability of DesK to assume different signaling states in response to changes in membrane fluidity. A random mutagenesis approach of the transmembrane segments of DesK was initiated to unveil the mechanistic details of membrane fluidity sensing. The aim of this work was to develop an easy screen system in B. subtilis for analysis of punctual mutations in DesK, based on sporulation as reporter phenotype. RapA is a phosphatase of the Spo0F response regulator, a key component of sporulation initiation cascade. Sporulation is impaired when RapA is expressed without its inhibitor (PhrA), so we built a rapA-phrA null mutant expressing ectopically rapA under the Pdes promoter (VRA2). As the Des pathway is negatively regulated by unsaturated fatty acids, des gene of VRA2 was disrupted, rendering VRA2des-. In sporulation medium this strain showed a Spo+ phenotype at 37º C and Spo- after a downshift from 37 to 25º C. This results support the concept of a useful screening system to be used in spore forming bacteria.