IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Screening of DesK mutants in Bacillus subtilis exploiting a novel reporter gene assay
Autor/es:
DÍAZ, ALEJANDRA; MANSILLA, MC; WULFF, JP; TROBIANI, G; DE MENDOZA, D
Lugar:
Puerto Madryn
Reunión:
Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010
Institución organizadora:
SAIB
Resumen:
The histidine kinase DesK from B. subtilis is a membrane thermosensor that sense a decrease in membrane fluidity. Together with the response regulator DesR, constitutes a two-component system that regulates the transcription of the des gene, coding for the acyl lipid desaturase delta5-Des. To uncover the mechanism of membrane fluidity sensing we performed a random mutagenesis approach of the transmembrane segments of DesK. The phenotypic selection of mutants was carried out using the screening system that we have previously developed in B. subtilis, based on sporulation as reporter phenotype. So, as sporulation is impaired when RapA is expressed without its inhibitor, PhrA, we used a rapA-phrA null mutant expressing ectopically Pdes-rapA to build a derivative des and desK null mutant that expresses isotopically desR under Pxyl promoter. The resulting strain, ARDCM2, showed Spo+ phenotype in sporulation medium after a temperature downshift to 23º C. A vector carrying Pxyl-desKC-end was built, which is suitable to clone desKN-end alleles upstream to desKC-end domain, regenerating full-length desK. ARDCM2 transformed with the vector expressing DesKwt showed reversion to Spo- phenotype. ARDCM2 transformants expressing mutated versions of DesK that showed Spo+ phenotype at 23ºC or Spo- phenotype at 37ºC were isolated.