INVESTIGADORES
MANSILLA Maria Cecilia
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.