Development of a novel reporter gene assay using the sporulation program of Bacillus subtilis

DIAZ, ALEJANDRA R; DE MENDOZA, D; MANSILLA, C

Montecatini Terme

Conferencia; 8th International Conference on Gram Positive Microorganisms; 2015

Two component systems regulate crucial cellular processes in microorganisms, and each comprises a receptor histidine kinase and a cytoplasmic response regulator. When exposed to low temperatures Bacillus subtilis is able to regulate their membrane fluidity by modifying the level of unsaturation of their phospholipids. After a cold shock this bacterium induces the expression of the des gene, coding for the Δ5 acyl lipid desaturase, mediated by a two-component system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR. Induction of this pathway is brought about by the ability of the kinase to assume different signaling states in response to changes in membrane fluidity. However, the molecular detail of how the input signal is sensed by the transmembrane segments of DesK and transmitted to the cytoplasmic catalytic domain is completely unknown. Here we describe a new experimental strategy that allows rapid identification of functional critical amino acids in the sensing ability of DesK. This approach involves random mutagenesis of the entire sensor domain of DesK coding sequence, followed by the application of a new screening procedure, based in the sporulation developmental program of B. subtilis. This method permitted us the recovery of DesK mutants defective in temperature sensing. The screening strategy described here should be applicable to all histidine kinases of B. subtilis and those that can be expressed functionally in this organism. Moreover, this reporter assay could be expanded to develop reporter assays in a variety of transcriptionally regulated systems.