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

DIAZ, A. R.; DE MENDOZA, D; MANSILLA, MC

Montecatini Terme

Congreso; 8th Conference on Functional Genomics of Gram-Positive Microorganisms - 18th International Conference on Bacilli; 2015

Two component systems regulate crucialcellular processes in microorganisms, and each comprises a receptor histidinekinase and a cytoplasmic response regulator. Whenexposed to low temperatures Bacillussubtilis is able to regulate their membrane fluidity by modifying the levelof unsaturation of their phospholipids. After a cold shock this bacteriuminduces the expression of the desgene, coding for the Δ5 acyl lipid desaturase, mediated by a two-componentsystem composed of a membrane-associated kinase, DesK, and a solubletranscriptional regulator, DesR. Induction of this pathway is brought about by theability of the kinase to assume different signaling states in response tochanges in membrane fluidity. However, themolecular detail of how the input signal is sensed by the transmembranesegments of DesK and transmitted to the cytoplasmic catalytic domain iscompletely unknown. Here we describe a new experimentalstrategy that allows rapid identification of functional critical amino acids inthe sensing ability of DesK. This approach involves random mutagenesis of theentire sensor domain of DesK coding sequence, followed by the application of anew screening procedure, based in the sporulation developmental program of B. subtilis. This method permitted usthe recovery of DesK mutants defective in temperature sensing. The screeningstrategy described here should be applicable to all histidine kinases of B. subtilis and those that can beexpressed functionally in this organism. Moreover, this reporter assay could beexpanded to develop reporter assays in a variety of transcriptionally regulatedsystems.