Proline residues of DesK as transmission elements of cold stimulus

PORRINI, L; ALBANESI, D; DE MENDOZA, D; MANSILLA, MC

Montecatini Terme

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

Temperature sensing is essential for the survival ofliving cells. When exposed to lowtemperatures Bacillus subtilis isable to regulate their membrane fluidity by modifying the level of unsaturationof their phospholipids. After a cold shock this bacterium induces the expressionof the des gene, coding for the Δ5acyl lipid desaturase, by a two-component system composed of amembrane-associated kinase, DesK, and a soluble transcriptional regulator,DesR. DesK is a bifunctional histidin kinase/phosphatase that senses thebiophysical state of the membrane and transmits this signal to thetranscriptional apparatus. To date the molecular detail of how the input signal is sensedby the transmembrane segments (TMS) of DesK and transmitted to the cytoplasmiccatalytic domain is completely unknown. In order to answer this fundamentalissue, we decided to identify residues critical for cold sensing ortransmission. DesK contains five proline residues in its TMS which areconserved in several membrane-bound bacterial thermosensors. To investigate whetherthese residues play a role in the function of DesK, we mutated each proline individually to alanine. All DesKPAmutants were unable to activate des transcriptionupon a temperature downshift, whereas their phosphatase activity was retained. By applying error prone PCR mutagenesis to the desKPA alleles we obtained secondarymutations that restored the wild type behavior. These results strongly suggest that prolineresidues of DesK TMS play an important role in sensing and transmitting cold stimulus by kinking the helices inorder to bring cytoplasmic domains into optimal position for catalysis.