SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

The SlyA transcriptional regulator controls the expression of genes involved in virulence and production of surface components inS. TyphimuriumandE. coli. Its mode of action is mainly explained by its antagonism with the H-NS repressor for the same DNA binding regions. Interestingly, it has been reported that the alarmone ppGpp promotes SlyA dimerization and DNA binding at the promoter ofpagC, enhancing the expression of this gene inSalmonella. A recurring problem in the field of stringent response has been to find a way of following ppGpp levelsin vivoin real time. We thought that SlyA, as a ppGpp responsive ligand, was a perfect candidate for the development of a specific ppGpp biosensor. Therefore, we decided to characterize in depth this SlyA control by ppGpp. However, using various genes whose expression is activated by SlyA, as reporters, we showed that ppGpp does not affect SlyA regulationin vivo. In addition, modulating ppGpp levels did not affect SlyA dimerizationin vivo, and did not impact its binding to DNAin vitro. We finally showed that ppGpp is required for the expression ofhlyEinE. coli, a gene also activated by SlyA, and propose that both regulators are independently required forhlyEexpression. The initial report of ppGpp action on SlyA might be explained by a similar action of SlyA and ppGpp onpagCexpression, and the complexity of promoters controlled by several global regulators, such as the promoters ofpagCinSalmonellaorhlyEinE. coli.