ACETYLATION AT LYS-92 ENHANCES SIGNALING BY THE CHEMOTAXIS RESPONSE REGULATOR PROTEIN CHEY

When Escherichia coli cells lacking all chemotaxis proteins except the response regulator CheY are exposed to acetate, clockwise flagellar r otation results, indicating the acetate stimulus has activated signali ng by CheY, Acetate can be converted to acetyl-CoA by either of two di fferent metabolic pathways, which proceed through acetyl phosphate or acetyl-AMP intermediates. In turn, CheY can be covalently modified by either intermediate in vitro, leading to phosphorylation or acetylatio n, respectively, Either pathway is sufficient to support the CheY-medi ated response to acetate ill viva, Whereas phosphorylation of Asp-57 i s a recognized mechanism for activation of CheY to stimulate clockwise flagellar rotation, acetylation of CheY is less well characterized. W e found evidence for multiple CheY acetylation sites by mass spectrome try and directly identified Lys-92 and Lys-109 as acetylation sites by Edman degradation of peptides from [C-14] acetate-labeled CheY, Repla cement of CheY Lys-92, the preferred acetylation site, with Arg has li ttle effect on chemotaxis but completely presents the response to acet ate via the acetyl-AMP pathway, Thus acetylation of Lys-92 activates c lockwise signaling by CheY in vivo. The mechanism by which acetylation activates CheY apparently is not simple charge neutralization, nor do es it involve enhanced binding to the FliM flagellar switch protein. T hus acetylation probably affects signal generation by CheY at a step a fter switch binding.