Evidence suggesting that eukaryotes and archaea use reversible N(epsilon)-lysine (N(epsilon)-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(epsilon)-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsB(Ac)), demonstrating that N(epsilon)-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac) and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(epsilon)-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.