Protein lysine acetylation is one of the abundant post-translational modifications in eukaryotes, but found in prokaryotes as well. Despite the evidence of the lysine acetylation in the prokaryotic RNA polymerases (RNAPs), its function remains unrevealed. We revealed that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of Streptomyces venezuelae. The 259th lysine (K259) located between 1.2 and 2 regions was determined as the acetylated residue of HrdB in vivo by LC-MS/MS analyses