pgsB encodes gamma-poly glutamic acid (gamma-PGA) synthetase and constitutes an operon with pgsC, pgsAA, and pgsE. Genetic analysis revealed that degQ and swrA, the known regulators of pgsB, are not required for pgsB expression when high cellular concentrations of phosphorylated form of the response regulator DegU (DegU-P) are present. However, swrA appeared still to be required for gamma-PGA synthesis under the conditions we tested. Since genetic analysis suggested that DegU-P activates pgsB directly, we performed gel retardation and footprint analyses using purified His-tagged DegU and the pgsB promoter. The in vitro experiments revealed that His-tagged DegU bound to the immediate upstream region of the -35 region of the pgsB promoter. A six-base deletion within the sequence (the -44 to -39 region) abolished DegU-binding to the pgsB promoter and pgsB transcription, confirming the importance of the sequence for DegU-dependent regulation of pgsB. Hence we conclude that DegU is a direct activator of the pgsB operon.