The putative stationary-phase sigma factor (sigma S) encoded by rpoS is essential for glycogen synthesis, but is not required for the transcription of glgC and glgA, which encode ADP-glucose-pyrophosphorylase and glycogen synthase, respectively. Using a mini-Mu random chromosomal library and a screen for glycogen overproduction, we identified a novel gene (glgS) involved in glycogen synthesis. glgS maps at 66.6 min (3247 kb) on the chromosome and constitutes a monocistronic operon. It encodes a hydrophilic and highly charged small protein, with a molecular weight of 7886, which is strongly expressed in minicells. Experiments with single-copy chromosomal glgS::lacZ gene fusions indicated that glgS expression is controlled by sigma S as well as by cAMP. Two transcriptional start sites were mapped in the upstream regulatory region of glgS. The glgSp1 transcript was absent in a cya mutant, whereas an rpoS mutant did not synthesize the glgSp2 transcript. Although glycogen synthesis is strongly stimulated by overproduction of GlgS and is inhibited by a glgS null mutation, glgS does not affect the expression of the glgCAP operon. Its potential role in the metabolic control of glycogen synthesis is discussed. Also, evidence is presented to show that the amount of glycogen accumulated in vivo in early stationary-phase cells is mainly determined by sigma S-controlled gene expression and allosteric activation of GlgC, whereas the absolute levels of expression of glgCAP as well as the intracellular concentration of cAMP are of minor importance.