Abstract
Starvation causes cells in a dense population of Myxococcus xanthus to change their gliding movements and construct mounds. Short-range C-signaling between rod-shaped cells within mounds induces gene expression that promotes differentiation into spherical spores. Several C-signal-dependent genes have been shown to be regulated by cooperative binding of two transcription factors to the promoter region. These FruA- and MrpC2-regulated genes (designated fmg) each exhibit a different arrangement of binding sites. Here, we describe fmgE, which appears to be regulated by three sites for cooperative binding of FruA and MrpC2. Chromatin immunoprecipitation analysis showed that association of MrpC2 and/or its longer form, MrpC with the fmgE promoter region, depends on FruA, consistent with cooperative binding of the two proteins in vivo. Electrophoretic mobility shift assays with purified His(10)-MrpC2 and FruA-His(6) indicated cooperative binding in vitro to three sites in the fmgE promoter region. The effects of mutations on binding in vitro and on expression of fmgE-lacZ fusions correlated site 1 (at about position -100 relative to the transcriptional start site) with negative regulation and site 2 (just upstream of the promoter) and site 3 (at about position +100) with positive regulation. Site 3 was bound by His(10)-MrpC2 alone, or the combination of His(10)-MrpC2 and FruA-His(6), with the highest affinity, followed by site 1 and then site 2, supporting a model in which site 3 recruits MrpC2 and FruA to the fmgE promoter region, site 1 competes with site 2 for transcription factor binding, and site 2 occupancy is required to activate the promoter but only occurs when C-signaling produces a high concentration of active FruA.