Combinatorial Regulation by MrpC2 and FruA Involves Three Sites in the fmgE Promoter Region during Myxococcus xanthus Development

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.

Combinatorial Regulation of thedevOperon by MrpC2 and FruA during Myxococcus xanthus Development

“…In each case, binding of the two proteins cooperatively to a site located immediately upstream of the promoter appears to activate transcription. However, insertion mutations in these genes cause, at most, mild defects in fruiting body formation (34,(39)(40)(41). In contrast, insertions in the dev operon impair sporulation dramatically (3,5).…”

“…MrpC2 binds cooperatively with FruA to the promoter regions of several C-signal-dependent genes in vitro (34,(39)(40)(41). Mutations in the cooperative binding sites affect promoter activity in vivo.…”

“…The expression of several C-signal-dependent genes has been shown to be under the combinatorial control of FruA and MrpC2 (34,(39)(40)(41). MrpC2 is an N-terminally truncated form of MrpC (42), a protein similar to transcription factors in the cyclic AMP receptor protein (CRP) family (43).…”

Myxococcus xanthus Developmental Cell Fate Production: Heterogeneous Accumulation of Developmental Regulatory Proteins and Reexamination of the Role of MazF in Developmental Lysis

“…Increased activation of FruA is proposed to induce FruA-dependent transcription of several target genes. In many cases, FruA acts in combination with MrpC2 (22,33,34,55). One important target of FruA/MrpC2 is the dev locus (62), which is expressed at high levels in aggregation centers (16), is necessary for efficient sporulation (59), and additionally promotes upregulation of fruA, inducing a positive-feedback loop (7).…”

The dev Operon Regulates the Timing of Sporulation during Myxococcus xanthus Development

“…Since the transcription of fruA depends on MrpC (15), and the two transcription factors bind DNA promoter regions cooperatively to regulate the transcription of many genes (16)(17)(18)(19)(20), including the dev operon (9), we also measured the MrpC level during development of the ΔdevT mutants. Consistent with an earlier blockage of the developmental program in the original ΔdevT408 -502 mutant, MrpC accumulation was reduced relative to wild-type DK1622 (Fig.…”