Mutational Analysis of the Myxococcus xanthus Ω4406 Promoter Region Reveals an Upstream Negative Regulatory Element That Mediates C-Signal Dependence

Abstract

ABSTRACT C signaling plays a key role in coordinating cell movement and differentiation during the multicellular developmental process of Myxococcus xanthus . C signaling regulates expression of genes induced after about 6 h into development, when cells are forming mounds. One gene whose expression depends absolutely on C signaling was identified by insertion of a transposable element at site Ω4406 which generated a transcriptional fusion between lacZ and an upstream promoter. We have investigated regulation of the Ω4406 promoter. A 5′ deletion revealed a negative regulatory element located between bp −533 and −100 relative to the transcriptional start site. In the absence of this element, the promoter was still developmentally regulated but about fourfold more active. Also, the truncated promoter region retained normal dependence on two developmental regulators, FruA and DevS, but lost its dependence on the C-signaling protein CsgA. We infer that C signaling partially overcomes the negative effect of the upstream element on activity of the Ω4406 promoter. Deletion of downstream DNA between bp 50 and 140 caused a threefold loss in expression, suggesting that a positive regulatory element lies in this region. Additional positive and negative regulatory elements are present in the region from bp −69 to −49, based on the effects of multiple-base-pair mutations. Within this region, a 5-bp element and a C-box-like sequence resemble sequences found in other developmentally regulated M. xanthus promoter regions, but the effects of single-base-pair changes in these sequences suggest that each functions uniquely. We conclude that regulation of the Ω4406 promoter involves multiple positive and negative regulatory elements located upstream and downstream of the region typically bound by RNA polymerase.