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

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 ⍀4406 which generated a transcriptional fusion between lacZ and an upstream promoter. We have investigated regulation of the ⍀4406 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 ⍀4406 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 ⍀4406 promoter involves multiple positive and negative regulatory elements located upstream and downstream of the region typically bound by RNA polymerase.

Identification of the Ω4406 Regulatory Region, a Developmental Promoter of Myxococcus xanthus , and a DNA Segment Responsible for Chromosomal Position-Dependent Inhibition of Gene Expression

“…7). The inhibitory effect of the 0.8-kb segment on developmental lacZ expression from fusions integrated at Mx8 attB has also been demonstrated with a vector (pREG1727) bearing a Km r gene (55). In contrast, the 0.8-kb segment does not inhibit developmental expression of lacZ from fusions integrated by homologous recombination at the native ⍀4406 site (Fig.…”

“…5) precisely localized the 5Ј end. Mutational analysis has confirmed that the 5Ј end reflects the transcriptional start site of the ⍀4406 promoter (55). However, we discovered that expression of a lacZ reporter fused transcriptionally to the ⍀4406 promoter depends strongly on the position of the fusion in the M. xanthus chromosome.…”

A combination of unusual transcription factors binds cooperatively to control Myxococcus xanthus developmental gene expression

“…The cis-regulatory element to which MrpC2 binds in the fmgA promoter region includes a 5-bp element and a C box. These 2 sequences are similarly arranged upstream of other C-signal-dependent promoters and are important for promoter activity (21)(22)(23), suggesting that MrpC2 may bind to these sites. Indeed, in the promoter region of the operon identified by Tn5 lac ⍀4499 (22), MrpC2 binds near a 5-bp element, and in combination with FruA, formation of shifted complexes in EMSAs is greatly enhanced (unpublished data).…”

“…1A) (18), and subsequent analysis showed that the FruA DNA-binding domain (FruA-DBD) binds to the sequence between Ϫ86 and Ϫ77 (17). The sequence between Ϫ63 and Ϫ46 contains 2 elements found in other C-signal-dependent promoter regions; a 5-bp element (consensus GAACA) and a C box (consensus CAYYCCY; Y means C or T) (21)(22)(23). Mutations in this region nearly abolish fmgA promoter activity ( Fig.…”

Combinatorial Regulation by a Novel Arrangement of FruA and MrpC2 Transcription Factors during Myxococcus xanthus Development

“…The finding that MrpC2 and FruA bind cooperatively to crucial cis-regulatory sequences upstream of the fmgA promoter suggests that these transcription factors coordinate starvation signaling and cell death with positional information via short-range C-signaling to govern gene expression and cell fate during M. xanthus development (35). This novel mechanism of fmgA combinatorial control was predicted to be conserved because similar cis-regulatory sequences have been found upstream of other developmentally regulated M. xanthus promoters (6,31,52,(59)(60)(61)67).…”