Identification of the Ω4400 Regulatory Region, a Developmental Promoter of Myxococcus xanthus

Abstract

Ω4400 is the site of a Tn5 lac insertion in theMyxococcus xanthus genome that fuses lacZexpression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac Ω4400. The DNA upstream of the Ω4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac Ω4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by β-galactosidase production in cells containing Tn5 lacΩ4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of −101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to −76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M. xanthus sigB orsigC gene did not affect Tn5 lac Ω4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M. xanthus promoter transcribed by a known form of RNA polymerase. However, the Ω4400 promoter does contain the sequence 5′-CATCCCT-3′ centered at −49 and the C-signal-dependent Ω4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the −10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.

Transcription factor MrpC binds to promoter regions of hundreds of developmentally-regulated genes in Myxococcus xanthus

“…These predicted MrpC binding sites might be involved in regulation of MXAN_2883, which lies upstream of fmgA in divergent orientation. The sites are not involved in fmgA regulation based on 5’ deletion analysis of the promoter region fused to a lacZ reporter [ 54 ]. No significantly-enriched ChIP-seq peaks were found in the fmgE promoter region, despite evidence for 3 MrpC2 binding sites in this region [ 48 ].…”

Identification of the Ω4499 Regulatory Region Controlling Developmental Expression of a Myxococcus xanthus Cytochrome P-450 System

“…Inspection of the DNA sequence upstream of the apparent ⍀4499 transcriptional start site revealed sequences similar to those found in two other M. xanthus promoters. Figure 6 shows the positions of three sequences in the ⍀4499 promoter region that are similar to the sequence 5Ј-CATCCC T-3Ј, which is found in the ⍀4403 and ⍀4400 promoters, centered at Ϫ49 bp (6,13). The similar sequences in the ⍀4499 promoter region are centered at Ϫ55, Ϫ33, and Ϫ1 bp.…”

“…6 is a 9-bp sequence centered at Ϫ65 bp that matches a sequence in the ⍀4400 promoter in eight of nine positions. The sequence in the ⍀4400 promoter is in the opposite orientation and is centered at Ϫ80 bp, within a region (between Ϫ101 and Ϫ76 bp) that was shown previously to be essential for activity of this promoter (6).…”

“…Below the consensus in Table 2 are four additional sequences found in promoters that have been reported to be C signal dependent. Two of these sequences occur in the ⍀4400 promoter region, centered at Ϫ5 and Ϫ80 bp (6). The other two sequences are centered at Ϫ51 and Ϫ63 bp in the fruA (46) and csgA (42) promoters, respectively.…”

“…Mutational analysis suggests that FruA must be phosphorylated to act (12), but neither an upstream kinase nor an immediate downstream target gene in this signaling pathway has been identified. Two potential targets for direct regulation by phosphorylated FruA are promoters identified by Tn5 lac insertions ⍀4403 (13) and ⍀4400 (6). These are among the first known promoters to be expressed in response to C signaling.…”