⍀4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac ⍀4403. We have cloned DNA upstream of the ⍀4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac ⍀4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or ␤-galactosidase accumulation in cells containing Tn5 lac ⍀4403. The putative transcriptional start site was mapped, and sequences centered near ؊10 and ؊35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli 70 RNA polymerase. However, deletions showed that an essential promoter element lies between ؊80 and ؊72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of ؊80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression.

When starved, Myxococcus xanthus cells send signals to each other that coordinate their movements, gene expression, and differentiation. C-signaling requires cell-cell contact, and increasing contact brought about by cell alignment in aggregates is thought to increase C-signaling, which induces expression of many genes, causing rod-shaped cells to differentiate into spherical spores. C-signaling involves the product of the csgA gene. A csgA mutant fails to express many genes that are normally induced after about 6 h into the developmental process. One such gene was identified by insertion of Tn5 lac at site ⍀4406 in the M. xanthus chromosome. Tn5 lac fused transcription of lacZ to the upstream ⍀4406 promoter. In this study, the ⍀4406 promoter region was identified by analyzing mRNA and by testing different upstream DNA segments for the ability to drive developmental lacZ expression in M. xanthus. The 5 end of ⍀4406 mRNA mapped to approximately 1.3 kb upstream of the Tn5 lac insertion. A 1.0-kb DNA segment from 0.8 to 1.8 kb upstream of the Tn5 lac insertion, when fused to lacZ and integrated at a phage attachment site in the M. xanthus chromosome, showed a similar pattern of developmental expression as Tn5 lac ⍀4406. The DNA sequence upstream of the putative transcriptional start site was strikingly similar to promoter regions of other C-signal-dependent genes. Developmental lacZ expression from the 1.0-kb segment was abolished in a csgA mutant but was restored upon codevelopment of the csgA mutant with wild-type cells, which supply C-signal, demonstrating that the ⍀4406 promoter responds to extracellular C-signaling. Interestingly, the 0.8-kb DNA segment immediately upstream of Tn5 lac ⍀4406 inhibited expression of a downstream lacZ reporter in transcriptional fusions integrated at a phage attachment site in the chromosome but not at the normal ⍀4406 location. To our knowledge, this is the first example in M. xanthus of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region.

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