Identification of the Ω4514 Regulatory Region, a Developmental Promoter of Myxococcus xanthus That Is Transcribed In Vitro by the Major Vegetative RNA Polymerase

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development (11, 12). When starved at high cell density on a solid surface, cells move in a coordinated fashion into aggregation centers, where they form mound-shaped fruiting bodies that each contain approximately 105 cells. Within the fruiting bodies, some of the rod-shaped cells differentiate into dormant, ovoid spores that are heat and desiccation resistant. Cell-cell interactions play a critical role in this multicellular developmental process (47). At least five extracellular signals, known as the A, B, C, D, and E signals, appear to be involved (10, 19). Mutants defective in the production of any one of these signals are arrested in development at a particular stage but are rescued by codevelopment with wild-type cells or cells that are defective in the production of a different signal. To study the role of cell-cell interactions in controlling gene expression during M. xanthus development, Tn5 lac, a transposon containing a promoterless Escherichia coli lacZ gene, has been used to identify developmentally regulated genes (32). Transposition of Tn5 lac into the M. xanthus chromosome can generate a transcriptional fusion between lacZ and an M. xanthus promoter. Among 2,374 Tn5 lac insertions, 29 of them were activated during M. xanthus development (34). The dependence of developmental gene expression on cell-cell interactions was examined by monitoring β-galactosidase expression of the lacZ fusions in cell interaction mutants. A and B signaling are required for normal developmental gene expression at the onset of development (17, 33, 35), D and E signaling are required at 3 to 5 h into development (7, 10), and C signaling is required at about 6 h into development (33, 38). To begin to understand the mechanism by which C signaling regulates developmental gene expression, the promoter regions of transcriptional units identified by Tn5 lac insertions Ω4403, Ω4400, and Ω4499 have been characterized (5, 14, 15). The promoters are not similar in the −10 and −35 regions and do not resemble promoters that are transcribed by E. coli σ70 or σ54 RNA polymerase (RNAP). However, both Ω4403 and Ω4400 have the sequence 5′-CATCCCT-3′ centered at −49 bp. Similar sequences are also found in the Ω4499 promoter region and other C-signal-dependent promoters at positions near −50 bp (14). Hence, a sequence with the consensus 5′-CAYYCCY-3′ (known as the C box) has been proposed to be important for C-signal-dependent gene expression. Here, we report studies on the regulation of Ω4514, a Tn5 lac insertion that does not depend upon C signaling for expression and yet is expressed with timing during development similar to that of promoters that do depend upon C signaling. The Ω4514 promoter is different from other M. xanthus development-specific promoters that have been characterized in that it resembles promoters transcribed by E. coli σ70 and it can be transcribed by σA RNAP isolated from vegetative M. xanthus cells. No C-box sequence is present near −50 bp. Regulation of the Ω4514 operon is complex, involving negative autoregulation by the product of the first gene of the operon and positive regulation mediated by upstream DNA elements. These studies provide the first example of a developmentally regulated M. xanthus gene that is transcribed by the major vegetative RNAP and establish a foundation for biochemical approaches toward identifying proteins that regulate Ω4514 expression.