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

Cell-cell interactions play a critical role in cellular differentiation in most multicellular organisms. The gram-negative bacterium Myxococcus xanthus undergoes multicellular development and differentiation in response to cell-cell interactions (10). Upon starvation at a high cell density on a solid surface, undifferentiated, rod-shaped cells move in a coordinated fashion into aggregation centers, where they build a mound-shaped fruiting body containing approximately 105 cells. Some cells within a nascent fruiting body differentiate into dormant, ovoid spores that are resistant to heat and desiccation. The processes of development and differentiation in M. xanthus are controlled by a program of gene expression which is highly coordinated in response to cell-cell interactions (27). At least five cell-cell signals are required for normal development. Mutants unable to produce any one of these signals are arrested in their development but can develop when mixed with either wild-type cells or cells that are defective in the production of a different signal (8, 19, 35). C-signaling mutants are one class of such developmental signaling mutants. All existing members of the C-signaling class have resulted from mutations in a single gene called csgA and are defective in both fruiting body formation and sporulation (20, 51, 52). Mutations in csgA also block rippling, the synchronous movement of waves of developing cells (46, 53). CsgA appears to mediate C signaling by acting as a developmental timer that, at successively higher concentrations, triggers the normal developmental sequence of rippling, aggregation, fruiting body formation, and sporulation (31, 37). Different levels of C signaling are also necessary to trigger the expression of different developmental genes (31). Sixteen C-dependent genes have been identified by using a transposon, Tn5 lac, that contains a promoterless lacZ gene near one end (32–34). Transposition of Tn5 lac into the M. xanthus chromosome can generate a transcriptional fusion between lacZ and an M. xanthus promoter. Tn5 lac insertions in C-dependent genes were identified as insertions whose lacZ expression was increased during the development of wild-type cells, but expression was delayed, reduced, or abolished in a csgA mutant background (33). Nearly all genes that begin to be expressed after 6 h into development appear to be at least partially C dependent. To begin to understand how C signaling regulates M. xanthus developmental gene expression, the DNA regulatory regions of C-signal-dependent genes must be characterized. This report describes the cloning and initial characterization of the regulatory region controlling the transcriptional unit identified by the insertion of Tn5 lac at site Ω4400 in the M. xanthus chromosome (34). Expression of Tn5 lac Ω4400 begins 6 to 12 h into development and is partially dependent on C signaling (33). Deletions were generated to localize the sequences required for promoter activity, the nucleotide sequence of the promoter region was determined and compared to those of other M. xanthus developmental promoters, and the transcriptional start site was mapped. Activity of the minimal promoter was reduced in csgA mutant cells, but normal expression was restored upon codevelopment with wild-type cells, indicating that the cloned promoter retains its partial dependence upon extracellular C signaling. Promoter activity required sequences relatively far upstream of the transcriptional start site, implying that a regulatory protein(s) in addition to RNA polymerase is involved in promoter activation. Comparison of the Tn5 lac Ω4400 promoter to that of the absolutely C-dependent Tn5 lac Ω4403 insertion (14) revealed features common to these genes which might lead to the identification of regulatory proteins involved in the activation of these genes in response to C signaling.