Regulation of the Myxococcus xanthus C-Signal-Dependent Ω4400 Promoter by the Essential Developmental Protein FruA

Understanding how cell-cell signaling coordinates gene expression during multicellular development is a fundamental problem in biology. Myxococcus xanthus is a bacterium that provides an attractive experimental system to elucidate molecular mechanisms of signal transduction leading to developmental gene expression (24, 32). When starved at a high cell density on a solid surface, rod-shaped M. xanthus cells move in a coordinated fashion to form mounds, each containing approximately 105 cells. Within these nascent fruiting bodies, some of the cells differentiate into heat- and desiccation-resistant spherical myxospores. This multicellular developmental process requires several extracellular signals (9, 16). Two of these signals, called A and C, are believed to be understood at the molecular level. A-signal appears to be amino acids and peptides that result from cleavage of outer membrane proteins by proteases secreted early in development (39, 47). A-signaling is thought to measure cell density (40) and if it is high enough, trigger expression of early developmental genes (4, 38) via a three-component system consisting of a sensor kinase (SasS), response regulator (SasR), and negative regulator (SasN) (reviewed in reference 26). C-signal appears to be a 17-kDa protein associated with the cell surface and possibly anchored in the outer membrane (31, 42, 50). It is derived by proteolytic cleavage of the 25-kDa product of the csgA gene (31, 42). C-signaling requires cell-cell contact (28, 29, 31), but a receptor has not yet been identified. FruA plays a key role in the C-signal transduction pathway, governing branches that control motility and gene expression leading to sporulation (10, 46, 51, 53). FruA is similar to response regulators in the FixJ subfamily (10, 46), but its putative cognate sensor kinase has not been identified. The N-terminal domain of FruA has a putative phosphorylation site, D59, and this residue is critical for function, suggesting that phosphorylation activates FruA (10). The molecular mechanism by which activated FruA would regulate motility is unknown, but somehow C-signaling increases methylation of FrzCD (51), a cytoplasmic chemoreceptor homolog that controls the frequency of reversal of the direction of gliding cell movement (3, 45). A low level of C-signaling results in rippling, the coordinated movement of cells that gives the appearance of traveling waves in movies made by time-lapse microscopy, while a higher level of C-signaling causes cell streaming, resulting in mound formation (reviewed in references 23 and 52). FruA's C-terminal domain has a putative helix-turn-helix motif (46) and has recently been shown to bind to specific DNA sequences upstream of two developmentally regulated M. xanthus promoters (55, 56). The results suggest that FruA directly activates transcription of these genes. One of these genes is expressed independently of C-signaling (55), and the other appears to exhibit partial dependence on C-signaling from one of its three promoters (56). Other potential direct targets of FruA transcriptional activation in response to C-signaling have been identified using Tn5 lac, a transposon that fuses transcription of the Escherichia coli lacZ gene to the promoter of a gene into which it inserts (33-35). One such insertion, at site Ω4400 in the M. xanthus chromosome, fuses lacZ expression to a promoter induced at about 6 h into development (34). Expression from the Ω4400 promoter is reduced about two- to threefold in a csgA mutant that is defective in producing C-signal (34), but expression can be restored by codeveloping the csgA mutant with wild-type cells, which supply C-signal (6). Therefore, activity of the Ω4400 promoter depends partially on extracellular C-signaling. A detailed mutational analysis of the Ω4400 promoter region revealed two regulatory elements upstream of the −35 region (58). DNA between bp −63 and the promoter −35 region was found to be absolutely essential for expression. This element contains two DNA sequences that are also found upstream of other developmentally regulated M. xanthus promoters (11, 57): a C box (consensus sequence CAYYCCY, in which Y means C or T) centered at bp −49, and a 5-bp element (consensus sequence GAACA) centered at bp −61. The second important regulatory element is located slightly farther upstream and does not appear to be found in other promoter regions examined so far. Mutations between bp −86 and −81 decrease Ω4400 promoter activity two- to fourfold and eliminate or reduce dependence on C-signaling (58). How any of these regulatory elements stimulate Ω4400 promoter activity was unknown. Here, we show that fruA is absolutely required for Ω4400 expression in vivo and that the C-terminal FruA DNA-binding domain binds in a sequence-specific manner to DNA upstream of the Ω4400 promoter in vitro. Mutations between bp −86 and −77 impair DNA binding in vitro, suggesting that FruA binds near bp −80 and activates Ω4400 transcription in response to C-signal during M. xanthus development. In support of this hypothesis, we devised a procedure for chromatin immunoprecipitation (ChIP) analysis of developing M. xanthus cells and used it to show that FruA associates with the Ω4400 promoter region in vivo. Moreover, we found that FruA was associated with the Ω4400 promoter region even in the absence of C-signaling. The results support a model in which FruA directly activates transcription from the Ω4400 promoter both before and after C-signaling. This model explains partial dependence of Ω4400 expression on C-signaling and may apply to other genes induced after 6 h into M. xanthus development, because many of these also exhibit partial dependence on C-signaling (30, 34, 41, 56).