Identification of the Ω4406 regulatory region, a developmental promoter of Myxococcus xanthus, and a DNA segment responsible for chromosomal position-dependent inhibition of gene expression

Myxococcus xanthus is a gram-negative, primarily soil-dwelling bacterium (6). It moves by gliding over solid surfaces. Swarms of these bacteria secrete enzymes that lyse prey bacteria and degrade macromolecules to provide nutrients. When nutrients become scarce, cells alter their behavior, piling on top of one another to form mounds. Within the mounds, cells differentiate from metabolically active rods to dormant spheres, resulting in mature, spore-filled fruiting bodies. Outside the fruiting bodies, cells differentiate into another type called peripheral rods (38, 39). Spores are resistant to environmental insults and allow M. xanthus to survive in unfavorable conditions. When nutrients again become available, spores in the fruiting body germinate to produce a swarm of rod-shaped cells capable of growth. The developmental process of M. xanthus has long been studied as a model to understand how cells interact with each other (reviewed in references 19, 21, 48, and 50). These studies are of broad significance because many, if not most, bacteria exist in microbial communities called biofilms, and within biofilms cells send signals to each other and respond by changing the expression of certain genes (reviewed in reference 41). Likewise, M. xanthus cells send signals to each other during development that coordinate changes in gene expression, which cause cells to alter their movement, metabolism, and shape. M. xanthus mutants defective in signaling were first described more than 25 years ago (14), but much remains to be learned about the molecular mechanisms of signaling and response. Among five classes of signaling mutants identified so far (5, 14), the signaling mechanism is well understood in two cases, A- and C-signaling. A-signaling involves the production of extracellular proteases early in development, which release peptides and amino acids that allow cells to assess the population density (reviewed in reference 21). Only if the number of cells per unit area is sufficient will development proceed. C-signaling involves the product of the csgA gene, a 25-kDa protein that appears to be associated with the outer membrane, where it is thought to be cleaved to a 17-kDa form that serves as the C-signal (25, 34). Transmission of the C-signal requires cell movement (23), possibly to allow cells to make end-to-end contacts (44). Upon transmission, C-signal modifies the motility behavior of the recipient cell (reviewed in references 19 and 50). At the population level, a low level of C-signaling causes rippling (accumulation of cells in parallel ridges that demonstrate movement in time-lapse microscopy), a higher level is needed for mound formation, and extensive cell-cell contacts within mounds, resulting in very high C-signaling, is believed to trigger sporulation (24, 31). Hence, C-signaling appears to coordinate cell behaviors during the middle to late stages of development. Given the importance of A- and C-signaling for morphological development to proceed, it is not surprising that many developmentally regulated genes depend on these signaling interactions for expression. Many of the developmentally regulated genes that have been studied were identified by transposition of Tn5 lac into the M. xanthus chromosome (28). Tn5 lac can generate a transcriptional fusion between an M. xanthus promoter and the Escherichia coli lacZ gene (26). Among 21 such fusions to developmentally regulated genes, 18 failed to be expressed in asg mutants defective in A-signaling (29). The results suggested that A-signaling is required in the first few hours of development for the expression of most developmental genes. The response to A-signaling involves the SasS-SasR-SasN histidine kinase-response regulator-negative regulator three-component signal transduction system (59, 60). By measuring expression of Tn5 lac fusions in a csgA mutant, it was revealed that C-signaling begins to be required at about 6 h into development (27). Genes induced after 6 h exhibited either reduced expression in a csgA mutant, or no expression. This partial or absolute dependence on csgA for expression appears to reflect dependence on extracellular C-signaling, because in every case tested, expression in the csgA mutant could be restored by codevelopment with wild-type cells, which provide C-signal in the mixture (4, 10, 11, 27). Only one component of the C-signal transduction pathway is known, FruA (8, 40). This protein is similar to response regulators of two-component signal transduction systems. It has a critical aspartate residue that is thought to be phosphorylated in response to C-signal, although the putative kinase has not been identified. The direct targets of FruA regulation are also unknown. Just as rippling, mound formation, and sporulation require different levels of C-signaling, the expression of different developmental genes requires distinct threshold levels of C-signal (24, 31). How the C-signal transduction pathway establishes such thresholds to ensure proper temporal and spatial regulation of C-signal-dependent genes is an important question. To investigate the regulation of C-signal-dependent genes, the promoter regions of several have been identified and characterized. Mutational analyses of the Ω4403 promoter region (56), which exhibits absolute dependence on C-signaling (11, 27), and of the Ω4400 and Ω4499 promoter regions (63, 64), which exhibit partial dependence (4, 10, 27), have defined critical cis-acting DNA elements. One motif found in all three promoter regions is a C box (consensus sequence CAYYCCY, in which Y means C or T) preceded 6 to 8 bp upstream by a 5-bp element (consensus sequence GAACA) (56, 63, 64). Although these sequences are crucial for the activity of all three promoters, the pattern of effects on expression of single base pair mutations in these sequences is different for each of the three promoters, indicating that the motif functions differently in each case. It has been proposed that a family of related transcription factors might recognize the C box and 5-bp element (and in some cases the sequence in between), each binding to a specific promoter region and activating transcription (63, 64). Here, we report the identification of the Ω4406 promoter region. Expression of the Ω4406 promoter depends absolutely on C-signaling, like that of the Ω4403 promoter, and both are expressed with similar timing during development. Interestingly, expression of both depends strongly on the location in the M. xanthus chromosome. We show that a DNA segment located 500 to 1,300 bp downstream of the Ω4406 promoter inhibits expression of a lacZ reporter when a transcriptional fusion is integrated into the M. xanthus chromosome by site-specific recombination at a phage attachment site but not when the fusion is present at the native Ω4406 location. We believe this is the first case in M. xanthus of a chromosomal position-dependent effect on gene expression attributable to a DNA segment outside the promoter region. We discuss possible mechanisms of the position effect, and we discuss the sequence of the Ω4406 promoter region in comparison with other C-signal-dependent promoter regions.