Regulating Pilin Expression Reveals a Threshold for S Motility in Myxococcus xanthus

The polar type IV pili of the gram-negative δ-proteobacterium Myxococcus xanthus help it to swarm over the surface of a solid growth medium, like agar (4, 5, 8). The pili also help M. xanthus build fruiting bodies when it begins to starve (7). In addition to the pili, which are called the social (S) engine and which are located at the leading pole of a gliding cell, M. xanthus has engines for adventurous motility located at the trailing pole of the cell (21). Swarming and fruiting body construction depend on coordinating these two polar gliding engines. Pili are long filamentous appendages that are helical polymers that contain tens of thousands of copies of the protein subunit pilin, the pilA gene product (15). To glide, the polar type IV pili of a M. xanthus cell attach to fibrils on other cells and then retract, which pulls the piliated cell forward (14). On both sides of the pilA gene, 16 other pil genes cluster together in the M. xanthus genome. The cluster includes two two-component systems, the pilS-pilR and pilS2-pilR2 systems (18). Little is known about the role of these regulators except that pilR is necessary for pilA expression (24), and a pilR2 knockout mutant is unable to swarm (2). M. xanthus is particularly useful for investigating the molecular mechanisms that underlie gliding motility and its coordination during fruiting body development (7). Transposon insertions of transcriptional reporters and nonpolar in-frame gene deletions have permitted the assignment of functions to several motility genes. Conditional expression of proteins with a nontoxic inducer has been lacking, however, which has limited expression studies and has hindered investigation of the pilus assembly process. Previous studies of the Escherichia coli tac promoter/lac repressor/operator in M. xanthus showed that isopropyl-β-d-thiogalactopyranoside (IPTG) could be used as an inducer of gene expression in M. xanthus (12). Unfortunately, expression of the tac promoter is expected to depend on σA, the major sigma factor of M. xanthus, and σA is lost during the development of M. xanthus (3). The native CarQRS system has also been tested as a means for controlling gene expression in M. xanthus (12). However, this system, which is induced by high-intensity light, interferes with development (13). Here we report a new adaptation of the E. coli lac repressor and operator that was used to construct a chromosome-based system for inducible regulation of gene expression that can function during both growth and development in M. xanthus. With this system, a single-copy gene can be removed from its endogenous regulation by placing it under the transcriptional control of an IPTG-inducible promoter. To test the regulation, we employed the well-characterized pilA (pilin) promoter that depends on the alternative sigma factor σ54 for transcription. This promoter was chosen because it is expressed at a high level during both growth and development (24). To quantify promoter activity, we tested the proportionality among the level of pilA expression, the amount of pili assembled, and the rate of S-motile swarming in M. xanthus.