Genetic and Molecular Analysis of cglB, a Gene Essential for Single-Cell Gliding in Myxococcus xanthus

Myxococcus xanthus is a rod-shaped, gram-negative soil bacterium that translocates on solid surfaces in the direction of the cell’s long axis, in a mode of movement called gliding motility (8, 30). Translocation by gliding motility is found in many phylogenetically unrelated prokaryotes. In M. xanthus, the microscopic gliding movements of individual cells are coordinated, leading to macroscopically visible expansion of swarms on an agar plate (14). These swarms originate from the edge of a colony and are observed as flare-like projections, with single cells moving ahead of groups. Thus, a wild-type colony exhibits an undefined edge. Genetic analyses of gliding motility, studies on swarm expansion, and single-cell tracking experiments have revealed that cellular movements of M. xanthus are controlled by two extensive gene systems, the A (adventurous)- and S (social)-motility systems as well as mgl and frz genes (3, 6, 11, 12, 14, 30, 32). The A-motility system controls gliding motility of individual cells (11), while the S-motility system is essential for movement of cells in groups (12). The A- and S-motility systems are complementary in the sense that colonies of cells deficient in both systems (A− S− double mutants) do not swarm (11). The systems differ in that close cell-to-cell proximity is required only for S-motility to operate. It has been suggested that the A- and S-motility systems encode for two different motors promoting surface translocation (11, 12, 31, 36, 38–40). Recent genetic and molecular studies on the S-motility system have shown the sglI region of M. xanthus to encode the genes required for structure, export, and function of type IV pili (13, 36–39). In a variety of diverse bacteria, type IV pili have been postulated to be involved in a mode of surface translocation known as twitching motility (5, 9, 27). Thus, S-motility in M. xanthus may be related to type IV pilus-dependent twitching movements. Gliding movements of individual M. xanthus cells occur in the absence of any visible cell organelle. The molecular structure of the gliding motor and the physics of force generation remain unknown. Since the A-motility system controls gliding of individual, isolated cells (11) (see below), it is likely that the genes of the A-motility system include those genes that encode components of the gliding motor. More than 37 genes are known to affect A-motility, and mutations in these genes, in contrast to those in S-motility genes, cause defects primarily in gliding motility rather than in gliding as well as in fruiting body formation (11, 20, 21). Colonies of such A− S+ cells exhibit a sharp, highly delineated edge, with no single cells present at the colony’s perimeter. This characteristic colony morphology is due to the inability of single cells to glide when separated from other cells by more than 2 μm (11) (see below). A-motility genes can be further divided into two subclasses, the agl genes and the cgl genes, based on an additional mutant phenotype (10, 11). If the defect in single-cell gliding of an A-motility mutant can be complemented by extracellular rescue, i.e., by mixing with wild-type cells or mutant cells of another motility class, then the gene is designated cgl (for contact or conditional gliding). Typically, this extracellular rescue is only transient though sufficient to allow microscopic observation of flare formation at swarm edges. A-motility mutants that cannot be rescued are called agl (adventurous gliding) mutants. The peculiar phenotype of cgl mutants suggests that the cgl gene products may function either as mechanical elements of the gliding motor that are associated with the outer membrane, as regulators of its activity, or as both. Five cgl loci, cglB, cglC, cglD, cglE, and cglF, have been identified, and these loci have been mapped by using transposon mutagenesis in conjunction with Mx8 cotransduction experiments (11, 29). During preliminary experiments, we identified cglB mutants as having a strong motility defect and a clear stimulation response. We have exploited these properties to conduct a molecular analysis of cglB in M. xanthus.