A defined medium to investigate sliding motility in a Bacillus subtilis flagella-less mutant

We have recently shown that undomesticated strains of Bacillus subtilis can extensively colonize the surfaces of rich, semi-solid media, by a flagellum-independent mechanism and suggested that sliding motility is responsible for surface migration. Here we have used a flagella-less hag null mutant to examine and confirm sliding motility. Using a defined semi-solid medium we determined that a B. subtilis hag mutant colonized the surface in two stages, first as tendril-like clusters of cells followed by a profuse pellicle-like film. We determined the levels of macro- and micro-nutrients required for the tendril-to-film transition. Sufficient levels of each of the macronutrients, glycerol, Na-glutamate, and Na-phosphate, and inorganic nutrients, K+, Mg2+, Fe2+ and Mn2+, were required for robust film formation. The K+ requirement was quantified in more detail, and the thresholds for complete tendril coverage (50 μM KCl) or film coverage (2–3 mM KCl) were determined. In addition, disruption of the genes for the higher affinity K+ transporter (KtrAB), but not the lower affinity K+ transporter (KtrCD), strongly inhibited the formation of both tendrils and films, and could be partially overcome by high levels of KCl. Examination of hag tendrils by confocal scanning laser microscopy revealed that tendrils are multicellular structures, but that the cells are not as highly organized as cells in wild-type B. subtilis pellicles. These results suggest that B. subtilis can use sliding motility to colonize surfaces, using a tendril-like growth mode when various macronutrients or micronutrients are limiting. If nutrients are balanced and sufficient, the surfaces between tendrils can be colonized by robust surface films. Sliding motility may represent a strategy for nutrient-deprived cells to colonize surfaces in natural environments, such as plant roots, and the media described here may be useful in investigations of this growth phenotype.