A screen for nonsliding mutants of Mycobacterium smegmatis yielded 20 mutants with transposon insertions in the mps gene, which is involved in glycopeptidolipid biosynthesis. One mutant had an insertion in a gene predicted to encode a membrane transport protein. All mutants lacked glycopeptidolipids and were unable to form biofilms on polyvinyl chloride.Recently, it has been shown that in spite of being nonflagellated microorganisms, mycobacteria can spread on the surface of solid growth medium by a sliding mechanism (9). This form of surface motility is produced by the expansive forces of the growing bacterial population, in combination with cell surface properties that favor reduced friction between the cells and the substrate, and it results in the slow movement of a uniform monolayer of cells as a unit (8). A time-lapse movie of sliding Mycobacterium smegmatis can be seen at http://gasp.med.harvard.edu/smegmatis/sliding.html. Both the fast-growing nonpathogenic M. smegmatis and the slow-growing opportunistic pathogen Mycobacterium avium are able to slide, and in both species this ability correlates with the presence of GPLs (9), a class of glycosylated peptidolipids present in the outermost layer of the cell envelope (11). However, no direct link between GPLs and sliding could be established, since the GPLdeficient strains in previous studies were not characterized genetically. Here we report the results of the first genetic analysis of sliding motility. Mutants unable to slide on motility plates lack GPLs and are also unable to form biofilms on polyvinyl chloride (PVC) plates.Genetic screen for M. smegmatis nonsliding mutants. In order to gain more insight into the mechanism driving sliding motility in M. smegmatis mc 2 155, a screen was set up to look for genes that when disrupted would result in the inability to slide on the surface of plates containing 0.2% glucose-M63 salts medium solidified with 0.5% agarose (sliding medium). A mariner-based transposon (17) was used that contained a kanamycin resistance gene (kan) as a selectable marker. The temperature-sensitive plasmid pMycoMar, harboring the marinerderived transposon, has been shown to work as an efficient transposon delivery system in M. smegmatis, resulting in random insertions in the genome (17). The temperature-sensitive mycobacterial replicon allows for the direct selection of transposition events on kanamycin-containing plates incubated at 40°C. A screen of 4,000 random transposon insertion mutants yielded a total of 21 nonsliding mutants that grew on the sliding medium but did not form the translucent halo surrounding the point of inoculation characteristic of wild-type mc 2 155 (Fig. 1A). All the nonsliding mutants had rough colony morphology and showed no detectable levels of GPLs by thin-layer chromatography (TLC) analysis (results not shown).Nonsliding mutants have transposon insertions in mps. The DNA sequence of the transposon insertion site for 20 of the nonsliding mutants was obtained using the arbitrary PCR technique, as previously ...

Mycobacteria are nonflagellated gram-positive microorganisms. Previously thought to be nonmotile, we show here thatMycobacterium smegmatis can spread on the surface of growth medium by a sliding mechanism. M. smegmatis spreads as a monolayer of cells which are arranged in pseudofilaments by close cell-to-cell contacts, predominantly along their longitudinal axis. The monolayer moves away from the inoculation point as a unit with only minor rearrangements. No extracellular structures such as pili or fimbriae appear to be involved in this process. The ability to translocate over the surface correlates with the presence of glycopeptidolipids, a mycobacterium-specific class of amphiphilic molecules located in the outermost layer of the cell envelope. We present evidence that surface motility is not restricted to M. smegmatis but is also a property of the slow-growing opportunistic pathogen M. avium. This form of motility could play an important role in surface colonization by mycobacteria in the environment as well as in the host.