The lipid-rich cell wall is a defining feature of Mycobacterium species. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant of Mycobacterium smegmatis mc 2 155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to the lsr2 gene. First identified as an immunodominant T-cell antigen of Mycobacterium leprae, lsr2 orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that the M. smegmatis lsr2 mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolatecontaining compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (␣-or ␣-mycolate) molecule is esterified to a glycerol. Upon complementation with an intact lsr2 gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation of M. smegmatis.The cell wall is a defining feature of mycobacteria. This complex, lipid-rich, hydrophobic structure is responsible for the acid-fast staining properties, distinctive colony morphology, and innate antibiotic resistance of Mycobacterium species (12,25,28). Among pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis, cell wall components contribute to virulence, persistence within macrophages, and modulation of the host immune response (16, 45).The cell wall forms an asymmetric lipid bilayer (25,28). The inner leaflet is composed of mycolic acids that are covalently bound to arabinogalactan, which is further linked to peptidoglycan via a phosphodiester bridge (12). The outer leaflet contains a variety of lipid components (26,28). In total, lipids comprise 60% (wt/wt) of the cell wall (12, 25). In addition to mycolic acids, various types of complex lipids are present in the cell wall. These include lipoglycans (e.g., lipoarabinomannan [LAM]), trehalose-containing glycolipids, phthiocerol dimycocerosates, phenolic glycolipids, and glycopeptidolipids (GPLs) (12, 25). The distribution of these lipids varies among mycobacterial species (12). Triacylglycerols (TAGs) are also present in the mycobacterial cell wall (35) and are thought to fill the gap between the meromycolate arm and the shorter ␣-chain of mycolic acids (28). Different lipids appear to have different roles. For example, LAM from M. tuberculosis, but not the structurally distinct LAM of nonpathogenic mycobacteria,...

SummaryLipooligosaccharides (LOSs) are antigenic glycolipids that are present in some species of Mycobacterium including the Canetti strain of M. tuberculosis. The core LOS structures from several mycobacterial organisms have been established, but the biosynthetic pathways of LOSs remain unknown. In this study, we describe two transposon insertion mutants of M. marinum that exhibit altered colony morphology. Cell wall analysis reveals that the MRS1271 mutant is defective in the synthesis of LOS-II, whereas the MRS1178 mutant accumulates an intermediate between LOS-I and -II. The genetic lesions were localized to two genes, MM2309 and MM2332. MM2309 encodes a UDP-glucose dehydrogenase that is involved in the synthesis of D-xylose. MM2332 is predicted to encode a decarboxylase. These two genes and a previously identified losA gene are localized in a gene cluster likely to be involved in the biosynthesis of LOSs. Our results also show that LOSs play an important role in sliding motility, biofilm formation, and infection of host macrophages. Taken together, our studies have identified, for the first time, a LOS biosynthetic locus. This is an important step in assessing the differential distribution of LOSs among Mycobacterium species and understanding the role of LOSs in mycobacterial virulence.