The acid-fastness of all mycobacteria is based upon a shared universal cell wall core structure. The mycobacterial cell wall consists of an outer lipid layer and an inner peptidoglycan layer. The outer layer is highly impermeable and is composed of unique 70 -90 carbon-containing lipids, known as mycolic acids. The mycolic acids are esterified to the non-reducing terminal arabinosyl residues of the polysaccharide arabinogalactan (1-5). The reducing end of arabinogalactan is connected to the peptidoglycan via the disaccharide linker, ␣-L-Rha-(133)␣-DGlcNAc-(13phosphate). Structural analyses showed that the integrity of the whole two-layer mycolic acid peptidoglycan assembly hinges on the presence of the rhamnosyl moiety as depicted in Fig. 1A. The complete structure of the linker is illustrated in Fig. 1B, and the reaction catalyzed by the enzyme, dTDP-Rha:␣-D-GlcNAc-pyrophosphate polyprenol, ␣-3-Lrhamnosyltransferase (referred to as rhamnosyltransferase in this study) is shown in Fig. 1C. The rhamnosyl residue and much if not all of the arabinogalactan polysaccharide are synthesized on GlcNAc-P-P-decaprenyl carrier lipid (6). The eventual transfer of the arabinogalactan-Rha-GlcNAc-phosphate unit to the O-6 of a muramic acid places the polysaccharide in mass onto the peptidoglycan. Finally, at some still to be defined point, the mycolic acids are attached to arabinofuranosyl residues at the non-reducing end of arabinogalactan.To further define and characterize the essential steps involved in the synthesis of the mycobacterial cell wall core, the classic microbial approach of isolating conditional lethal mutants was undertaken. Our strategy was to isolate temperature-sensitive (TS) 1 mutants in the genetically amenable and relatively fast growing Mycobacterium smegmatis mc 2 155 (7). A preferred large temperature range that would support growth precluded Mycobacterium tuberculosis from serving as the host for the generation of TS mutants. TS mutants would be genetically complemented with M. tuberculosis genomic DNA in hopes of identifying essential genes encoding cell wall biosynthetic enzymes. Herein, we describe the isolation of a TS cell wall mutant and the independent genetic complementation of that mutant with a M. tuberculosis gene and an E. coli gene. We report biochemical characterization of the TS mutant, the deduced amino acid change due to the mutation, the genetic complementation of an E. coli mutant to confirm the function of a M. tuberculosis gene, and the effect of the mutation on mycobacterial viability after exposure to non-permissive temperatures. EXPERIMENTAL PROCEDURESIsolation of TS Mutants-The strategy for the isolation and enrichment of bacterial TS mutants in a culture as outlined by A. Morris Hooke (8) was adapted for use in this study. M. smegmatis mc 2 155 (7) was inoculated into Middlebrook 7H9 with ADC supplement (Difco) (7H9) and grown at 37°C to ϳ10 8 colony-forming units/ml. Nitrosoguanidine (Sigma) was added to a final concentration of 0.1 mg/ml, and cultures were incubated at 37°C...