The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of d -arabinofuranose (Ara f ) residues. The immediate precursor of the polymerized Ara f is decaprenylphosphoryl- d -Ara f , which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Ara f is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-β- d -5-phosphoribose. (ii) Decaprenylphosphoryl-β- d -5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-β- d -ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-β- d - erythro -pentofuranose. (iv) Decaprenylphosphoryl-2-keto-β- d - erythro -pentofuranose is reduced to form decaprenylphosphoryl-β- d -Ara f . Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

MmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis ; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis . Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis , including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.

Summary: Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non‐replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low μM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non‐replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Background: Sensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis. Results: We demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway. Conclusions: The Wag31_Mtb phosphorylation is a novel molecular mechanism by which Wag31_Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria.

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N -glycolylmuramic rather than N- acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis . The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N- acetyl and N- glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N -acylmuramyl-pentapeptide precursor molecules also contain a mixture of N- acetyl and N- glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d -cycloserine consisted entirely of N- glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d -cycloserine contained only N- glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis , consisted of the N -glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N- glycolyl and N- acetyl products. Thus, treatment with vancomycin and d -cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N- acetylmuramyl- l -alanine- d -glutamate- meso -diaminopimelic acid- d -alanyl- d -alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N -acetylmuramic acid, N- glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso -diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal d -alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.