We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast-and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.The growth and nutritional requirements of mycobacteria have been intensely studied since the discovery of Mycobacterium tuberculosis (32). This resulted in an overwhelming body of literature on the physiology of mycobacterial metabolism in the years before the dawn of molecular biology (20,53,54). Carbon metabolism of mycobacteria has attracted renewed interest since the discovery that M. tuberculosis relies on the glyoxylate cycle for survival in mice (36,41). This observation indicates that M. tuberculosis uses lipids as the main carbon source during infection. On the other side, genes that encode a putative disaccharide transporter were essential for M. tuberculosis during the first week of infection, indicating that M. tuberculosis may switch its main carbon source from carbohydrates to lipids with the onset of the adaptive immune response (61). However, the nutrients and the corresponding uptake proteins are unknown for M. tuberculosis inside the human host. Surprisingly, this is also true for M. tuberculosis growing in vitro and for Mycobacterium smegmatis, which is often used as a fast-growing, nonpathogenic model organism to learn more about basic mycobacterial physiology. There is no doubt that the uptake pathways have been adapted to the habitats of M. tuberculosis and M. smegmatis, the human body and soil, respectively. Thus, much can be learned about the lifestyles of both organisms by a comparison of the complements of specific nutrient uptake proteins. Previously, 38 ATP-binding cassette (ABC) transport proteins...

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MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic ␤-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surfaceexposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic ␤-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nmlong part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic ␣-helices or ␤-sheets are integrated into a lipid membrane.Mycobacteria are surrounded by an inner membrane and a giant macromolecule consisting of peptidoglycan, arabinogalactan, and very long chain fatty acids, the mycolic acids (1). Minnikin (2) originally proposed that the mycolic acids, which are covalently bound to the arabinogalactan-peptidoglycan co-polymer, form the inner leaflet of an unique outer membrane (OM).3 Experimental evidence for this model was provided by x-ray diffraction studies, which showed that the mycolic acids are oriented in parallel and perpendicular to the plane of the cell envelope (3). The presence of an organized structure of extremely low fluidity in an isolated mycobacterial cell wall was confirmed by differential scanning calorimetry (4). The vast abundance of non-covalently bound lipids in mycobacteria is assumed to constitute the outer leaflet of a supported asymmetric lipid bilayer. The analysis of spin-labeled fatty acids inserted into isolated mycobacterial cell walls by electron paramagnetic resonance supported the existence of a moderately fluid outer leaflet, whereas the inner leaflet had an extremely low fluidity (4, 5). This interpretation is consistent with observations that mutants with defects in the production of some of the major extractable lipids (glycopeptidolipids, phthiocerol dimycocerosate) showed an increased OM permeability (6, 7). Thus, the mycobacterial OM resembles a supported asymmetric lipid bilayer ...

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.Phosphorus is indispensable for the biosynthesis of nucleic acids and phospholipids and for the energy supply of any cell. Bacteria employ sophisticated transport mechanisms to acquire phosphorus-containing nutrients from the environment. In gramnegative bacteria, phosphates first need to cross the outer membrane (OM). To this end, Escherichia coli produces the two general porins, OmpF and OmpC, under conditions of phosphate excess. Under phosphate-limiting conditions, these porins are partially replaced by the pore protein PhoE (30), which preferentially allows the diffusion of anions (1), in contrast to the cation preference of OmpF and OmpC (29). Hence, the diffusion of phosphates through PhoE pores is more efficient and is the prevalent pathway for phosphates across the outer membrane under phosphate-limiting conditions (17). While inorganic phosphate is the preferred source of phosphorus, many bacteria can also take up organic phosphates and release phosphate by the action of periplasmic phosphatases such as PhoA. E. coli possesses four transport systems, Pst, Pit, GlpT, and UhpT, that translocate inorganic phosphate across the inner membrane (48). Part of the Pst system is the periplasmic protein PstS, which binds and transfers phosphate to the transmembrane components PstA and PstC. PstB hydrolyzes ATP and delivers energy for the translocation of phosphate across the inner membrane by PstA/PstC. Pst systems bind and transport phosphate with bindin...

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