SummaryMycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis . In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis . This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis . Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the Δ Δ Δ Δ mspA and Δ Δ Δ Δ mspA Δ Δ Δ Δ mspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15-and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA , mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli . These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis .

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...

Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of Mycobacterium smegmatis lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the mspA gene caused high-level resistance of M. smegmatis to 256 g of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the mspA mutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were increased 2-to 10-fold. This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the mspA mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the mspA mutant highlights the prominent role of outer membrane permeability for the sensitivity of M. smegmatis to antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.The prevalence and spread of antibiotic resistance are increasingly serious problems that hamper the effective treatment of infectious diseases (26). The search for new antibiotics is mainly based on novel bacterial targets and high-throughput screening assays (10). However, many lead compounds discovered in vitro may fail because they do not reach their targets at sufficiently high concentrations in vivo (7). This is true in particular for gram-negative bacteria, which, in contrast to grampositive bacteria, are protected from the toxic actions of certain antibiotics, dyes, and detergents and host defense factors such as lysozyme by an additional outer membrane (OM) (49). The OM can be crossed by at least two general pathways: the hydrophobic (or lipid) pathway, which is characterized by the nature and the interactions of the membrane lipids, and the hydrophilic (or porin) pathway, whose properties are determined by water-filled channel proteins, the porins, which span the OM of gram-negative bacteria (49). It has been shown by the pioneering work of Nikaido and collaborators (21, 46) that Escherichia coli and Salmonella porins play a major role in the transport of ␤-lactam antibiotics. Subsequent studies showed that porin-deficient mutants of gram-negative bacteria were also more resistant to quinolones, tetracyclines, chloramphenicol, nalidixic acid, and trimethoprim (6,15,25,52). These data suggest that porins are involved in the transport...

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin – the second most abundant polysaccharide on earth – is a decisive evolving advantage as it is a source for both elements. Streptomycetes are high-GC Gram-positive soil bacteria that are equipped with a broad arsenal of chitinase-degrading genes. These genes are induced when the streptomycetes sense the presence of chitooligosaccharides. Their expression is repressed as soon as more readily assimilated carbon sources become available. This includes for example glucose or N-acetylglucosamine, the monomer subunit of chitin. Historically, the first cis-acting elements involved in carbon regulation in streptomycetes were found more than a decade ago upstream of chitinase genes, but the transcriptional regulator had so far remained undiscovered. In this work, we show that these cis-acting elements consist of inverted repeats with multiple occurrences and are bound by the HutC/GntR type regulator DasR. We have therefore designated these sites as DasR-responsive elements (dre). DasR, which is also the repressor of the genes for the N-acetylglucosamine-specific phosphotransferase transport system, should therefore play a critical role in sensing the balance between the monomeric and polymeric forms of N-acetylglucosamine.

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