The number of porins per cell determines the influx rate of hydrophilic solutes across the OM and was calculated for wild-type M. smegmatis and the porin mutants MN01 (ΔmspA) and ML10 (ΔmspAΔmspC) to 2400, 800 and 150 pores, respectively, using a surface area of 2.4 µm2 for an exponentially growing cell (see Experimental procedures). A striking observation was that the growth rate of M. smegmatis, both in rich and minimal medium and on agar plates, dropped drastically with its porin-mediated OM permeability (Fig. 8). This is in contrast to porin mutants of E. coli and Salmonella typhimurium, which did not show a growth defect in rich media (Henning and Haller, 1975; Nurminen et al., 1976). Only under glucose limitation and in competition experiments, a growth advantage of wild-type E. coli over an ompF mutant was observed (Liu and Ferenci, 1998). Loss of both the OmpF and OmpC porins increased the doubling time of E. coli by 15% (Bavoil et al., 1977). Why do porin mutants of M. smegmatis show a severe growth defect, but porin mutants of E. coli do not? Bavoil and Nikaido found that a 1000-fold reduced residual porin level is sufficient to support growth of E. coli at maximal rate with 0.2% glucose (Bavoil et al., 1977). By contrast, in M. smegmatis a modest 3- and 15-fold reduced number of porins increased the generation time by 30% and 80% respectively. In minimal medium the growth rate of M. smegmatis declined more rapidly with the number of porins than in rich medium (Fig. 8) underlining the greater importance of porins at low nutrient concentration. This has been observed very early for E. coli (Bavoil et al., 1977) and is a consequence of the first Fick's law of diffusion. Higher influx rates are achieved at higher nutrient concentrations and less porins are needed to maintain a constant nutrient influx into the cell under those conditions. The data fit perfectly to a straight line (Fig. 8, open triangles) demonstrating that porin-mediated nutrient influx across the OM is the sole determinant of the growth rate of M. smegmatis porin mutants at low carbon concentrations. This was confirmed by complementation experiments, which restored both the OM permeability to hydrophilic solutes and the growth rate to wild-type levels. However, it is not clear whether the low efficiency of the porin pathway limits the growth rate of wild-type M. smegmatis. Experimental proof of this hypothesis would require to show that a larger number of porins in the wild-type strain would increase the growth rate. However, different mspA expression vectors using strong mycobacterial promoters only increased the initial amount of mspA transcript, but not the number of MspA porins in the OM (D. Hillmann and M. Niederweis, unpublished) indicating that unknown mechanisms limit integration of MspA into the OM. Acceleration of growth was observed after heterologous expression of mspA in M. bovis BCG (Mailaender et al., 2004; Sharbati-Tehrani et al., 2004). Taken together these results establish the slow porin-mediated uptake of hydrophilic nutrients is an important determinant of the slow growth of mycobacteria as proposed earlier by Nikaido and coworkers (Jarlier and Nikaido, 1990). It is likely that slow nutrient uptake across the OM is not the only growth rate-limiting step and that the rates of other cellular processes have been adapted. Indeed, it was observed for E. coli that the effective permeabilities of the inner and the outer membranes are well matched for galactosides for concentrations in the range of 100–200 µM (West and Page, 1984). It should be noted that an excess of porins may also be dangerous for M. smegmatis as its sensitivity to drugs (Stephan et al., 2004a) and to toxic compounds generated by immune cells (Sharbati-Tehrani et al., 2005) is increased in the wild-type compared with porin mutants. These observations underline the importance for M. smegmatis to balance its porin-mediated OM permeability between two mutually exclusive needs of rapid uptake of nutrients and protection from toxic compounds.