5 (buffered with 100 mM Tricine). No difference was found between the wild-type and pitA mutant strains (not shown). An E. coli pitA mutant Bucladesine price displayed increased resistance to toxic divalent cations (Zn2+ and Cd2+), due to reduced uptake of these ions [22]. The M. smegmatis pitA mutant and wild-type strain were therefore grown on solid media (ST agar, 50 mM MES [pH 7], 1 mM phosphate) containing 1-15 mM

ZnSO4 or CuSO4. Both strains were able to grow in the presence of 1 mM of either salt, but could not grow at concentrations of 5 mM or higher. Taken together, the data presented here suggest that either PitA of M. smegmatis does not transport MeHPO4, or that one or both of the high-affinity systems also recognize such a complex GM6001 mw as substrate. It should be noted that no substrate specificities have been determined to date for a Pst system from a Gram-positive bacterium, or for a Phn system. The pitA mutant displays no defect in phosphate uptake We next determined the rates and kinetics of uptake of [33P]ortho-phosphate, to assess whether the pitA deletion strain had a defect in phosphate uptake. To prevent induction of the Pst or Phn systems, cells were grown in LBT medium as

described in the methods section. As shown in figure 3, maximum uptake rates were 12.9 ± 1.6 nmol min-1 mg protein-1 for the wild-type, and 9.9 ± 1.0 nmol min-1 mg protein-1 EPZ015938 manufacturer for the pitA strain. Kd values were similar between the strains, with 50.1 ± 26 μM phosphate for the wild-type and 27.9 ± 16.4 μM phosphate for the pitA strain. Slight differences in transport rates at the higher phosphate concentrations were not significant (p > 0.2 in unpaired, two-tailed t-test). Figure 3 Kinetics of phosphate uptake. Initial uptake rates of ortho-phosphate (33P, > 92.5 TBq mmol-1) into LBT-grown whole cells of M. smegmatis mc2155 (solid squares) and the pitA deletion strain (open squares) were measured over 60 s at phosphate concentrations between 25 μM and 500 μM. Rates are expressed as nmol phosphate min-1 mg mycobacterial protein extract-1, and data are shown as the mean ± standard error of Sclareol the mean from two to five

independent measurements per point. These kinetic parameters suggest that the rates of transport determined are due to activity of the high-affinity systems, because Kd values of phosphate uptake under phosphate-starved (i.e. Pst and Phn systems induced) conditions were found to be between 40 and 90 μM phosphate [13]. The rates of transport in the present study are about ten-fold lower than those in phosphate-starved cells, consistent with the previously described 20-fold lower expression from the pst and phn promoters under these conditions [13]. PitA of M. smegmatis therefore appears to be either not active, or to have a very low activity, which cannot be detected over the background of the high-affinity systems using the assay employed here.