In a tributyrin assay, we observed that mutation D47A or D47E did not affect the halo (Fig. 5), strongly suggesting that this position is not phosphorylated during signalling. In contrast, mutation of D52 to alanine abrogates lipase activity, pointing out that D52 needs

to be phosphorylated for LipR regulatory activity. Interestingly, mutation D52E restores the halo. This indicates that the glutamate can mimic IWR-1 chemical structure the phosphorylated aspartate as was suggested for NtrC (Klose et al., 1993). In conclusion, transcription of lipA in P. alcaligenes is regulated by the combined action of σ54 and response regulator LipR. Further analysis of the system is likely to offer RG-7204 new possibilities to steer the lipase production levels to a higher scale and will contribute to a further understanding of this important class of

bacterial enhancer-binding proteins. We thank Dr Susanne Wilhelm for providing us the pTZ110 vector, and Valerie R. Wiersma and Marjolein Garsen for their assistance in mutagenesis and analysis of LipR. This research was sponsored by EU grant QLK3-CT-2002-02086 and MEST-CT-2005-020. R.H.C. was partly supported by the European Community Initiative Interreg IIIA and W.Q. partly by KOP-EFRO. Part of this work was published as a dissertation: (http://dissertations.ub.rug.nl/FILES/faculties/science/2009/j.k.krzeslak/04c4.pdf) “
“Because of the emergence of strains of Mycobacterium tuberculosis resistant to first-line antituberculosis agents, one of the second-line drugs, p-aminosalicylate (PAS), has regained importance

in the treatment of tuberculosis. The mode of action of PAS, however, remains controversial as to whether it inhibits mycobactin or folate biosynthesis. To unravel this, we have studied the effect of PAS on wild-type Mycobacterium smegmatis and its mutants (gene knockouts of the salicylate pathway –trpE2, entC and entD). The wild type had no sensitivity to PAS (MIC>400 μg mL−1), whereas the mutants were hypersensitive, with 1 μg mL−1 inhibiting growth. The sulphonamides, trimethoprim and dapsone, had little effect on the growth of either the mutants ifenprodil or the wild type. In addition, PAS at 0.5 μg mL−1 increased the accumulation of salicylate with the wild type and mutants. These results support our hypothesis that PAS targets the conversion of salicylate to mycobactin, thus preventing iron acquisition from the host. Because of the emergence of strains of Mycobacterium tuberculosis that are resistant to currently used drugs, both singly and as multidrug combination therapies, new chemotherapeutic targets need to be identified and thus new drugs need to be created.