subtilis 168, YH2M (MW) and the double

mutant 8R. As shown in Fig. 6a, the half-life of 168 and single-mutant MW was ≈1.5 min, whereas the half-life of 8R was calculated to be ≈3 min. This twofold increase of the half-life of the double-mutant must be due to a contribution of single-mutant WM at position +6, demonstrating that this mutation leads to the stabilization of the bmrA mRNA. Figure 6b shows the mRNA secondary structures predicted for the bmrA 5′ untranslated region. The transition at position +6 leads to a change of the predicted structure and a decrease in Gibbs free energy ΔG. According to http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi, the first stem–loop is stabilized. This is in accordance with previous observations on the mRNA-stabilizing function of 5′-terminal stem–loops (Hansen et al., 1994; Hambraeus et al., 2000). Because antibiotic resistance is most often only transiently advantageous to bacteria, an efficient way to escape the Silmitasertib molecular weight lethal action of drugs BKM120 mw is the regulation of resistance gene expression at the transcriptional or the translational level following mutations or the movement of mobile genetic elements (Depardieu et al., 2007). Piddock (2006) reported that chromosomally encoded efflux pumps may be overexpressed due to mutations in the local repressor, mutations

in global regulatory genes, promoter mutations or insertion sequences. In an induction experiment, we confirmed the finding of Steinfels et al. (2004) that bmrA is not inducible by any specific substrate. Furthermore, using EMSA and a radioactively labelled fragment of the bmrA upstream region, no specific binding protein acting as an activator or a repressor could be identified in crude protein extracts of the mutant or the wild-type strain (data not shown). Instead, we identified a mechanism of adaptation without fine-tuning, resulting in antibiotic resistance by constitutively upregulated expression of a specific protein. Such proteins may encompass ABC transporters, permeases, transcription

factors or sigma factors. For instance, Stirrett et al. (2008) reported the upregulated expression of several efflux pumps in Y. pestis by overexpression of the transcriptional regulator RobAYp from a multicopy plasmid. So far, spontaneous constitutively resistant mutants in Gram-positive bacteria revealing overexpression due to promoter mutations have only ifenprodil been detected in a few cases (Piddock, 2006). For instance, the triclosan efflux pump of Pseudomonas aeroginosa was upregulated by a mutation in the −35 region of the promoter (Mima et al., 2007), while in M. smegmatis a G to T transversion in the −10 region of the promoter increased the copy number of the d-alanine racemase conferring resistance to d-cycloserine (Cáceres et al., 1997). Similar data were obtained by Ohki & Tateno (2004), who reported the increased expression of the bmr3 efflux transporter due to a +4 mutation that also resulted in the stabilization of the corresponding bmr3 mRNA.