2). We also ruled out the influence of RpoS on the stimulatory effect of the tolC mutation because the expression of the ΔsbmA∷lacZY fusions in the tolC rpoS double mutant remained as high as in the single tolC mutant (Fig. 2). Taking into account that the tolC mutation increases micF expression, a sRNA that exerts its regulatory effect at a post-transcriptional level, it was unlikely that this was responsible for a direct effect on sbmA transcription. However, an indirect regulation would be possible through a putative protein, whose translation could be affected by MicF. We therefore assayed the expression of the ΔsbmA∷lacZY fusion in a tolC micF double-mutant strain. The strain

was constructed KU-57788 mw by P1 transduction with a micF mutant strain SM3001 (Matsuyama & Mizushima, 1985) as a donor and MC4100 sbmA∷lacZY tolC strains as recipients. Figure this website 2 shows that the tolC mutation was able to affect the sbmA expression even in the absence of micF. Moreover, no difference was found in the expression of sbmA when micF was overexpressed in the strain MC4100 sbmA∷lacZY carrying the micF plasmid (pCX28) (Mizuno et al., 1984) (Fig. 2b). These results led us to suggest that the tolC mutation stimulates the sbmA expression in a micF-independent manner. In order to estimate SbmA levels in the absence of tolC, we evaluated changes in one of the phenotypes

attributed to this protein, the MccB17 uptake. Liothyronine Sodium For this, we determined the MIC to MccB17 of a tolC mutant and compared it with the isogenic wild-type strain. As shown in Table 1, the mutant was 128-fold more sensitive than the wild-type strain. Even though there is no report of TolC’s involvement in MccB17 export, we ruled out this possibility by means of an assay that compared the production and exportation of this microcin in the presence and absence of TolC and no difference was observed (data not shown). The acrB mutant showed the same sensitivity level as the wild-type MC4100 strain (Table 1). This is an expected result,

given that this locus had no effect on the expression of sbmA (Fig. 3). In a tolC mutant, the amount of OmpF is drastically reduced and this effect is due to the activation of micF (Misra & Reeves, 1987). It was also observed that a tolC mutation increases the OmpC levels present in the membrane (Morona & Reeves, 1982). As it was postulated that OmpF plays a role in the MccB17 uptake, the tolC mutation should cause an increase in resistance to MccB17 and not hypersensitivity as we observed (Table 1). Lavina et al. (1986) showed that the OmpC protein is only partially able to replace OmpF with respect to MccB17 importation. Thus, we had to exclude an increased sensitivity to MccB17, in a tolC mutant, as a consequence of a MicF-mediated OmpC enhancement.