2). FK77 (ΔbceRS), MM02 (ΔbceAB) and MM03 (ΔvraDE) strains reduced their resistance level, revealing hetero-resistance to bacitracin. MM03

in particular showed a significant reduction compared with two other mutants, FK77 and MM02. In wild-type MW2 strain, the expression of bceA and vraD was rapidly induced by the addition of bacitracin into the medium (Fig. 3). This induction was found to occur after 5 min of bacitracin exposure. The level of vraD transcript was more than 100-fold that of the wild type. Interestingly, a low concentration of bacitracin (0.5, 1 μg mL−1) significantly induced the expression of two genes (bceA and vraD) until 15 min, after which the expression of both decreased. In contrast, bacitracin (above 8 μg mL−1) continued Afatinib to induce these two genes even after 15 min. The level of the TCS (bceR) transcript itself did not increase by addition of bacitracin after 30 min, but slightly increased after 60 min (data not shown). The level of vraF transcript was not increased

by bacitracin (data not shown). The expressions of bceA and vraD in MM08 (ΔbceS) were not induced by addition of bacitracin, while the induction of their expressions by bacitracin were observed in the strain (MM09) which complemented with the gene for bceS in MM08 (Fig. 3). Also, in FK77 (ΔbceRS) strain, induction of the expression of bceA and vraD upon the addition of bacitracin was completely inhibited. check details In this study, we identified one uncharacterized TCS (MW2545-44) that has the ability to sense bacitracin and positively regulates two transporters responsible for bacitracin resistance. Interestingly, this TCS regulates not only a downstream gene coding for ABC transporter (MW2543-42), but also another transporter (MW2620-2621) known as vraDE, which is located separately

from the genes coding for the TCS. These two ABC transporters, especially VraDE, showed a high similarity with B. subtilis ABC transporter Nintedanib (BIBF 1120) BceAB responsible for bacitracin efflux (Ohki et al., 2003). In addition, similar transporters showing homology with BceAB have been reported in several Gram-positive bacteria, such as BcrAB of B. licheniformis (Podlesek et al., 1995), BcrAB of Enterococcus faecalis (Manson et al., 2004) and MbrAB of Streptococcus mutans (Tsuda et al., 2002). This type of ABC transporter is considered to pump out bacitracin to the exterior of the cell directly. Upstream of bceAB in B. subtilis, the gene coding for TCS, known as BceRS, is located (Ohki et al., 2003; Rietkötter et al., 2008). BceRS has been demonstrated to sense bacitracin and induce the expression of BceAB, leading to resistance to bacitracin. Like bceRSAB in B. subtilis, the results in this study showed that S. aureus has the same system, so we designated this TCS as BceRS (MW2545-2544) and the downstream transporter as BceAB (MW2543-2542). In S. aureus, another gene, MW2546, was closely located upstream of bceRS, speculating three genes were in a same operon (Fig. 1).