For reason of clarity, we limited our analysis to genes induced ≥threefold and repressed ≥fivefold by rhamnolipids. Genes controlled by the same regulator form discrete clusters based on their expression pattern under different stress conditions (Fig. 2a). Genes belonging to the cell envelope stress response of B. subtilis are grouped in three clusters and can be assigned to two regulators, σM and the LiaRS TCS (Fig. 2b).

They are induced by cell wall antibiotics and rhamnolipids, but not by secretion stress (with the exception of liaH). One of these three clusters contains the target operon of the LiaRS TCS as well as the downstream genes gerAAAB. The other two clusters include mostly target genes of σM. Noteworthy, within the σM regulon, there is a subset of genes, including the mreBCDminCD operon involved in cell division, that is not induced by vancomycin (upper part of σM1 cluster in Fig. 2b). Differences in the induction check details profiles of subsets of σM-dependent

genes have been observed previously (Eiamphungporn & Helmann, 2008). Genes mediating the secretion stress response also cluster together (Fig. 2b). The CssRS-dependent target genes htrA and htrB are ICG-001 datasheet not only induced by secretion stress and rhamnolipids, but also weakly by vancomycin and bacitracin. Genes repressed by rhamnolipids show almost unchanged expression under the other conditions tested (Fig. 2c). One exception is the pyr operon, which is strongly repressed by rhamnolipids,

but weakly induced by friulimicin and vancomycin. Taken together, the hierarchical clustering analysis indicates that rhamnolipids induce a combination of two different stress responses: the cell envelope stress response represented by the LiaRS TCS and the ECF σ factor σM, and the heat and secretion stress response mediated by CssRS. Simultaneous induction of the LiaRS TCS and σM is common for cell wall antibiotics such as daptomycin, vancomycin, or bacitracin (Mascher et al., 2003; Hachmann et al., 2009; Wecke et al., 2009). But none of the σM-dependent target genes is induced by secretion stress, while both the CssRS and LiaRS TCS are induced by cell wall antibiotics, rhamnolipids, and secretion stress, but with different intensities OSBPL9 (Fig. 2d). Bacteria use signal transducing systems to detect harmful compounds and alter gene expression to protect the cell. We hypothesize that the signal transducing systems activated by rhamnolipids confer resistance and counteract cell damage caused by this antimicrobial compound. Therefore, we compared the growth behavior of B. subtilis wild-type cultures exposed to different rhamnolipid concentrations with strains carrying gene deletions leading to ‘ON’ or ‘OFF’ states of the induced signal transducing systems, which results either in no or constitutively high expression of the corresponding target genes.