Those harboring a clean deletion of liaR were identified using PCR. Bacillus subtilis wild-type and mutant strains were inoculated from fresh overnight cultures and grown aerobically in LB medium until an OD600 nm of c. 0.5. The cultures were split into 1 mL samples and different concentrations of
rhamnolipids were added. The effect of rhamnolipids GW-572016 cell line on cell density of each sample was monitored over a period of 7 h. Genome-wide expression profiling is a powerful approach to characterize the response to a certain stimulus, such as the presence of antimicrobial compounds. It has also been used to gain insights into inhibitory mechanisms and to differentiate between different modes of action of novel antibiotics (Hutter et al., 2004; Fischer & Freiberg, 2007; Wecke et al., 2009). We used genome-wide DNA microarray analysis to investigate the response of the model organism B. subtilis to the presence of rhamnolipids, which have been shown to affect cell envelope integrity (Vasileva-Tonkova et al., 2011). B. subtilis was treated with sublethal concentrations (50 μg mL−1) of rhamnolipids, which is sufficient to induce a transcriptional response, but does not impair growth of the culture, as can be demonstrated NVP-BGJ398 mw by concentration-dependent lysis curve experiments (see below and Fig. 3). After 10 min of induction,
total RNA was prepared and DNA microarray analysis performed. Expression of 40 loci was ≥fivefold increased by rhamnolipids compared Montelukast Sodium with the mRNA levels of an uninduced culture (Table 3 and Fig. 1a). Almost half
of these loci can be assigned to known regulons of TCS or ECF σ factors. The most strongly induced locus was the liaIHGFSR operon (c. 640-fold), which is autoregulated by the LiaRS TCS (Mascher et al., 2004). The first two genes of this locus, liaIH, represent the main targets of LiaRS-dependent signal transduction and liaH encodes a phage-shock protein homolog. The LiaRS TCS is activated by cell wall antibiotics, especially lipid II-interacting compounds, but it does not mediate resistance against most of its inducers (Mascher et al., 2004; Wolf et al., 2010). Strong expression of the lia locus also resulted in significant read-through transcription of the downstream located gerAAABAC operon, which has been observed previously for both B. subtilis and Bacillus licheniformis (Mascher et al., 2003; Wecke et al., 2006). The genes htrA (c. 60-fold) and htrB (c. 25-fold), both encoding serine proteases, were also strongly induced by rhamnolipids (Table 3 and Fig. 1a). Expression of both genes is controlled by the TCS CssRS, which is activated by heat and secretion stress. Expression of cssRS itself was not induced by rhamnolipids, similar to the effect of heat stress, although moderately increased expression of this operon can be observed under secretion stress conditions caused by overexpression of the secretory protein α-amylase (Darmon et al.