cereus ATCC14579 genome sequence d Domains detected using SMART search http://​smart.​embl-heidelberg.​de/​ (Letunic et al., 2006 Nucl Acid Res 34: D257-D260). PR; PadR domain; SS, signal sequence; TMS(n), transmembrane AZD2171 nmr segment (n is the number of such domain); PPD, periplasm domain. Proteins with TMS are highlighted in bold Figure 1 Conservation of BC4206-BC4207 operon and surrounding genes in fully sequenced B. cereus, B. thuringiensis and B. weihenstephanensis species. BC4203-BC4212 numbers are depicted as genes are labelled in the genome of B. cereus ATCC14579. Arrows indicate the BC4207 homologue (in grey), PadR

homologues (in black), conserved proteins with putative function: BC4203, BC4205, BC4209 and BC4210 encoding for putative hydrolase, spore lyase, lypoate-protA ligase and rhodase,

respectively (dashed lined), putative conserved regulators: BC4204, BC4211 and BC4212 for putative iron dependent repressor, LacI type regulator and TetR like regulator, respectively (stripped) and other putative genes (in white). Validation of array experiments Real-time RT-PCR was performed on independent samples to validate our array results. To verify that upregulation of the genes were the result of specific treatment with AS-48 and not a general response, we also applied samples that were incubated in the presence of sublethal LY3023414 amount of bacitracin (25 μg/ml) or nisin (2 μg/ml), bacteriocins that both affect cell wall biosynthesis through blocking the lipid II cycle by interaction with C55-isoprenyl pyrophosphate [16] or forming pores in cell membrane during interaction with lipid II [17, 18], respectively. Two genes were selected (BC4207 and BC4028), both coding for a putative membrane protein and both located downstream of a PadR like regulator (BC4206 and BC4029, respectively), for quantitative real time RT-PCR. Quantitative real time PCR showed 26 ± 6 and 18 ± 4 times upregulation of BC4207 and BC4028 in samples treated with AS-48 compared with control samples, respectively (Figure 2). Similar analysis of samples treated

with AS-48 for 15 min showed less O-methylated flavonoid then 2 times induction of the BC4207 and BC4028 genes, in agreement with the lack of significant changes after 15 min of AS-48 treatment in microarray experiments. Samples incubated in the presence of bacitracin showed slightly enhanced expression of target genes, while addition of nisin did not significantly change the transcription of these genes. Figure 2 RT-qPCR detection of B. cereus BC4207 and BC4028 genes. Relative expressions of BC4207 (grey bars) and BC4028 (white bars) were determined in AS-48, bacitracin and nisin treated B. cereus ATCC14579 cultures (see Methods for concentrations). Transcript levels of genes were normalized to the level of house keeping rpoA gene and compared to untreated samples (dashed line). Overexpression of BC4207 increases resistance against AS-48 in B. cereus and B.