FIGURE 2.

Redox-regulated efflux mediates Burkholderia susceptibility to PYO and its collateral effects on antibiotic resilience. (a) SoxR-mediated regulation of RND-9 in bcc. The example is based on the Burkholderia multivorans ATCC 17616 genome locus structure and orientation. SoxR is oxidized by redox active metabolites (RAMs), triggering its activation (left) (Dietrich et al., 2008; Imlay, 2013; Perry et al., 2021). In bcc, SoxR is commonly found adjacent to the RND-9 efflux system; the SoxR box (Dietrich et al., 2008) is found upstream to RND-9 genes, and the sequence displayed is the consensus found in this genomic region for the bcc strains studied (center). Following other examples of RND efflux systems, proteins derived from Bmul_3930 and Bmul_3931 are likely associated with the inner membrane, while the one from Bmul_3932 is likely an outer membrane protein (Du et al., 2018). SoxR-mediated induction of the system allows efflux of toxic molecules by the bacterial cell (right). (b) Expression levels of the second gene in the RND-9 operon (Bmul_3931) measured by qRT-PCR in different B. multivorans strains in the presence or absence of PYO (n = 3). ΔsoxR comp means complementation of ΔsoxR. Data are shown as normalized cDNA (see Materials and methods). Mean fold differences between control (− PYO) and treatment (+ PYO) are also displayed for all three strains. For additional qRT-PCR results, see Figure S2. (c) Effect of PYO on tolerance to ciprofloxacin (CIP, 10 μg/ml) in the same three B. multivorans strains (n = 4). (d) Phylogenetic relationship between the Burkholderia species used in this study (gray shading highlights species within the bcc group). For full tree detailing different strains, see Figure S4. For broader phylogenetic placement of these species within the Burkholderia genus, see Depoorter et al. (2016). (e) PYO effect on growth rates of distinct strains of the different Burkholderia species studied. Data for three different concentrations are shown (10, 100, and 200 μM). The results are shown as a ratio of the growth rates for each strain under different PYO concentrations by their growth rates in the “no PYO” condition (i.e., values close to 1 mean no inhibition, while values close to 0 mean severe growth inhibition by PYO). Presence or absence of the genomic locus containing SoxR and RND-9 next to each other in these strains is indicated by the gray or white boxes respectively. Growth rates were estimated based on growth curves under the different conditions (Figure S5). In panels b and c, the black dots mark the means and error bars represent 95% confidence intervals.