Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS

Abstract

Two-component systems are widely used by bacteria to mediate adaptive responses to a variety of environmental stimuli. The CusR/CusS two-component system in Escherichia coli induces expression of genes involved in metal efflux under conditions of elevated Cu(I) and Ag(I) concentrations. As seen in most prototypical two-component systems, signal recognition and transmission is expected to occur by ligand binding in the periplasmic sensor domain of the histidine kinase CusS. Although discussed in the extant literature, little experimental evidence is available to establish the role of CusS in metal homeostasis. In this study, we show that the cusS gene is required for Cu(I) and Ag(I) resistance in E. coli and that CusS is linked to the expression of the cusCFBA genes. These results show a metal-dependent mechanism of CusS activation and suggest an absolute requirement for CusS in Cu(I)- and Ag(I)-dependent upregulation of cusCFBA expression in E. coli.

Figure 1. Open Reading Frames within the cus locus

The cusRS locus encodes the two-component system and cusC, cusF, cusB and cusA form the transmembrane efflux channel in E. coli. The operons are divergently transcribed.

Figure 2. Copper sensitivity of E. coli wild-type, E. coli ΔcusS and E. coli ΔcusS/pBADcusS to copper

The normalized cell densities of the different strains of E. coli were compared after 15 hours of growth under anaerobic conditions in media containing different concentrations of CuSO₄. The data are plotted as an average of three independent experiments including their standard error of mean (SEM).

Figure 3. Minimum inhibitory concentration (MIC) of silver for E. coli wild-type, E. coli ΔcusS and E. coli ΔcusS/pBADcusS

E. coli cells were on MM9 plates containing different concentration of AgNO₃ for 16 hours. The MIC values are shown on the right and are an average of three independent experiments.

Figure 4. Metal accumulation in E. coli cells grown in medium containing copper

The graph represents copper accumulation in cells at 0 (black), 2 (diagonal lines) and 4 hours (gray) after addition of 7.5 µM CuSO₄ respectively. The table below the graph represents the actual concentration of copper measured using ICP-MS. Each bar represents the average of three independent experiments and their SEM. ANOVA analysis with multiple comparisons shows that in the absence of cusS, E. coli cells accumulate higher concentrations of copper (P < 0.001).

Figure 5. CusS is required for expression from cusC

Data shows the relative expression from the cusC gene +/− SEM, as determined in E. coli wild-type (gray), E. coli ΔcusS (checkered) and E. coli ΔcusS/pBADcusS (diagonal lines) after exposure to 5 µM AgNO₃ for 0, 2 and 4 hours. Total RNA from cells was extracted, reverse transcribed and cDNA was subjected to real-time PCR using primers specific for cusC. ANOVA analysis with multiple comparisons showed that transcription from cusC is absent when cusS is disrupted (P < 0.0001).