, 2001; Yamamoto & Ishihama, 2005). The E. coli cus system consists of two operons,
one of which encodes the proteins of the CusCFBA efflux pump. The second operon is divergently transcribed from the cusCFBA genes and encodes the CusR/CusS two-component system (TCS) (Fig. 1). The CusR/CusS TCS is involved in the regulation of transcription from the cusCFBA genes upon the onset of silver or copper stress (Munson et al., 2000; Franke et al., 2001). There is at least a twofold increase in transcription from cusR and cusS genes upon induction by Ag(I) or Cu(I) ions (Yamamoto & Ishihama, 2005). The central role of CusS is seen in its occurrence in association with metal efflux genes in different species of Gram-negative bacteria (Pontel & Soncini, 2009). In Pseudomonas putida, the CusS homolog CinS activates the transcription of the cinR and RG-7388 chemical structure cinS genes in response to both Cu(I) and Ag(I) (Quaranta et al., 2009). On the basis of the sequence homology to other histidine kinases of two-component systems, E. coli CusS is predicted to be a membrane-bound protein, which forms a two-component system with the response regulator CusR (Munson et al., 2000; Yamamoto et al., 2005). Under the conditions of elevated concentrations of Cu(I)/Ag(I), CusS
and CusR are essential for the induction of the copper efflux genes cusCFBA (Munson et al., 2000; Franke et al., 2003). Signal recognition by ligand binding in selleck chemicals llc the periplasmic sensor domain of CusS is expected to elicit downstream transmembrane and cytoplasmic signaling events, and thus, CusS is predicted to play an important role in cell adaptation to changes in extracytoplasmic levels of copper and silver ions. This study establishes the role of the cusS gene in Cu(I) and Ag(I) resistance in E. coli. Additionally, we report that the presence of the cusS gene is essential for the upregulation of the cusCFBA genes in the bacterium. All strains were grown at
37 °C in modified Luria broth (MLB) (1% tryptone and 0.5% yeast extract), MLB agar plates or modified M9 broth (MM9) (0.1% ammonium sulfate as the source of nitrogen and no sodium chloride), or MM9-agar plates. Antibiotics (ampicillin 100 μg mL−1 and kanamycin Sodium butyrate 30 μg mL−1) were added to the growth media for purposes of strain selection. All overnight cultures containing the pBAD24 vectors were grown in the presence of 0.02% d-glucose to prevent expression from the arabinose promoter. To promote expression from the genes on the pBAD24 vector, 0.2% l-arabinose was added to the growth media. Reagents and chemicals were obtained from Sigma, and MLB components were obtained from Difco. Bacterial strains and plasmids used in this study are listed in Table 1. Knockout strains were made using the lambda-Red-mediated gene recombination technique as detailed by Datsenko and Wanner (Datsenko & Wanner, 2000).