However, in an extended analysis
using different strains and growth conditions, the ability of CusCFBA to confer resistance to these substances could not be verified. These results strongly suggest a narrow substrate specificity for the CusCFBA system. Differing results between the Biolog and the MIC assays may be due to differences in the preparation of the tested learn more compounds. The native Biolog multiwell plate contained dry deposited chemicals and the concentration range of the chemicals covered orders of magnitude. It is possible that in the Biolog assay certain hydrophobic analytes were not fully soluble, such that the bacteria were not exposed to the intended concentration. In the MIC assays, organic solvents or ionic mixtures were used to solubilize the compounds to a particular concentration. Thus, some differences may be seen if the end concentrations are different between the two assays. Narrow substrate specificity can be attributed to the metal-binding sites of CusB and
CusF. X-ray absorption spectroscopy data show that Cu(I) is bound to CusB in a three-coordinate environment, indicative of Cu–S interactions I-BET-762 nmr (Bagai et al., 2007). CusB does not contain any cysteine residues; consequently, the sulfur-containing species in CusB that coordinate Cu(I) are methionine residues. Through site-directed mutagenesis and subsequent isothermal titration calorimetry data, Bagai and colleagues showed that three methionine residues, M21M36M38, are important in metal binding and subsequent copper efflux. Moreover, CusF, a metallochaperone of the Cus complex, has been shown to bind metal via a primarily three-coordinate metal-binding site (Loftin et al., 2007) and directly transfers the metal ion to the periplasmic component, CusB (Bagai et al., 2008). Here, Cu(I) is coordinated with two sulfurs from M47 and M49 and a nitrogen from
H36, with W44 capping the metal site. These methionine residues in CusB and CusF are essential in the extrusion of copper and silver from the periplasm to the extracellular space. To determine Phosphoprotein phosphatase the prevalence of these metal-binding motifs, blast analysis was performed against all sequenced gammaproteobacterial genomes (Altschul et al., 1990). The number of sequences that contained these specific metal-binding motifs is shown in Fig. 1. All orders within the Gammaproteobacteria class, except one, Pasteurellaceae, contain genes encoding CusB- and CusF-like proteins with the metal-binding motifs. Interestingly, when performing blast analysis on the MFP GesA, no highly conserved residues were found. Consequently, the narrow substrate specificity for the CusCFBA complex may be attributed to the conserved residues for metal binding in CusB and CusF. Analysis of CusA showed that it belongs specifically to a group of efflux pumps responsible for the extrusion of heavy metals. CusA shares high sequence identity to SilA (S.