Abstract
The trace element copper serves as cofactor for many enzymes but is toxic at elevated concentrations. In bacteria, the intracellular copper level is maintained by copper efflux systems including the Cue system controlled by the transcription factor CueR. CueR, a member of the MerR family, forms homodimers, and binds monovalent copper ions with high affinity. It activates transcription of the copper tolerance genes copA and cueO via a conserved DNA-distortion mechanism. The mechanism how CueR-induced transcription is turned off is not fully understood. Here, we report that Escherichia coli CueR is prone to proteolysis by the AAA+ proteases Lon, ClpXP, and ClpAP. Using a set of CueR variants, we show that CueR degradation is not altered by mutations affecting copper binding, dimerization or DNA binding of CueR, but requires an accessible C terminus. Except for a twofold stabilization shortly after a copper pulse, proteolysis of CueR is largely copper-independent. Our results suggest that ATP-dependent proteolysis contributes to copper homeostasis in E. coli by turnover of CueR, probably to allow steady monitoring of changes of the intracellular copper level and shut-off of CueR-dependent transcription.
Highlights
Copper is a trace element required as cofactor for full functionality of several enzymes, such as cytochrome c oxidase of the respiratory chain (van der Oost et al, 1994)
For construction of pBO3687, a plasmid encoding constitutively expressed CueR, the cueR gene was amplified from genomic E. coli K12 DNA and cloned into pACYC184 via primer-derived restriction sites
The basal level of the copper efflux regulator CueR always present in the cell is elevated at increasing copper concentrations (Yamamoto and Ishihama, 2005)
Summary
Copper is a trace element required as cofactor for full functionality of several enzymes, such as cytochrome c oxidase of the respiratory chain (van der Oost et al, 1994). To analyze the stability of different CueR variants, cells containing inducible expression plasmids encoding for corresponding CueR proteins were grown overnight in M9 minimal medium containing corresponding antibiotics for selection at 30◦C. For analyses of Strep_CueR stability over the entire growth curve cells were grown in LB medium + Amp at 37◦C to different growth phases and in vivo degradation experiments were performed in every growth phase as described above.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
