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Journal of Bacteriology, November 2007, p. 7669-7680, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00745-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Requirement of the Zinc-Binding Domain of ClpX for Spx Proteolysis in Bacillus subtilis and Effects of Disulfide Stress on ClpXP Activity

Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon

Received 11 May 2007/ Accepted 27 August 2007

Spx, a transcriptional regulator of the disulfide stress response in Bacillus subtilis, is under the proteolytic control of the ATP-dependent protease ClpXP. Previous studies suggested that ClpXP activity is down-regulated in response to disulfide stress, resulting in elevated concentrations of Spx. The effect of disulfide stress on ClpXP activity was examined using the thiol-specific oxidant diamide. ClpXP-catalyzed degradation of either Spx or a green fluorescent protein derivative bearing an SsrA tag recognized by ClpXP was inhibited by diamide treatment in vitro. Spx is also a substrate for MecA/ClpCP-catalyzed proteolysis in vitro, but diamide used at the concentrations that inhibited ClpXP had little observable effect on MecA/ClpCP activity. ClpX bears a Cys4 Zn-binding domain (ZBD), which in other Zn-binding proteins is vulnerable to thiol-reactive electrophiles. Diamide treatment caused partial release of Zn from ClpX and the formation of high-molecular-weight species, as observed by electrophoresis through nonreducing gels. Reduced Spx proteolysis in vitro and elevated Spx concentration in vivo resulted when two of the Zn-coordinating Cys residues of the ClpX ZBD were changed to Ser. This was reflected in enhanced Spx activity in both transcription activation and repression in cells expressing the Cys-to-Ser mutants. ClpXP activity in vivo is reduced when cells are exposed to diamide, as shown by the enhanced stability of an SsrA-tagged protein after treatment with the oxidant. The results are consistent with the hypothesis that inhibition of ClpXP by disulfide stress is due to structural changes to the N-terminal ZBD of ClpX.

Published ahead of print on 7 September 2007.

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