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Journal of Bacteriology, September 2008, p. 5738-5745, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00576-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor

Sumarin Soonsanga ,^1,, Jin-Won Lee,^1,2, and John D. Helmann^1*

Department of Microbiology, Wing Hall, Cornell University, Ithaca, New York 14853-8101,¹ Department of Life Science, Hanyang University, Seoul, South Korea²

Received 25 April 2008/ Accepted 14 June 2008

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.

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* Corresponding author. Mailing address: Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu

Published ahead of print on 27 June 2008.

S.S. and J.-W.L. contributed equally to this work.

Present address: Microbial Engineering Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.

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Journal of Bacteriology, September 2008, p. 5738-5745, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00576-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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