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Journal of Bacteriology, November 1998, p. 5906-5912, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of a Peroxide-Resistant Mutant of the Anaerobic Bacterium Bacteroides fragilis

Edson R. Rocha and C. Jeffrey Smith*

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina

Received 14 May 1998/Accepted 3 September 1998

A Bacteroides fragilis mutant resistant to hydrogen peroxide and alkyl peroxide was isolated by enrichment in increasing concentrations of hydrogen peroxide. The mutant strain was constitutively resistant to 100 mM H2O2 and 5 mM cumene hydroperoxide (15-min exposure). In contrast, the parent strain was protected against <10 mM H2O2 when the peroxide response was induced with a sublethal concentration of H2O2, and no protection was observed in untreated cells. In addition, catalase activity in the mutant strain was not repressed in anaerobic cultures as reported previously for the parent strain. Comparison of the protein profile of crude extracts of the B. fragilis strains revealed that at least three oxidative stress-induced proteins in the parent strain were constitutively expressed in the mutant as detected by nondenaturing polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these overexpressed proteins confirmed the presence of a deregulated catalase (KatB), an alkyl hydroperoxidase reductase subunit C (AhpC), and a Dps/PexB homologue. Northern blot analysis and katB::cat transcriptional fusion studies revealed that in the mutant, katB was deregulated compared to the parent and that katB was controlled by a trans-acting regulatory mechanism. Moreover, constitutive expression of KatB and of the AhpC and Dps homologues in the H2O2-resistant mutant suggests that these proteins may share a common oxidative stress transcriptional regulator and may be involved in B. fragilis peroxide resistance.

* Corresponding author. Mailing address: East Carolina University School of Medicine, Dept. of Microbiology and Immunology, Greenville, NC 27858-4354. Phone: (252) 816-3127. Fax: (252) 816-3535. E-mail: jsmith{at}brody.med.ecu.edu.

Journal of Bacteriology, November 1998, p. 5906-5912, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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