ABSTRACT
The peroxide response-inducible genes ahpCF,dps, and katB in the obligate anaerobeBacteroides fragilis are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria.oxyR and dps were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe,Porphyromonas gingivalis. Northern blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and that dps mRNA was approximately 500 bases in length.dps mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion ofoxyR abolished the induction of ahpC andkatB following treatment with hydrogen peroxide or oxygen exposure. However, dps expression was induced approximately fourfold by oxygen exposure in ΔoxyR strains but not by hydrogen peroxide. This indicates that dps expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of ΔoxyR mutant strains with wild-type oxyR and oxyR(Con) restored the inducible peroxide response and the constitutive response of theahpCF, katB, and dps genes, respectively. However, overexpression of OxyR abolished the catalase activity but not katB expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of oxyR expression in the parents and in ΔoxyR and overexpressing oxyR strains by Northern blotting and oxyR′::xylBfusions revealed that B. fragilis OxyR does not control its own expression.
A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast