Abstract
In this study we report the identification and role of the alkyl hydroperoxide reductase (ahp) gene in Bacteroides fragilis. The two components of ahp, ahpC, and ahpF, are organized in an operon, and the deduced amino acid sequences revealed that B. fragilis AhpCF shares approximately 60% identity to orthologues in other gram-positive and gram-negative bacteria. Northern blot hybridization analysis of total RNA showed that the ahpCF genes were transcribed as a polycistronic 2.4-kb mRNA and that ahpC also was present as a 0.6-kb monocistronic mRNA. ahpC and ahpCF mRNAs were induced approximately 60-fold following H(2)O(2) treatment or oxygen exposure of the parent strain but were constitutive in a peroxide-resistant strain. Further investigation using an ahpCF'::beta-xylosidase gene transcriptional fusion confirmed that ahpCF had lost normal regulation in the peroxide-resistant strain compared to the parent. The ahpCF mutant was more sensitive to growth inhibition and mutagenesis by organic peroxides than the parent strain, as determined by disk inhibition assays and the frequency of mutation to fusidic acid resistance. This finding suggests that the ahp genes play an important role in peroxide resistance in B. fragilis. Under anaerobic conditions, we observed increases in the number of spontaneous fusidic acid-resistant mutants of five- and sevenfold in ahpCF and ahpF strain backgrounds, respectively, and eightfold in the ahpCF katB double mutant strain compared to the parent and katB strains. In addition, ahpCF, ahpF, and ahpCF katB mutants were slightly more sensitive to oxygen exposure than the parent strain. Moreover, the isolation of a strain with enhanced aerotolerance and high-level resistance to alkyl hydroperoxides from an ahpCF katB parent suggests that the physiological responses to peroxide toxicity and to the toxic effects of molecular oxygen are overlapping and complex in this obligate anaerobe.