Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.     

A Xanthomonas alkyl hydroperoxide reductase subunit C (ahpC) mutant showed an altered peroxide stress response and complex regulation of the compensatory response of peroxide detoxification enzymes

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A Xanthomonas ahpC mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H(2)O(2) killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional ahpC gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an oxyR mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in ahpC appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in Xanthomonas differed from that in other bacteria.     

Compensatory functions of two alkyl hydroperoxide reductases in the oxidative defense system of Legionella pneumophila

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were approximately 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.