Insights into the Function of a Second, Nonclassical Ahp Peroxidase, AhpA, in Oxidative Stress Resistance in Bacillus subtilis

Abstract

Organisms growing aerobically generate reactive oxygen-containing molecules, such as hydrogen peroxide (H 2 O 2 ). These reactive oxygen molecules damage enzymes and DNA and may even cause cell death. In response, Bacillus subtilis produces at least nine potential peroxide-scavenging enzymes, two of which appear to be the primary enzymes responsible for detoxifying peroxides during vegetative growth: a catalase (encoded by katA) and an alkylhydroperoxide reductase (Ahp, encoded by ahpC). AhpC uses two redox-active cysteine residues to reduce peroxides to nontoxic molecules. A specialized thioredoxin-like protein, AhpF, is then required to restore oxidized AhpC back to its reduced state. Curiously, B. subtilis has two genes encoding Ahp: ahpC and ahpA. Although AhpC is well characterized, very little is known about AhpA. In fact, numerous bacterial species have multiple ahp genes; however, these additional Ahp proteins are generally uncharacterized. We seek to understand the role of AhpA in the bacterium's defense against toxic peroxide molecules in relation to the roles previously assigned to AhpC and catalase. Our results demonstrate that AhpA has catalytic activity similar to that of the primary enzyme, AhpC. Furthermore, our results suggest that a unique thioredoxin redox protein, AhpT, may reduce AhpA upon its oxidation by peroxides. However, unlike AhpC, which is expressed well during vegetative growth, our results suggest that AhpA is expressed primarily during postexponential growth. IMPORTANCEB. subtilis appears to produce nine enzymes designed to protect cells against peroxides; two belong to the Ahp class of peroxidases. These studies provide an initial characterization of one of these Ahp homologs and demonstrate that the two Ahp enzymes are not simply replicates of each other, suggesting that they instead are expressed at different times during growth of the cells. These results highlight the need to further study the Ahp homologs to better understand how they differ from one another and to identify their function, if any, in protection against oxidative stress. Through these studies, we may better understand why bacteria have multiple enzymes designed to scavenge peroxides and thus have a more accurate understanding of oxidative stress resistance.

AhpA is a peroxidase expressed during biofilm formation inBacillus subtilis

“…In contrast to that seen for ahpC , we previously found that expression of ahpA is not altered upon mutation of the other peroxide‐scavenging enzymes (Broden et al., ). Furthermore, ahpA is not substantially expressed during exponential phase and does not appear to be required to protect cells from peroxides during vegetative growth (Broden et al., ). Instead, the expression of ahpA is regulated by the transition‐state regulatory protein AbrB (Broden et al., ; Chumsakul et al., , ).…”

“…Studies in B. subtilis demonstrate that its two primary peroxide scavengers are AhpC and the vegetative catalase KatA; strains lacking both ahpC and katA grow poorly under aerobic conditions (Broden et al., ; Bsat et al., ). Consistent with this, both ahpC and katA are expressed moderately well during exponential growth (Broden et al., ; Bsat et al., ; Faulkner, Ma, Fuangthong, & Helmann, ; Fuangthong, Herbig, Bsat, & Helmann, ). Furthermore, mutation of either ahpC or katA results in the increased expression of other proteins responsible for protecting cells against peroxides (Antelmann et al., ; Bsat et al., ; Chen, Keramati, & Helmann, ).…”

“…AhpA (encoded by ykuU ), on the other hand, is a nonclassical Ahp and lacks this cognate AhpF (Broden et al., ). Instead, AhpA appears to use a thioredoxin‐like protein AhpT (encoded by ykuV ) as its reductase (Broden et al., ; Rasmussen, Nielsen, & Jarmer, ; Zhang et al., ). In contrast to that seen for ahpC , we previously found that expression of ahpA is not altered upon mutation of the other peroxide‐scavenging enzymes (Broden et al., ).…”

“…Instead, AhpA appears to use a thioredoxin‐like protein AhpT (encoded by ykuV ) as its reductase (Broden et al., ; Rasmussen, Nielsen, & Jarmer, ; Zhang et al., ). In contrast to that seen for ahpC , we previously found that expression of ahpA is not altered upon mutation of the other peroxide‐scavenging enzymes (Broden et al., ). Furthermore, ahpA is not substantially expressed during exponential phase and does not appear to be required to protect cells from peroxides during vegetative growth (Broden et al., ).…”

“…Furthermore, ahpA is not substantially expressed during exponential phase and does not appear to be required to protect cells from peroxides during vegetative growth (Broden et al., ). Instead, the expression of ahpA is regulated by the transition‐state regulatory protein AbrB (Broden et al., ; Chumsakul et al., , ). AbrB primarily represses the expression of its target genes during exponential growth (Strauch & Hoch, ).…”