Here we provide a global overview of the major transcriptional changes elicited by various peroxidative stress conditions. Our findings indicate that PerR is the major regulator of genes induced by low levels of H2
, while OhrR regulates the single gene that is most rapidly and selectively induced by the model organic peroxide t-buOOH. However, the largest single group of genes induced by these treatments is the σB
-dependent general stress regulon (30
). Indeed, knowledge about the general stress response regulon, as deduced both from previous studies of the heat shock stimulon (22
) and from extensive characterization of the σB
), provided important background for our analysis.
Our work also illustrates the value of monitoring the kinetics of global changes in gene expression following application of a stress. Many of the most dramatic transcriptional effects were maximal within 3 min of application of the stress conditions, and there was an often rapid return to lower expression levels within 10 to 20 min. The transient nature of the σB-dependent general stress response is well known, but transient induction of the PerR and OhrR regulons has not been well documented previously. Thus, studies based on a single time point may miss major parts of the transcriptional response.
Our results emphasize the importance of control experiments for interpreting transcriptional profiles. We found, for example, that the vast majority of the transcriptional changes noted 40 min after H2O2 treatment were also observed in mock (H2O)-treated samples and were due to a growth phase transition rather than any effect of the peroxide treatment. Similarly, the comparison of the t-buOOH and t-buOH stimulons (Fig. ) highlighted the need to use appropriate controls when the effects of particular classes of compounds are investigated. Indeed, we cannot exclude the possibility that t-buOOH is an inducer of the σB regulon because it is reduced in vivo to t-buOH. However, the rapid kinetics of these transcriptional responses make it seem unlikely that reduction to the alcohol is necessary for induction.
While transcriptional profiling is an extremely powerful tool for monitoring changes in gene expression after a change in culture conditions, the comparison of mutant and wild-type strains presented additional challenges. In this case, the perR
mutant and wild-type strains differed significantly in growth rate and physiological state. Despite these differences, the perR
mutant clearly revealed derepression of known PerR-repressed target genes. Remarkably, the extent of derepression in the samples corresponded very well with the fold induction 3 min after treatment with H2
(Fig. ). This correlation suggests that the magnitude of peroxide induction of different genes reflects the extent to which PerR represses gene expression. As noted elsewhere, the fur
gene is unusual among PerR-repressed genes in that it is not peroxide inducible under these or any other conditions tested (18
). While not apparent from the transcriptional profiling data, PerR does mediate an approximately fourfold repression of fur
in response to Mn(II) (18
It is instructive to compare the B. subtilis
and E. coli
peroxide stress responses. In E. coli
, transcriptional profiling was performed with cells treated with much higher levels of H2
(1 mM), in part because lower levels led to only transient changes in gene expression (39
). Under these conditions 140 genes were induced approximately fourfold in the wild-type strain, and an even greater number of genes were induced in an oxyR
mutant strain. The OxyR regulon includes genes with protective and detoxification functions, such as katG
(hydroperoxidase I), ahpCF
(alkyl hydroperoxide reductase), and dps
(DNA-binding protein). In addition, OxyR activates transcription of genes that maintain intracellular thiols in their reduced states, including gorA
(gluthathione reductase), grxA
(glutaredoxin), and trxA
(thioredoxin 2). In aerobically growing E. coli
the concentration of intracellular H2
is maintained at levels near 20 nM by the potent peroxidase activity of Ahp (10
). Catalase peroxidase appears to be most important in detoxifying higher levels of peroxides and has the added advantage of being active even in energy-depleted cells that may lack sufficient reducing capacity to maintain optimal Ahp activity. Ahp itself was originally characterized as the major resistance factor protecting cells against alkyl peroxides. However, Ahp is also active with H2
, and this may be the physiologically relevant substrate (10
The PerR regulon differs from the regulon controlled by OxyR in several respects. PerR does not appear to control functions that might be involved in maintaining intracellular thiols in a reduced state. Although B. subtilis encodes several predicted peroxiredoxins, including a thiol-dependent peroxidase (tpx) and the product of ygaF (the gene immediately upstream of perR), only the ahpCF genes are members of the peroxide stimulon characterized in this study. In future work, it will be important to define the roles of catalase, Ahp, peroxiredoxins, and the organic hydroperoxide resistance proteins in B. subtilis. While all of these enzymes may act to detoxify reactive oxygen species in the cell, genetic studies indicate that they are not redundant.