Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität Greifswald, D-17487 Greifswald, Germany
Correspondence
Georg Homuth
georg.homuth{at}uni-greifswald.de
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ABSTRACT |
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J. Mostertz and C. Scharf contributed equally to this work.
The datasets for all genes significantly up- and downregulated by peroxide and paraquat stress including all expression level ratios are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org. The complete dataset of the transcriptome analysis can be obtained from the authors upon request.
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INTRODUCTION |
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The anion is known to attack enzymes with exposed [4Fe4S] clusters, which results in the release of iron and loss of activity (Gardner & Fridovich, 1991
; Brown et al., 1995
). Furthermore,
directly inhibits the synthesis of cysteine and aromatic amino acids in Escherichia coli (Carlioz & Touati, 1986
; Benov et al., 1996
; Benov & Fridovich, 1999
). By spontanous dismutation or in the course of its enzymic detoxification,
is rapidly converted to H2O2.
Peroxides are weak oxidizing agents known to react with cysteinyl-thiols in proteins by formation of disulfide bonds or sulfonic acid derivatives (Imlay, 2002). H2O2 is able to generate carbonyl groups in lysine, arginine, threonine and proline residues and to oxidize methionine to sulfoxide adducts (Stadtman, 1993
). Peroxidation of membrane lipids is triggered by peroxides, at least in mammalian systems (Imlay, 2002
). Most significantly, H2O2 reacts with reduced iron ions to form OH
, which in turn oxidizes most cellular compounds at diffusion-limited rates (Farr & Kogoma, 1991
; Henle & Linn, 1997
). Since the phosphodiester backbone and nitrogenous bases both provide ligands that can tightly bind adventitious iron, DNA damage after peroxide exposure may be caused by metal-catalysed local conversion of H2O2 to OH
(Henle & Linn, 1997
).
In aerobically growing cells, significant amounts of and H2O2 are generated by enzymic misdirection of electrons to dioxygen. It is assumed that flavin-dependent transfer reactions of the respiratory chain are primarily responsible for the generation of ROS (Imlay & Fridovich, 1991
; Messner & Imlay, 1999
). In addition to the autoxidation of cellular components, exposure to ionizing radiation, redox-active compounds or transition metals and depletion of antioxidants may contribute to the intracellular formation of ROS. If the amount of ROS increases to toxic levels, cells encounter oxidative stress.
In bacteria, oxidative stress is sensed by specific transcriptional regulators able to activate defence mechanisms when the ROS concentration exceeds a critical level (Farr & Kogoma, 1991; Storz & Zheng, 2000
; Pomposiello & Demple, 2002
). In E. coli, peroxides are monitored by the response regulator OxyR (Christman et al., 1989
). In the presence of peroxides, an intramolecular thioldisulfide switch of cysteine residues causes activation of OxyR, which subsequently leads to the expression of peroxide defence proteins including the H2O2-detoxificating catalase HPI (VanBogelen et al., 1987
; Zheng et al., 1998
). After superoxide treatment, the oxidation of a [2Fe2S]+ cluster of the SoxR regulatory protein to [2Fe2S]2+ activates the SoxRS system and induces the superoxide-specific response (Gaudu et al., 1997
; Hidalgo et al., 1997
). Among other targets of SoxRS, a Mn-dependent superoxide dismutase catalysing the dismutation of
to H2O2 is synthesized at elevated levels (Walkup & Kogoma, 1989
; Greenberg et al., 1990
; Pomposiello & Demple, 2002
).
In the soil bacterium Bacillus subtilis, protection against inorganic peroxides is primarily mediated by the induction of specific stress proteins which are controlled by the repressor protein PerR (Chen et al., 1995; Bsat et al., 1998
). In contrast to OxyR of E. coli, the Fur-homologous PerR regulator requires a metal cofactor for DNA binding (Herbig & Helmann, 2001
). H2O2 is suggested to react at a metal centre of the protein, thereby impairing the DNA-binding ability (Herbig & Helmann, 2001
), which leads to increased synthesis of the vegetative catalase KatA, the alkyl hydroperoxide reductase AhpC/AhpF, the DNA-protecting protein MrgA and the haem biosynthesis proteins HemA, HemX, HemC, HemD, HemB and HemL (Bsat et al., 1996
). In addition, the genes encoding the iron-uptake regulator Fur, the zinc-uptake system ZosA (formerly YkvW) and PerR itself are members of the PerR regulon (Herbig & Helmann, 2002
; Helmann et al., 2003
). The observed induction of genes encoding peroxide stress proteins after challenging the cells with low levels of H2O2 causes a nearly complete resistance of B. subtilis to otherwise lethal concentrations of H2O2 (Dowds et al., 1987
; Murphy et al., 1987
). In contrast to the PerR-dependent H2O2 protection, resistance against organic peroxides is mediated by the OhrR repressor (Fuangthong et al., 2001
). Up to now, no
-specific regulator has been found in B. subtilis. Previous studies using 2D PAGE revealed that addition of paraquat, which causes
stress, triggers the enhanced synthesis of a set of specific proteins which are not induced by H2O2 (Antelmann et al., 1997
; Bernhardt et al., 1999
).
In this study, we analysed the global gene expression of B. subtilis in response to oxidative stress. Variations in gene expression after treatment with H2O2 or the -generating agent paraquat were monitored at the level of transcription using DNA macroarray hybridization and at the level of protein synthesis by 2D gel electrophoresis. The results allowed classification of oxidative-stress-responsive genes into two groups: those that exhibited similar expression patterns in the presence of both ROS, and those that responded more strongly to one stimulus or the other. Many genes encoding proteins of so far unknown functions could be assigned to one of these groups, suggesting functions in the context of coping with oxidative stress conditions. The present study represents a first step towards the functional characterization of these genes.
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METHODS |
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Transcriptome analysis.
Cultivation of B. subtilis 168 and induction of oxidative stress were performed as described for the proteome analysis. Cell harvesting, preparation of RNA, and macroarray analysis with Panorama B. subtilis gene arrays and specific cDNA labelling primers (Sigma-Genosys) were performed as described by Eymann et al. (2002). The total RNA was checked by Northern blot analysis. For each condition, four macroarray experiments were carried out using two independently isolated RNA preparations and two different array batches. Quantification of the hybridization signals using the ArrayVision software (version 5.1, Imaging Research) was performed as described by Eymann et al. (2002)
. Gene-specific expression level ratios were calculated by dividing the mean of the normalized, artifact-reduced volumes of the parallel replica spots on the stress array (H2O2 or paraquat) by the corresponding values of the control array for upregulated expression or reciprocally for downregulated expression. Expression level ratios
3 in at least three of four experiments per condition were considered as significant. Final evaluation of the macroarray data included the consideration of putative operons derived from the genome sequence using the SubtiList database (http://genolist.pasteur.fr/SubtiList/) as well as previously known transcriptional units.
To facilitate presentation of the expression data, arithmetic means and standard deviations calculated from all four expression level ratios are given for every significantly upregulated gene in Table 1. The datasets for all genes significantly up- and downregulated by peroxide and paraquat stress including all expression level ratios are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org. The complete dataset of the transcriptome analysis can be obtained from the authors upon request.
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RESULTS AND DISCUSSION |
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Characterization of transcriptome and proteome after exposure to H2O2 and paraquat
The transcriptome analysis revealed 92 genes exhibiting significantly elevated and 97 genes exhibiting significantly reduced mRNA levels after H2O2 challenge. After exposure to paraquat, the expression of 129 genes was found to be induced at the level of mRNA while the expression of 169 genes was decreased. From these genes, 51 were up- and 87 were downregulated by peroxide as well as superoxide stress. The accompanying proteome analysis demonstrated enhanced synthesis for about 55 proteins after peroxide challenge and decreased synthesis for about 150 proteins (Fig. 1A). About 65 proteins were found to be synthesized at higher rates and 200 at lower rates after exposure to paraquat (Fig. 1B
). Around 20 proteins were present in higher and 140 proteins in lower amounts after peroxide as well as after paraquat treatment. The expression data for upregulated genes are summarized in Table 1
; results for the various classes of genes are discussed below.
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The primary defence against the effects of inorganic peroxides involves the induction of the PerR-dependent stress response. The activation of this peroxide-specific response after exposure to observed in our analysis may reflect the dismutation of
to H2O2 either by spontaneous dismutation or as part of antioxidant strategies. Alternatively, direct inactivation of PerR by
cannot be excluded. In E. coli, the gene expression patterns after superoxide and H2O2 treatment also show a partial overlap, including all peroxide-specific stress proteins belonging to the OxyR regulon (Greenberg & Demple, 1989
). The overlap of the
and peroxide responses in B. subtilis including members of the PerR regulon has already been demonstrated by means of proteomics (Antelmann et al., 1997
; Bernhardt et al., 1999
).
Fur regulon
The fur gene encoding the ferric uptake regulator was found to be induced after oxidative stress, most probably due to PerR-dependent derepression, which was previously demonstrated (Fuangthong et al., 2002). The fur induction could indicate a shut-down of cellular iron uptake to prevent further generation of OH
, since intracellular iron promotes formation of OH
from H2O2 via Fenton chemistry in the presence of ROS. However, the Fur repressor protein itself appeared to be inactive under the conditions tested. In our study, 11 of the 25 probable transcriptional units described to be upregulated in a fur knockout mutant (Baichoo et al., 2002
) exhibited increased mRNA levels after peroxide and
stress, indicating derepression of the Fur regulon. In E. coli, fur was also reported by Zheng et al. (1999)
to show increased expression after H2O2 and
stress, which is mediated by OxyR and SoxRS. The observation of high Fur protein amounts after oxidative stress prompted these authors to speculate that iron uptake regulation might not be the only function of Fur. It was proposed that the Fur protein could sequester iron in order to deprive DNA of metal ions or to catalyse the breakdown of H2O2 directly (Zheng et al., 1999
). This could also be the case for the B. subtilis Fur protein.
On the other hand, the induction of genes with functions in iron uptake which was detected in our analysis could represent an adaptive response to iron limitation. The activity of many defence proteins, e.g. catalases, requires metal ion incorporation. In an approach similar to our study, Helmann et al. (2003) did not detect induction of the Fur regulon using a comparable H2O2 concentration. Since the experiments reported by these authors were performed in rich medium in contrast to the synthetic medium used in our experiments, we speculated that the different expression patterns might have resulted from differing metal concentrations in these media. To verify this hypothesis, cells were grown in the synthetic medium used in our transcriptome analysis (Belitsky Minimal Medium, BMM) as well as in the rich medium used by Helmann and coworkers (MuellerHinton Broth, MHB) and RNA was prepared before and after addition of H2O2. These RNA preparations were used in Northern hybridizations with probes for mRNA specified by dhbF, fhuD, ykuP and yxeB. These genes were previously demonstrated to represent direct targets of Fur (Baichoo et al., 2002
) and were significantly upregulated after addition of H2O2 in our transcriptome study. The results of these Northern experiments are shown in Fig. 2
.
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The results of the Northern hybridizations unambiguously demonstrated induction of the Fur regulon by H2O2 in synthetic as well as in rich medium. Therefore, it can be excluded that different metal concentrations in the media were responsible for the differences between our results and those obtained by Helmann et al. (2003). It has to be supposed that the use of different techniques for transcriptome analysis (macroarrays versus microarrays) accounts for the observed differences, perhaps as a result of differential sensitivities.
B regulon
Although oxidative stress does not represent a typical inducer of the B-dependent general stress response, 12 probable transcriptional units described to be regulated by
B (Petersohn et al., 2001
) were found to have significantly elevated expression levels after peroxide treatment, among them the csbD-, gspA- and yjgD-specific mRNAs. In addition, several of the
B-dependent genes were also induced by paraquat treatment. Inspection of the induction factors of the other known
B-dependent genes revealed that the majority were slightly induced after peroxide treatment even though the ratios did not match the criterion of significance. This observation is in accordance with the findings of Helmann et al. (2003)
, who also reported induction of
B-dependent genes after H2O2 treatment. Further studies will reveal if the activation of the
B regulon is accomplished by limitation of energy via the RsbP regulatory protein or if peroxide stress activates the general stress response by physical stress via the RsbU-dependent signalling cascade (Hecker & Völker, 2001
).
CtsR regulon
The expression of the CtsR-regulated operon ctsR-mcsA-mcsB-clpC, which belongs to class III of heat-shock genes (Krüger & Hecker, 1998), was increased by both oxidative stimuli. The two genes, radA and yacK, which are located immediately downstream of the clpC operon also exhibited oxidative stress induction, indicating transcriptional readthrough of the terminator structure downstream of clpC under these conditions. The other members of class III of heat-shock genes, the monocistronic transcribed genes clpE and clpP, showed around twofold increased mRNA amounts, thereby missing the criterion of significance, under one or both conditions. It was previously shown that class III heat-shock genes controlled by CtsR are strongly induced by the disulfide-stress-generating agent diamide (Leichert et al., 2003
). Disulfide stress may be considered as a subgroup of oxidative stress since peroxides are assumed to act as disulfide-generating agents and could represent the link between oxidative and heat stress.
Sulfur-limitation-regulated genes
Several genes encoding proteins involved in sulfur assimilation and synthesis of the sulfur-containing amino acids cysteine and methionine were induced after exposure to paraquat. In contrast, no change or even a downregulation was found for most of these genes after H2O2 challenge. Significant induction after exposure to was observed for 6 of the 11 transcriptional units described to form the S box regulon. As the S box regulatory mechanism was recently reported to be based on a direct interaction of available S-adenosylmethionine with the mRNA leader regions, the induction of this regulon indicated methionine limitation (Grundy & Henkin, 1998
; Mandal et al., 2003
; McDaniel et al., 2003
). The genes yxjG and yxjH, which are also predicted to represent members of the S box regulon, exhibited an induction of around twofold, thereby missing the criterion of significance.
Elevated expression was also found for the ssuABCDygaN operon, which encodes proteins for uptake and utilization of aliphatic sulfonates in the context of sulfur assimilation (van der Ploeg et al., 1998). This operon is not regulated by the S box mechanism and is repressed by sulfate and cysteine, which also holds true for the two recently characterized operons ytmIJKLMNO-ytnI-ribR-ytnLM and yxeKLMNOPQ, encoding proteins predicted to be involved in the utilization of alternative sulfur sources (Coppee et al., 2001
; Auger et al., 2002
).
Altogether, the data strongly suggest an stress induced sulfur limitation in B. subtilis. As the ironsulphur cluster containing sulphite reductase is not inactivated by
in E. coli, this phenotype may not result from a specific damage of this enzyme (Messner & Imlay, 1999
). The reduction of paraquat within its
-generating redox-cycling process might cause NADPH/H+ exhaustion, which may lead to insufficient sulfite reduction and in turn could trigger a sulfur limitation response. On the other hand, impairment of cysteine biosynthesis by
stress in E. coli is correlated with damage to the cell envelope and subsequent leak of sulfite (Benov et al., 1996
). These results were obtained using an
-accumulating sodA mutant, allowing the exclusion of secondary effects. Many bacteria produce low-molecular-mass thiols for protection against oxygen toxicity, e.g. glutathione or mycothiol (Newton et al., 1996
). In B. subtilis, a comparable mechanism has not been described up to now. However, a protective function of low-molecular-mass thiols from sulfur sources under oxidative stress conditions, especially after superoxide stress, cannot be excluded. The increased requirement of such compounds could therefore also explain the observed enhanced expression of genes encoding proteins involved in sulfur assimilation and synthesis of the sulfur-containing amino acids after exposure to paraquat.
SOS regulon
The genes belonging to the SOS regulon are controlled by the RecA/LexA (RecA/DinR) regulators (Miller et al., 1996). Members of this regulon, which encode the DNA-damage inducible (Din) proteins, are transcriptionally induced after DNA damage (Love et al., 1985
). Nearly half of these genes exhibited significantly increased mRNA amounts after exposure to H2O2. The induction of e.g. recA, lexA, uvrABC and dinC (renamed tagC in the SubtiList database) suggests H2O2-induced DNA damage and subsequent RecA/LexA activation under the chosen conditions. Several of the other known members of the din regulon exhibited elevated expression but missed the criterion of significance. The yneABynzC operon also belongs to the SOS regulon of B. subtilis and was recently found to be induced after H2O2 challenge (Kawai et al., 2003
). The yneB gene exhibited one of the strongest induction factors after peroxide treatment detected in this study, without a concomitant higher induction by paraquat. While YneA represents the functional counterpart of the cell division inhibitor SulA of E. coli, YneB shares similarities to resolvases (Kunst et al., 1997
; Kawai et al., 2003
).
Interestingly, increased expression of members of the SOS regulon was mostly limited to the generation of oxidative stress by addition of H2O2, and was much less pronounced after paraquat treatment. This finding might be explained by a strictly controlled dismutation of after paraquat addition which is followed by efficient catalase-dependent detoxification of the generated H2O2. Thus, generation of hydroxyl radicals from
might be kept to a minimum after paraquat treatment. In contrast, H2O2, which might be available in much higher amounts after peroxide addition for a short period of time, could be partly converted to OH
via Fenton chemistry, resulting in larger amounts of this highly DNA-damaging agent.
Stringent-regulated genes
Preliminary data indicate that oxidative stress induces ppGpp accumulation in E. coli (VanBogelen et al., 1987). In our study, the majority of the genes which were previously identified to be up- or downregulated in the course of the stringent response induced by amino acid limitation (Eymann et al., 2002
) also responded to oxidative stress. Positively stringent-regulated genes encoding extracellular serine proteases (Epr, Vpr) and urease (UreABC) were found at elevated expression levels after one or both stimuli. Furthermore, both ROS effected the downregulation of genes encoding ribosomal proteins and translation factors, indicating an extensive downregulation of the protein synthesis apparatus. However,
stress provoked stronger effects than H2O2 stress. This more pronounced response to
stress most probably results from the strong growth inhibition caused by the addition of paraquat. Whereas H2O2 is assumed to be rapidly degraded by cellular detoxification mechanisms, the continuous generation of
by the redox-cycling agent paraquat may cause an extreme overload of the cellular capability to cope with oxidative stress. Most probably, these differences in the extent of the oxidative stress are responsible for the observed differences in the growth behaviour and the intensity of the stringent response activation.
Other genes upregulated by oxidative stress
Besides several genes encoding products without known function, the expression of genes encoding proteins with predicted roles as antioxidants was also found to be increased in response to both stimuli. Clearly antioxidant functions are assigned to the gene products of trxA, trxB, msrA and tpx. The thioredoxin TrxA and the corresponding reductase TrxB are suggested to function in defence against oxidative stress by directly detoxifying H2O2 (Spector et al., 1988), by acting as hydrogen donor for peroxidases (Chae et al., 1994
) or by reactivating oxidatively damaged proteins, notably those with non-native disulfide bonds (Fernando et al., 1992
). The peptidyl methionine sulfoxide reductase MsrA is predicted to reduce methionine sulfoxides to methionine, thus restoring protein function after oxidative stress in B. subtilis, analogous to the role of MsrA in E. coli (Moskovitz et al., 1995
). The tpx gene encodes a predicted thiol peroxidase that probably acts in peroxide detoxification. Furthermore, genes which encode proteins with similarities to NADH-dependent flavin oxidoreductases (yqiG, yqjM) and several dehydrogenases exhibited increased expression after H2O2 and after paraquat challenge. It was recently reported that the YqjM protein shares similarity with the yeast Old Yellow Enzyme and is rapidly induced by addition of H2O2 or trinitrotoluene, suggesting a role in detoxification (Fitzpatrick et al., 2003
). The upregulation of a putative nitro/flavin reductase encoded by nfrA after H2O2 and paraquat treatment indicates a function in the reduction of nitric oxides after oxidative stress (Zenno et al., 1998
; Kobori et al., 2001
). In E. coli, the nfrA homologue nfsA is induced by
in a SoxRS-dependent manner (Benov & Fridovich, 2002
).
About 30 genes of unknown function exhibited significantly elevated expression levels after paraquat treatment and were less or not at all induced after H2O2 challenge. Only a weak induction of the sodA gene encoding the Mn-dependent superoxide dismutase was detectable after stress. The homologous gene in E. coli was demonstrated to be strongly induced in a SoxRS-dependent manner (Pomposiello & Demple, 2002
). Two B. subtilis open reading frames (sodF and yojM) encoding proteins with similarities to Fe- and CuZn-dependent superoxide dismutases of E. coli (Kunst et al., 1997
) were not induced after paraquat treatment. The spontaneous dismutation of
is supposed to be a very slow process. For efficient detoxification, conversion of
to H2O2 should be based on an antioxidant activity (Fridovich, 1995
; Fridovich, 1997
). Within the group of antioxidants, manganese, Mn(II) complexes and manganous porphyrins are capable of eliminating
(Archibald & Fridovich, 1981
, 1982
). The relatively high amount of manganese in our growth medium may serve for
dismutation under the chosen conditions, thus making an induction of superoxide dismutase unnecessary. This is in accordance with studies of Inaoka et al. (1999)
, who reported Mn(II)-dependent
-scavenging activity after paraquat treatment in B. subtilis. However, no induction of intracellular superoxide dismutase activity was found with or without manganese after paraquat exposure by these authors (Inaoka et al., 1998
).
Several genes were found to be significantly upregulated following H2O2 exposure but to a lesser extent or not at all after paraquat challenge. For most of their products, the function in peroxide resistance, if any, is still unknown.
Genes downregulated by oxidative stress
The datasets for all genes significantly downregulated by peroxide and paraquat stress including all expression level ratios are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org. In addition to the stringent response (see above), both stimuli caused significant repression of genes encoding proteins involved in the biosynthesis of purines, pyrimidines, arginine and histidine. Furthermore, several genes which encode enzymes of glycolysis and the tricarboxylic acid cycle were downregulated. The finding that the expression of several genes was strongly decreased by paraquat treatment but only slightly or not at all by H2O2 challenge may be due to the stronger growth inhibition after paraquat exposure. In contrast, several genes were only downregulated after peroxide challenge, among them the members of the cysH and the srf operon.
Proteins up- or downregulated by oxidative stress
The results of the 2D PAGE analyses after H2O2 and paraquat treatment (Fig. 1A, B) were in good agreement with these obtained in the transcriptome analyses. While members of the PerR, Fur and CtsR regulons were found to be induced by both stimuli (e.g. KatA, AhpC, AhpF, MrgA; DhbB, DhbD; ClpC), proteins encoded by
B- and RecA-regulated genes were exclusively or more distinctly upregulated after H2O2 challenge (e.g. GsiB; RecA). The synthesis of the methionine synthase MetE was reduced after peroxide stress but continued after exposure to paraquat. Both stimuli caused an extensive downregulation of the protein synthesis apparatus. A huge number of vegetative proteins were no more or less synthesized after the exposure to peroxide or paraquat, e.g. Ef-Tu, Ef-G, PtsH, PdhA and PurB. This downregulation of the protein biosynthesis apparatus was more pronounced after
challenge than after exposure to H2O2.
Concluding remarks
Exposure of B. subtilis cells to oxidative stress causes major changes in the global gene expression pattern. Besides partially overlapping responses induced by H2O2 and paraquat, clearly stimulus-specific gene expression patterns were detected. Both stimuli strongly induced the PerR-dependent stress response, albeit to a different extent. Furthermore, derepression of the Fur regulon, induction of proteins with antioxidant functions and a slight activation of the CtsR regulon after both stimuli were observed. The negatively stringent-controlled genes were also downregulated by both stresses, although the response was more distinct after addition of paraquat. The SOS response was found to be activated primarily by H2O2, which also induced a slight B-dependent stress response. Genes encoding proteins involved in sulfur assimilation and the biosynthesis of cysteine and methionine were induced solely by paraquat challenge, indicating sulfur limitation. Only a weak induction of sodA was detected by the transcriptome and proteome studies, recommending a further analysis of the SodA function in oxidative stress resistance. Our data support the assumption that induction of the PerR regulon represents the primary stress response after inorganic peroxide stress, whereas the weak induction of the
B regulon in the absence of additional limitations or stimuli is of minor physiological importance. No induction of the OhrR-regulated ohrA (yklA) gene was observed after H2O2 treatment, emphasizing the previously reported function of this gene in organic peroxide resistance but not in H2O2 stress resistance (Fuangthong et al., 2001
).
The combination of transcriptome and proteome analysis established a panoramic view of the adaptational strategies of B. subtilis to oxidative challenge. Many genes with still unknown function were observed to be induced or repressed by one or both stimuli. The next step in the context of a systematic analysis should be to address the contribution of the products encoded by the induced genes to oxidative stress defence, with the goal of a comprehensive view of stress adaptation after exposure to ROS.
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ACKNOWLEDGEMENTS |
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Received 23 July 2003;
revised 27 October 2003;
accepted 30 October 2003.
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