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. 2009 Feb;71(4):876-94.
doi: 10.1111/j.1365-2958.2008.06568.x. Epub 2008 Dec 23.

Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR)

Thi Thu Huyen Nguyen  1 , Warawan Eiamphungporn, Ulrike Mäder, Manuel Liebeke, Michael Lalk, Michael Hecker, John D Helmann, Haike Antelmann
Affiliations

Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR)

Thi Thu Huyen Nguyen et al. Mol Microbiol. 2009 Feb.

Abstract

Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding a gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively.

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Figures

Fig. 1.
Fig. 1.
Thiol-dependent detoxification pathways for methylgloxal (A) and formaldehyde (B) in bacteria. A. MG reacts spontaneously with GSH to form hemithioacetal, followed by isomerization to S-lactoylglutathione by the glyoxalase I enzyme. S-lactoylglutathione is substrate for the glyoxalase II enzyme and is converted to D-lactate and free GSH. This scheme is adapted from Ferguson et al. (1998). B. In prokaryotes and eukaryotes FA is detoxified by GSH-dependent Fdhs (Harms et al., 1996). FA reacts spontaneously and reversibly with GSH to form S-hydroxymethylglutathione. Fdh-like enzymes such as class III alcohol dehydrogenases catalyse the NAD-dependent oxidation of S-hydroxymethylglutathione into S-formylglutathione (Jensen et al., 1998). This GSH thiol ester is reversibly hydrolysed by a S-formylglutathione hydrolase to GSH and formate. This scheme is adapted from Jensen et al. (1998).
Fig. 2.
Fig. 2.
Growth curves (upper panels) and survival ratios (lower panels) of B. subtilis wild-type cells in the presence of FA (left) and MG (right). B. subtilis wild-type cells were grown in minimal medium to an OD500 of 0.4 and exposed to 0.5, 1, 2, 5 and 10 mM FA or 1.4, 2.8, 5.6 and 11.2 mM MG at the time points that were set to zero. Appropriate dilutions were plated for viable counts (cfu ml−1).
Fig. 3.
Fig. 3.
Dual-channel images of the protein synthesis pattern of B. subtilis wild-type before (green image) and 10 min after the exposure to 5.6 mM MG (A, red image) or 1 mM FA (B, red image). Cytoplasmic proteins were labelled with L-[35S]methionine and separated by 2D PAGE as described in Experimental procedures. Image analysis of the autoradiograms was performed using the Decodon Delta 2D software. Proteins that are synthesized at increased levels in response to FA or MG stress in at least two independent experiments are indicated by white labels. Their respective induction ratios are listed in Table S1. Spot identification was performed using MALDI-TOF-TOF mass spectrometry from Coomassie-stained 2D gels as described in Experimental procedures.
Fig. 4.
Fig. 4.
Transcriptional analysis of selected genes that are strongly induced in the microarray analyses by FA and MG. Transcript analysis of clpE, nfrA, cysK, arsB and katA indicates the induction of the CtsR, Spx, CymR, ArsR and PerR regulons by FA and MG, which overlaps with the response to diamide and quinone-like electrophiles (Antelmann et al., 2008). Transcription of the HxlR-controlled hxlAB operon is specifically induced by aldehydes. The formate dehydrogenase operon yrhED and the formate transporter operon yrhFG–yrzI respond specifically to MG. The arrows point towards the size of the specific transcripts.
Fig. 5.
Fig. 5.
Hierarchical clustering analysis of gene expression in response to electrophiles in B. subtilis. The treatment with the electrophiles includes diamide (Dia), catechol (Cat), methylhydroquinone (MHQ), formaldehyde (FA) and methylglyoxal (MG). Genes expression data were clustered based on the induction ratios leading to ten defined groups (nodes). Nodes enriched for genes that belong to the electrophile stress responsive regulons (CtsR, Spx, CymR, ArsR, CzrA, CsoR, SigmaD), the quinone-responsive regulons [PerR, YodB, CatR (YvaP), MhqR] and the aldehyde-responsive regulons (HxlR, AdhR, LexA) are shown to the right of the cluster. Red indicates induction and green repression under the specific conditions of electrophile stress (see Experimental procedures for details).
Fig. 6.
Fig. 6.
Transcript analyses (A), transcriptional organization (B) and promoter alignments of the adhR, yraC and adhA–yraA operons which are regulated by the MerR-type transcriptional regulator AdhR in response to carbonyl compounds. A. For Northern blot experiments 5 μg of RNA each was isolated from the B. subtilis strains before (co) and 10 min after exposure to 1 mM FA, 2.8 mM and 5.5 mM MG. Northern blots were hybridized with yraA, adhA or adhR-specific mRNA probes (shown underlined). The arrows point towards the sizes of the adhA–yraA, yraA and adhR specific transcripts. B. Gene organization of adhR, yraC and adhA–yraA. Transcriptional start sites are indicated by bent arrows. C. The transcription start sites of the adhR, yraC and adhA–yraA specific mRNAs were determined by 5´ RACE and are indicated as +1. The −10 and −35 promoter elements are underlined. The conserved inverted repeat sequences are shown in upper case letters.
Fig. 7.
Fig. 7.
Binding of purified AdhR to the adhR, yraC and adhA promoters using DNA electrophoretic mobility shift assay. Increasing amounts of purified AdhR protein were added to the labelled adhR (A), yraC (B) and adhA (C) promoter probes. Approximately 10 mM of MG or FA was added to analyse the effect of these compounds on the DNA binding activity of AdhR to these target promoters. (D) AdhR does not bind to an unrelated promoter fragment (the yoeB promoter region).
Fig. 8.
Fig. 8.
The conserved Cys52 of AdhR is essential for activation of adhA–yraA transcription by aldehydes. A. For adhA-specific Northern blot analysis, RNA was isolated from strains before (co) and after treatment with 2% xylose for 20 min and subsequently with 2.8 mM MG or 1 mM FA for 10 min. B. Western blot analysis was performed using Anti-FLAG antiserum to confirm expression of FLAG–AdhR and FLAG–AdhRC52A proteins.
Fig. 9.
Fig. 9.
The ΔadhA and ΔhxlR mutants are sensitive to FA stress and the ΔyraAΔyfkM mutant is sensitive to FA and MG. B. subtilis wild-type (wt), ΔadhA, ΔyraAΔyfkM and ΔhxlR mutant strains were grown in minimal medium to an OD500 of 0.4 and treated with 0.5 and 1 mM FA or 1.4 mM MG. The growth rates of the strains after exposure to FA and MG are shown.
Fig. 10.
Fig. 10.
The LMW thiol cysteine protects against aldehyde toxicity in B. subtilis. A. Changes in the level of cysteine in the metabolome were measured using GC-MS in B. subtilis cellular extracts after exposure to FA and MG as described in Experimental procedures. Average values and standard deviations are quantified from at least three independent cultivation experiments and the ratios are related to the untreated control. B., C. B. subtilis wild-type cells were grown in minimal medium to an OD of 0.4 and different concentrations of extracellular cysteine were added prior to exposure of FA (B) or MG (C). These experiments indicate that 1 mM cysteine was able to protect against 2 mM FA and 3 mM cysteine titrates 3 mM MG which restored the growth.
Fig. 11.
Fig. 11.
Protective mechanisms against formaldehyde toxicity in B. subtilis. Exposure of B. subtilis to FA induces general electrophile-stress responsive regulons (CymR, Spx, CtsR), the peroxide specific PerR regulon, metal ion-efflux regulons (ArsR, CsoR, CzrA), the DNA damage inducible LexA regulon and aldehyde detoxification regulons (AdhR, HxlR). Pathways that have been demonstrated in this study are shown with solid arrows and predicted pathways are displayed with dashed arrays. The AdhR and HxlR regulons for specific detoxification are marked in yellow and the other FA-inducible stress regulons are marked in red. (1). FA reacts with low-molecular-weight thiols (R-SH) to S-hydroxymethylthiol, which is converted to S-formylthiol by the thiol-dependent AdhR-controlled AdhA and further by an unknown hydrolase to formate. (2). S-formylthiols could be also exported via the metal ion efflux systems such as ArsBC, CopA, CzcD, which are upregulated by FA. (3). In addition, FA is detoxified via the RuMP pathway by HxlA generating hexulose-6-phosphate (H6P) and HxlB producing fructose-6-phosphate (F6P). (4). The reaction of FA with thiol buffers (e.g. cysteine) results in depletion of reduced LMW thiols and an imbalanced thiol-redox homeostasis. This leads to derepression of the CymR regulon to increase cysteine biosynthesis. (5). Depletion of reduced thiol buffers also causes induction of the Spx regulon to induce thiol-disulphide reducing systems (TrxAB) and restore the thiol-redox balance. (6). FA causes DNA–protein cross-links resulting in DNA damage and induction of the LexA regulon to repair DNA damages. (7). FA reacts also with protein thiols leading to protein cross-links or S-hydroxymethyl modifications in proteins. These modified or aggregated proteins are proteolytically degraded by the CtsR-regulated ClpCP machinery and repaired or degraded by the YraA cysteine proteinase. (8). The reaction of FA with DNA and proteins could generate also secondary reactive intermediates that in turn could produce ROS which lead to the induction of PerR-regulated oxidative stress defence enzymes.
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