The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Jeffrey Green. Tel: +44 114 2224403. Fax: +44 114 2728697. e-mail: jeff.green{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: proteinmRNA interactions, proteomics, ironsulphur protein
Abbreviations: Acn, aconitase; IRP, iron regulatory protein; MV, methyl viologen; ODH, 2-oxoglutarate dehydrogenase; SCS, succinyl-CoA synthetase; SodA, superoxide dismutase; UTR, untranslated region
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INTRODUCTION |
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In higher organisms, the cytoplasmic aconitases (c-Acns) are remarkable bifunctional proteins that gain aconitase catalytic activity (c-Acn) by assembling [4Fe4S] clusters under conditions of iron sufficiency, but during iron-starvation and oxidative stress, when the clusters are disassembled, the catalytic activity is lost and the apo-proteins acquire site-specific RNA-binding activity (IRP1) (Beinert et al., 1996 ). The apo-proteins (IRP1s) recognize specific sequences [iron regulatory elements (IREs)], which form stemloop structures at the 5' or 3' untranslated regions (UTRs) of relevant mRNA transcripts, such as ferritin and the transferrin receptor. Binding of IRP1 at the 5' UTRs blocks translation, whereas binding at the 3' UTRs enhances translation by increasing transcript stability (Hentze & Kuhn, 1996
). The RNA binding activity of IRP1 increases in response to reagents such as H2O2, superoxide and nitric oxide, which induce oxidative stress, degrade the ironsulphur clusters and hence inactivate the enzyme (c-Acn) and generate IRP1.
It has been shown that the aconitases of E. coli (AcnA and AcnB) and the aconitase of B. subtilis (CitB) also have dual roles as enzymes and post-transcriptional regulators (Tang & Guest, 1999 ; Alen & Sonenshein, 1999
). Apo-AcnA and apo-AcnB have each been shown to interact at physiological concentrations with the 3' UTRs of acnA and acnB mRNA when their ironsulphur clusters are oxidatively disassembled. Consequently, AcnA and AcnB synthesis is enhanced in vitro by the apo-Acns and this enhancement is abolished by deletion of the 3' UTRs from the DNA templates, presumably through loss of acnmRNA stabilization by bound apo-Acn. In B. subtilis aconitase has been shown to recognize a rabbit ferritin IRE and IRE-like sequences/structures in operons encoding cytochrome oxidase and an iron uptake system (Alen & Sonenshein, 1999
). Furthermore, a mutant strain expressing an enzymically inactive aconitase that retains RNA binding activity was able to sporulate more efficiently than an aconitase-null mutant, suggesting that aconitase has a non-enzymic role in sporulation (Alen & Sonenshein, 1999
). More recently, the E. coli AcnA and AcnB apo-proteins have likewise been shown to bind site-specifically to the rabbit ferritin IRE; previous failures to induce binding were attributed to the low range of protein concentrations used in earlier tests (Y. Tang & J.R. Guest, unpublished observations). Analyses of virulence factor production in Pseudomonas aeruginosa (exotoxin A) and Xanthomonas campestris have also indicated that aconitase might perform a regulatory role (Somerville et al., 1999
; Wilson et al., 1998
).
As stated above, the two aconitases of E. coli have been adapted for different physiological roles, but both are sensitive to conditions of oxidative stress mediated by superoxide and by nitric oxide (Gardner & Fridovich, 1992 ; Gardner et al., 1997
). Restoration of activity after nitric oxide treatment is inefficient, probably due to nitrosylation of the [4Fe4S] cluster. However, after oxidation by oxygen aconitase activity can be efficiently restored by anaerobic reassembly of the ironsulphur cluster. Thus, the two aconitase proteins of E. coli have the potential to serve as oxidative stress sensors through the reversible assembly/disassembly of their [4Fe4S] clusters and the consequent interconversion between catalytic and RNA-binding functions. Furthermore, because of the different relative stabilities of the ironsulphur clusters of AcnA and AcnB the response can be poised at two levels, with AcnB sensing low/intermediate stress and AcnA reacting during exposure to severe stress. Here, the physiological and regulatory effects of acn mutations on the oxidative stress response of E. coli were investigated. It was found that acn mutants are hypersensitive to oxidative stress and that the synthesis of superoxide dismutase (SodA) is positively regulated by apo-AcnA and negatively regulated by apo-AcnB.
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METHODS |
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DNA was prepared and manipulated by standard procedures (Sambrook et al., 1989 ). Total bacterial RNA was extracted from W3110 by the hot acid/phenol method (Aiba et al., 1981
). The RNA (
100 µg) was resuspended in 50 µl of buffer containing 20 mM Tris/HCl (pH 8·0), 10 mM MgCl2, 1 mM DTT and 10% (v/v) glycerol before adding RNase-free DNase I (20 U) and incubating at 37 °C for 30 min to remove traces of DNA. The RNA was then isolated by phenol extraction followed by ethanol precipitation. Aconitase holo-proteins and apo-proteins were prepared according to Tang & Guest (1999)
.
Proteomic analyses.
Cultures (in 10 ml of L broth) of MC4100 (parent), JRG3509 (acnA), JRG3510 (acnB) and JRG3511 (acnAB) were grown with vigorous shaking (250 r.p.m.) in 100 ml flasks at 37 °C to exponential phase (OD600 0·81·0). The bacteria were then either harvested or, where stated, their transcription was arrested by adding rifampicin (0·2 mg ml-1) immediately prior to adding MV (0·3 mM). Incubation was then continued for a further 60 min. The harvested bacteria were resuspended in 20 mM Tris/HCl (pH 7·3) for ultrasonic disruption and cell-free supernatant extracts were recovered by centrifugation at 14000 g for 10 min at 4 °C and stored at -20 °C. Protein contents were assayed by the Bio-Rad method, with bovine serum albumin as the standard, and samples containing 100 µg protein were applied to immobilized pH gradient strips (110 mm in length; a linear pH 47 gradient, Amersham Pharmacia) for fractionation in the first dimension. Second-dimension fractionation was performed with ExcelGel SDS 818%, using a Pharmacia Multiphor II electrophoresis unit. In preliminary experiments pairs of identical gels were either stained with Coomassie blue R-250 for protein visualization or electroblotted onto ProBlott membranes for protein identification by N-terminal-amino-acid sequencing. In subsequent experiments, stained gels were analysed using ImageMaster 3.01 software (Amersham Pharmacia) and polypeptide spots that varied in intensity by at least 2·5-fold due to redox stress or to strain differences were excised, destained and digested with trypsin prior to peptide analysis by mass spectrometry. The MASCOT program was used to identify proteins from their peptide profiles.
Northern blotting and mRNA half-life.
The effects of MV on sodA mRNA content and stability were studied with equivalent exponential-phase cultures of parental and acn strains. Rifampicin (200 µg ml-1) was added 10 min after the addition of MV (0·3 mM); samples were then taken after a further 0, 2, 5, 10 and 20 min, and chilled immediately in liquid N2 for RNA extraction and quantitative Northern analysis. RNA (25 µg) was separated in a 1% agarose/formaldehyde gel (Sambrook et al., 1989 ), denatured and then transferred to a Hybond-N membrane (Amersham Pharmacia) for hybridization using a sodA probe labelled with [
-32P]dCTP [3000 Ci mmol-1 (111 TBq mmol-1); NEN] according to the manufacturers instructions (Ready to Go Labelling Kit; Amersham Pharmacia). The probe, containing the entire sodA-coding region flanked by two 300 bp segments, was PCR-amplified from E. coli W3110 chromosomal DNA. The relative amounts of sodA mRNA in each RNA sample and their corresponding half-lives were determined by direct analysis of the blot using a Packard Instant Imager with Electronic Autoradiography software (version 2.01).
In vitro transcriptiontranslation.
Plasmid DNA (pGEM-T containing the sodA-coding region and 300 bp of upstream and downstream sequences from W3110 genomic DNA) or linear DNA amplified from the pGEM-T sodA plasmid with the Boehringer Expand High Fidelity PCR System (1 µg) was mixed with 3·75 µl S30 extract for linear templates (Promega), 0·25 µl [35S]methionine [1000 Ci mmol-1 (37 TBq mmol-1); ICN], 1·25 µl amino acid mixture minus methionine, 5 µl S30 premix, apo-aconitase (0·2 and 2·0 µM final concentration prepared according to Tang & Guest, 1999 ) and H2O to 12·5 µl, before incubating at 37 °C for 90 min. Translation products were precipitated with 50 µl acetone and resuspended in loading buffer for SDS-PAGE separation, autoradiography and quantitative analysis of 35S-labelled components excised from the gels and counted in vials containing 3 ml of Safefluor S (Lumac-LSC) using a Beckman LS 1801 Liquid Scintillation System.
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RESULTS |
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In subsequent experiments, the effects of adding MV to each strain were investigated. Moreover, rifampicin was added to stressed and unstressed cultures in order to arrest transcription and thus to improve the detection of changes potentially stemming from the post-transcriptional (mRNA-binding) activities of the apo-aconitases. Representative protein profiles from redox-stressed cultures of the parent and acnB strains are shown in Fig. 3. Quantitative pairwise comparisons of the profiles of redox-stressed and unstressed cultures of each strain revealed a total of 17 polypeptides that both varied at least 2·5-fold under stress or between strains and were identifiable by tryptic digestion and mass spectrometry (Table 2
). Again there was a marked increase in SodA production by the acnB mutant and also in the direct response to redox stress in other strains (Table 2
; Fig. 3
). Hence, the SodA content increased 4·8-fold in the parental strain and threefold (to the same level) in the acnA mutant when exposed to MV (Table 2
). However, consistent with the preliminary findings, the acnB mutant produced ninefold more SodA than its parent in the absence of MV, reaching a level that was almost twice the level of SodA achieved by redox-stressed cultures of the parental and acnA strains (Table 2
). It was also apparent that despite having a relatively high level of SodA, the acnB mutant strain could still produce more under redox-stress conditions (Table 2
). The SodA profile of the acnA acnB double mutant resembled that of the acnB mutant in having an elevated basal level of SodA, but it no longer appeared to be further enhanced by redox stress conditions.
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Other changes included the lowering of chaperone protein levels (GroEL, DnaK and Tig) in the acnB mutant strain (Table 2). This implies that protein folding problems may arise when AcnB is absent. In the same context the modulations of oligopeptide-binding protein (OppA) and maltose-binding protein (MalE) synthesis may be related to protein folding, because both have been shown to interact with unfolded proteins, to promote the functional folding of citrate synthase and
-glucosidase, and to prevent aggregation of citrate synthase under heat shock conditions (Richarme & Caldas, 1997
). Alternatively, proteins such as MalE, OppA, ODH and succinyl-CoA synthetase (SCS; ß-subunit) that participate in substrate transport and the citric acid cycle may be responding to the absence of a fully functional citric acid cycle.
The greatly enhanced level of HdeB in the acnB mutant is interesting because HdeB is a periplasmic protein that may be involved in acid resistance. Its expression is enhanced in stationary phase and repressed by H-NS (Yoshida et al., 1993 ; Holland et al., 1999
) and the pattern of expression observed here suggests that AcnA (the stationary-phase enzyme) has a positive effect on HdeB synthesis, consistent with the view that Acn proteins assist in co-ordinating a stress response. Finally, the RpsA contents are lowered in all of the acn mutants (Table 2
). RpsA is a component of the 30S ribosomal subunit that is essential for the efficient translation of most bacterial transcripts and it negatively autoregulates translation probably by interacting with its own transcript (Skouv et al., 1990
; Boni et al., 2000
) not unlike the aconitases (Tang & Guest, 1999
). Hence, translation of rpsA mRNA is geared to ribosome synthesis because the former is inhibited by excess RpsA subunits. The lower levels of RpsA in acn mutants suggest that overall translation activity may be reduced in these mutants, and thus the increase in SodA and TrxB contents is all the more significant. This may be linked in some unknown manner to the increased levels of asparaginyl-tRNA synthetase (AsnS), N-acetylneuraminate lyase and dihydrofolate reductase.
From the proteomic studies, it was concluded that acn lesions affect the production of proteins associated with the oxidative-stress response, central metabolism, acid stress, protein translation, protein folding and DNA repair. Indeed, the overall pattern of changes suggests that the mutant profiles resemble those exhibited by the parental strain after exposure to oxidative stress. Also, it appeared that in particular AcnA and AcnB directly or indirectly affect the synthesis of SodA, even in the absence of transcription.
Studies with a sodAlacZ translational fusion
Further studies on the effects of acn lesions on SodA synthesis were made with the isogenic series of MC4100 (lac) derivatives containing acnA and acnB mutations and a sodAlacZ translational fusion (Table 1
). Expression of sodAlacZ in exponential cultures of the parental strain was approximately threefold higher when MV was added (Fig. 4
). Exactly the same expression profiles were observed for sodA expression in the acnA mutant (Fig. 4
). However, in unstressed cultures of the acnB mutant, sodA expression approached that of the stressed parental cultures and was enhanced a further
1·5-fold by adding MV (Fig. 4
). Compared to the acnB mutant sodA expression was lower than in comparable cultures of the acnAB double mutant, but not as low as in cultures of the parental or acnA strains (Fig. 4
). The pattern of sodA expression in exponential-phase cultures was consistent with the proteomic analysis and it indicates that both aconitase proteins affect SodA synthesis either directly or indirectly, but in opposite directions. Relative to the baseline provided by the double mutant it appears that AcnA enhances sodA expression, whereas AcnB is inhibitory, possibly via an inhibitory interaction between apo-AcnB and the 5' UTR of the sodA transcript. Furthermore, it would appear that in the parental strain the positive influence of AcnA is completely masked by the negative regulatory effects of AcnB. Any enhancement that might normally stem from Acn interaction with the 3' UTR of the sodA transcript would not be observed in these experiments because this region is deleted from the sodAlacZ reporter.
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Synthesis and stability of sodA mRNA
A remarkable feature of the apo-Acn proteins of E. coli is their ability to interact specifically with the 3' UTRs of both acn transcripts, thereby increasing translation and the intracellular contents of the Acn proteins by protecting the acn transcripts (Tang & Guest, 1999 ). To investigate whether aconitase-mediated post-transcriptional regulation contributes to the changes in SodA content, revealed by the proteomic and sodAlacZ fusion studies, the amounts and half-lives of sodA mRNA were compared in acn mutant and parental strains. Northern blotting showed that the sodA transcript increased 23-fold in the acnB mutant compared to the parent (Fig. 5
). The relative amounts of sodA mRNA also increased six- and threefold in the acnA and acnAB mutants, respectively. The half-lives of the sodA transcripts likewise increased from 5 min in the parental strain to 9, 11 and >20 min in the acnA, acnAB and acnB mutants, respectively (Fig. 5
). Hence, it would appear that the sodA transcript has a greatly increased half-life and is consequently more abundant in the acnB mutant than in the other strains. The relative amounts of sodA transcript matched the levels of the sodAlacZ expression exhibited by the corresponding unstressed fusion strains (Fig. 4
), at least insofar as the values for the acnB mutant were higher than those of the acnAB double mutant. Moreover, all of the strains responded to MV by increasing their sodA mRNA contents, the relative increases being greatest with the parental strain and least with the acnB mutant (not shown), which also agrees with the sodAlacZ fusion data (Fig. 4
). Therefore, the results indicate that, either directly or indirectly, AcnB reduces the stability of sodA transcripts and that at least one factor contributing to the increased expression of sodA in the acnB mutant is likely to be mRNA stabilization.
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DISCUSSION |
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The major exponential-phase aconitase, AcnB, appears to be particularly important for neutralizing the effects of superoxide generated during aerobic growth and in resisting H2O2. In contrast, the stationary-phase SoxRS-regulated enzyme, AcnA, seems to play a lesser role in aerobic exponential-phase cultures, as expected from the observed patterns of transcriptional regulation of the two acn genes (Cunningham et al., 1997 ). Potential targets affected by the absence of aconitase-mediated neutralization of reactive oxygen species were revealed by proteomic analysis. These targets include SodA and TrxB, which both have well-characterized roles in resisting oxidative stress, indicating that the accumulation of reactive oxygen species in the absence of Acn is sufficient to trigger a response. However, the degree of stress generated by the acn lesions was not sufficient to induce expression of the OxyR regulon because the catalase activities [0·075 to 0·081 U (mg protein)-1] and the amounts of KatG protein were unaffected in the acn mutants relative to the parental strain. Other consequences of the acn lesions were reduced levels of translation (RpsA, AsnS) and chaperone (GroEL, DnaK, Tig) proteins, and increased amounts of proteins of the citric acid cycle (ODH, SCS), substrate transport (OppA), acid stress (HdeB) and possibly DNA repair (YggX). Many of the corresponding genes have recently been reported to respond to the imposition of MV and/or sodium salicylate stress (Pomposiello et al., 2001
). The up-regulation of components of the ODH complex and of SCS is interesting because these enzymes are major control points in switching the citric acid cycle from its aerobic, mainly catabolic, role to its anaerobic, non-cyclic and mainly anabolic, role. The acn lesions studied here would clearly impair or abolish carbon flux through the citric acid cycle and lead to depletion of the NADH:NADPH pools, which would be further depleted by the presence of MV. The up-regulation of ODH and SCS might represent misguided attempts to restore the flow of carbon through the citric acid cycle, thereby generating reducing equivalents.
The regulation of sodA expression is complex because the activity of the sodA promoter is modulated by at least five transcription factors [SoxRS, Fur, FNR (fumurate and nitrate reduction regulator), ArcA and IHF (integration host factor)] (Compan & Touati, 1993 ). Expression is mainly controlled by SoxRS, which activates sodA expression in response to superoxide, whereas FNR, ArcA, Fur and IHF repress expression in response to oxygen and iron availability. This combination of regulators allows sodA expression to be geared to the degree of stress posed by the simultaneous exposure to iron and oxygen (superoxide), which can lead to the production of very toxic hydroxyl radicals. The present studies suggest another seemingly more immediate regulatory mechanism that controls SodA synthesis at the translational level. It would now appear that the aconitases can act both positively (AcnA) and negatively (AcnB) to control translation of the sodA message. This dual control may facilitate fine-tuning with respect to growth phase and environmental stress. Analysis of the sodA transcript for sequences/structures resembling those associated with Acn-binding to acn mRNA has revealed only one short sequence, ACGCG, located in the 5' UTR (12 bases downstream of the transcriptional start site and 42 bases upstream of the start codon) of the sodA transcript which is present in the 3' UTRs of the two acn transcripts (Tang & Guest, 1999
). The absence of stemloop structures resembling those identified in the 3' UTRs of the acnA and acnB transcripts in sodA suggests that interactions between apo-Acn proteins and sodA may be distinct from those observed previously, but the effects on translation of sodA occur at similar concentrations to those required for regulation of acn translation (Tang & Guest, 1999
).
The enhanced expression of sodA in E. coli acn mutants is a clear indication that by acting as a sink for superoxide and by regulating sodA translation the Acn proteins serve as a first line of defence against the superoxide produced during aerobic metabolism. Thus, it is suggested that the status of the [4Fe4S] cluster of AcnB reflects the degree of oxidative stress normally experienced by E. coli. When oxidative stress is low AcnB might serve both as an aconitase and as a repressor of sodA translation, but as the level of stress increases the more stable AcnA protein takes over the metabolic function of AcnB. This response is sufficient to manage the superoxide stress without resorting to enhanced SodA production, and thus remaining apo-AcnB continues to block sodA translation and destabilize the sodA transcript. If the level of superoxide stress continues to threaten the bacterium the SoxRS system is activated and transcription of sodA is enhanced. Consequently, the intracellular concentration of the sodA transcript increases relative to the concentration of AcnB and in combination with the generation of apo-AcnA, through the use of AcnA [4Fe4S] clusters as superoxide sinks, the sodA transcript is stabilized and translation is enhanced to detoxify remaining superoxide.
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ACKNOWLEDGEMENTS |
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Received 12 October 2001;
revised 4 December 2001;
accepted 7 December 2001.