The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: John R. Guest. Tel: +44 114 222 4406/3. Fax: +44 114 272 8697. e-mail: j.r.guest{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: conitase, mRNA stability, oxidative stress, post-transcriptional regulation, proteinmRNA interactions
Abbreviations: IRE, iron-responsive element; IRP, iron-regulatory protein; MV, methyl viologen; NEM, N-ethylmaleimide; UTR, untranslated region
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INTRODUCTION |
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In Escherichia coli, two aconitases (AcnA and AcnB) have been identified and the existence of a third aconitase (AcnC) has been inferred from the residual activity (5% of wild-type) detected in an acnAB double mutant (Prodromou et al., 1992
; Bradbury et al., 1996
; Gruer et al., 1997b
). Sequence comparisons show that AcnA is 53% identical to human IRP1 and 2729% identical to mitochondrial aconitases, whereas AcnB is only 1517% identical to AcnA, the IRPs and mitochondrial aconitases. The major IRE-binding site of IRP1 (DLVIDHSIQVD), identified by UV cross-linking (Basilion et al., 1994
), is likewise conserved at nine of the eleven positions in AcnA but only at two positions in AcnB. It is also apparent that the domain organization of AcnB (4-1-2-3) is cyclically permuted relative to the arrangement (1-2-3-linker-4) found in other aconitases and the IRPs (Gruer et al., 1997a
). Specific roles for AcnA and AcnB have been identified by physiological and enzymological studies with acn mutants (Gruer et al., 1997b
), regulatory studies with acnlacZ reporter fusions, and transcript analyses (Gruer & Guest, 1994
; Cunningham et al., 1997
). Thus, AcnB is the major aconitase synthesized during the exponential phase, whereas AcnA is a stationary-phase enzyme which is also specifically induced by iron and oxidative stress. The aerobic growth of an acnA mutant is unimpaired in glucose minimal medium, indicating that this lesion is fully complemented by acnB+, whereas the growth of an acnB mutant is severely impaired and only slightly improved by adding glutamate. Growth is virtually abolished in an acnAB double mutant, except in the presence of glutamate, where the spontaneous mutational inactivation of the citrate synthase gene (gltA) seems to eliminate the deleterious effects of citrate accumulation and leads to the proliferation of acnAB gltA triple mutants. The regulatory studies further showed that the acnA gene is expressed from independent
38 and
70 promoters, the latter being repressed by FNR and ArcA and activated (directly or indirectly) by CRP, Fur, iron, FruR and SoxRS, whereas the acnB gene is expressed from a single
70 promoter that is repressed by ArcA, FruR and Fis, and activated by CRP.
It is not known whether any bacterial aconitases have regulatory functions like the IRPs. Aconitase is needed in Bacillus subtilis for sporulation but its mode of action is unclear (Dingman et al., 1987 ), and in Xanthomonas campestris a gene (rpfA) which regulates pathogenicity factor production has recently been shown to encode an aconitase that may serve as an iron-responsive transcription regulator (Wilson et al., 1998
). The present work shows that the apo-forms of AcnA and AcnB of E. coli each bind to the 3'UTRs of acnA mRNA and acnB mRNA and enhance the synthesis of both proteins (in vitro), presumably by increasing mRNA stability. A direct correlation between enzyme inactivation, increased Acn protein synthesis and acn mRNA stability, observed with oxidatively stressed cultures, further indicates that mRNA binding by bacterial aconitases represents a novel component of the oxidative stress response. Brief accounts of these findings were communicated previously at a Gordon Research Conference (Microbial Response to Stress, 1998) and the World Congress on Iron Metabolism (BioIron99 Abs 5).
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METHODS |
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Overproduction, purification and assay of aconitases.
The enzymes were purified from shaken cultures (2x500 ml) of JRG4004 (acnA) and JRG3099 (acnB) grown to OD595 0·5 at 37 °C before induction with IPTG (1 mM) for 4 h at 25 °C (AcnA) or 2 h at 37 °C (AcnB). The bacteria were resuspended in 20 ml Tris/HCl buffer (40 mM, pH 8·0) for preparing ultrasonic extracts. Ammonium ferrous sulphate (0·25 mM, final concn) and DTT (2·5 mM, final concn) were added to the cell-free extracts to reactivate the aconitases. Extract containing 150 mg protein in 20 ml was loaded on a DEAE Sepharose column (16x35 mm), washed with 5 vols Tris/HCl (40 mM, pH 8·0), and eluted with a linear gradient (300 ml, 00·2 M sodium citrate in the same buffer) using an FPLC system (Pharmacia). Coloured fractions containing AcnA or AcnB were pooled and ammonium sulphate (1·7 M, final concn) was added before applying to an Ether 650S column (16x40 mm). The column was washed with 5 vols 40 mM Tris/HCl (pH 8·0) containing 1·7 M ammonium sulphate and the Acn protein was eluted with a linear gradient (200 ml, 1·70 M ammonium sulphate in the same buffer). Typically about 30 mg Acn protein (>95% pure) was recovered.
The apo-aconitases were obtained by incubating holo-enzyme solutions with a 50-fold molar excess of EDTA (pH 8·0) and a 20-fold molar excess of potassium ferricyanide for 6 min at 4 °C according to Kennedy & Beinert (1988) and the protein was recovered using a small Ether 650S column, as above. Aconitase activity was measured according to Kennedy et al. (1983)
: one unit of activity represents 1 µmol cis-aconitate formed min-1. The native (as isolated) aconitases retained 620% of the specific activity and approximately 70% of the iron of the fully reconstituted enzymes whereas the apo-proteins were completely lacking in iron and totally inactive.
Affinity chromatography and RT-PCR procedures
. Apo-AcnA and apo-AcnB (500 µg protein) were coupled to 100 µl of activated CH Sepharose 4B (Pharmacia) by incubating for 16 h at 4 °C in 0·1 M NaHCO3 buffer pH 8·0 containing 0·5 M NaCl (final vol., 500 µl). The affinity matrices were washed exhaustively and blocked with 0·1 M Tris/HCl (pH 7·5), whereafter 20 µl samples were used to prepare affinity columns. Columns were equilibrated with 20 µg total MC4100 RNA in 200 µl RNA-binding buffer (10 mM HEPES pH 7·5, 3 mM MgCl2, 0·1 % Triton X-100, 40 mM KCl and 5% glycerol), as well as 1% ß-mercaptoethanol, 200 µg BSA, 150 U RNasin (Promega) and 20 µg tRNA, for 30 min at 20 °C, and then washed with 5 ml of the RNA-binding buffer to remove unbound RNA. Bound RNA was eluted with 50 µl 0·3 M sodium acetate buffer (pH 4·5) and precipitated with ethanol.
Reverse transcription was performed with all of the affinity-enriched RNA sample or 2 µg of total RNA in 2 µl, mixed with 1 µl reverse primer (40 µM), 1 µl reverse transcriptase buffer (10x) and 2 µl dNTPs (5 mM each), and heated to 70 °C for 10 min before cooling slowly to room temperature. Thereafter, 0·5 µl sodium pyrophosphate (80 mM), 0·5 µl actinomycin D (500 µg ml-1), 1 µl placental ribonuclease inhibitor (Promega), 1 µl AMV reverse transcriptase (25 U; NBL) and H2O to 10 µl were added and the mixtures were incubated at 37 °C for 90 min before the product DNA was precipitated by adding 1 µl sodium acetate (3 M; pH 4·5) and 50 µl ethanol. Specific segments of cDNA (~300600 bp copied from relevant coding regions) were then amplified by PCR with Taq DNA polymerase (Promega) over 25 cycles using appropriate forward primers and the same reverse primers, and the products were analysed by agarose gel electrophoresis (1·5%) using a 0·2510·0 kb GeneRuler (MBI Fermentas) for calibration.
RNA Sepharose affinity columns were prepared by cross-linking in vitro transcription products of the A5 and B5 3'UTR fragments, or 0·3 µg tRNA, to 100 µl activated CH Sepharose as described above. The columns were equilibrated with Acn protein (50 µg in 200 µl RNA-binding buffer) for 15 min at 20 °C before washing (2x2 ml RNA-binding buffer), eluting with successive 2x300 µl aliquots of the same buffer containing increasing concentrations of NaCl (0·13·0 M), and analysing the fractions by SDS-PAGE.
Gel retardation analysis.
RNA probes were first denatured by heating at 70 °C for 5 min and cooled rapidly. RNA-binding reactions were performed in 1 µl 10xRNA-binding buffer (100 mM HEPES pH 7·5, 30 mM MgCl2, 1% Triton X-100, 400 mM KCl and 50%, glycerol), 1 µl ß-mercaptoethanol (10%), 0·5 µl BSA (10 mg ml-1), 0·5 µl RNasin (Promega), 0·2 µl tRNA (200 µg ml-1), RNA probe (0·1 ng in 1 µl), Acn protein and H2O to 10 µl, incubated at 4 °C for 30 min. The Acn protein was pre-incubated with N-ethylmaleimide (NEM), when present, in buffer (7 µl) for 30 min at 4 °C before completing the reaction mixture and adding the probe. The samples were resolved in 5% non-denaturing polyacrylamide gels, which were dried and autoradiographed.
In vitro transcription.
Specific RNA molecules for gel retardation analysis were obtained by in vitro transcription of PCR-amplified DNA templates into which T3 promoters had been incorporated by including a T3 promoter sequence at the 5' end of each forward primer. The source plasmids for DNA amplification were pGS447 (acnA) and pGS801 (acnB) and the locations of the primers used to generate specific DNA templates, A1A7 and B1B5, are shown in Fig. 2(a). Transcription in vitro was performed with 10 ng PCR-amplified DNA in the presence of 1 µl RNasin (Promega), 4 µl T3 transcription buffer (5x; Promega), 2 µl DTT (100 mM), 2 µl UTP (500 µM), 2 µl ATP, GTP and UTP mix (each 2·5 mM), 1 µl BSA (20 mg ml-1), 2 µl [
-32P]UTP [3000 Ci mmol-1 (111 TBq mmol-1); NEN], 2 µl T3 RNA polymerase (Promega; 40 U) and water to 20 µl. After incubation at 37 °C for 90 min, 10 U RNase-free DNase I was added to remove the DNA templates, and labelled RNA was purified by phenol and ethanol precipitation.
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Methyl-viologen-induced oxidative stress.
The effects of methyl viologen (MV) on Acn activity and Acn protein synthesis were studied with cultures of E. coli DJ901 (soxR) grown for 2 h to exponential phase. Rifampicin (200 µg ml-1) was added followed immediately by MV and incubation for a further 1 h before measuring total aconitase activity (without reactivation) and immunoblotting with specific anti-Acn sera. The effects of MV on acn mRNA content and stability were studied by adding MV (0·3 mM) 10 min before rifampicin (200 µg ml-1), to cultures of W3110. Samples taken at 0, 2, 5, 10 and 20 min were chilled immediately to 4 °C in liquid N2 for RNA extraction and quantitative Northern blotting analysis. The RNA samples (25 µg) were fractionated in 1% agarose formaldehyde gel, transferred to Hybond-N (Amersham), and hybridized with [
-32P]dCTP-labelled probes according to the Ready to Go labelling-kit instructions (Pharmacia). The acnA and acnB probes containing the intact 2·5 kb (acnA) and 2·8 kb (acnB) coding regions, were generated by PCR and the relative densities of hybridized bands were measured with an Instant Imager (Electronic Autoradiography; Packard).
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RESULTS |
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Apo-AcnA had a lower affinity (KD8 µM) for both A5 and B5 compared to Apo-AcnB (KD
1·3 µM), where KD denotes the protein concentration giving half-maximal initial retardation. Although relatively low, the binding affinities are physiologically significant when related to the intracellular concentrations of aconitase deduced from specific activity measurements and Western blotting analysis: 16 µM for AcnA and 212 µM for AcnB, at different stages of the growth cycle (Gruer et al., 1997b
). The shifts were correspondingly smaller for AcnA than those induced by AcnB (Fig. 2b
). This could reflect inherently different Acn-binding affinities based on binding-site recognition. Alternatively, the presence of 124 additional amino acid residues in domain 4 of AcnB and correspondingly fewer in domains 13 compared to AcnA (Gruer et al., 1997a
) might have a greater effect on the mobility of the apo-AcnBRNA complex, especially if RNA is bound in the active-site cleft.
In order to ensure that the natural acn transcripts extend across the regions of Acn binding, RT-PCR was used with total E. coli RNA to provide evidence for transcription of the corresponding 3'UTRs. Two series of reverse primers designed to generate and amplify cDNAs from 169, 269 and 336 bp downstream of the acnA stop codon, and 81, 199, 308, 482 and 528 bp downstream of the acnB stop codon, were paired with forward primers hybridizing within the respective coding regions. Amplified products were obtained in every case except in the controls lacking reverse transcriptase. Thus it was concluded that even though the 3' transcription termini were not defined, they are located downstream of the longest 3'UTR segments (A5 and B5) studied here.
Effects of the AcnA and AcnB proteins on acn gene expression in vitro
The regulatory effects of IRPs have been observed in transcriptiontranslation systems in vitro (Kim et al., 1996 ; Gray et al., 1993
). Analogous studies with an E. coli transcriptiontranslation system showed that the apo-forms of AcnA and AcnB increase the de novo synthesis of both aconitases by up to fourfold, based on plasmid-directed incorporation of 35S into AcnA and AcnB (Fig. 3
). The stimulatory effects were apparent at apo-Acn concentrations as low as 0·2 µM (20 µg protein ml-1) except in one case where higher concentrations (
2·4 µM; 240 µg protein ml-1) of apo-AcnB were needed to stimulate acnB expression. In the same experiments, expression of the plasmid-encoded bla and yacH genes was unaffected by the aconitases (Fig. 3
). No incorporation in the Acn region was observed with vector controls, nor was any enhancement observed with comparable concentrations of bovine serum albumin. It was therefore concluded that the Acn apo-proteins specifically enhance expression of the acn genes, possibly at the translational level by acn transcript stabilization (via 3'UTR binding), as proposed for IRP1 and transferrin receptor mRNA.
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Effects of stress factors on Acn enzyme activity, Acn synthesis and acn mRNA stability, in vivo
Oxidative stress factors abolish the catalytic activity with the concomitant acquisition of the mRNA-binding and stabilizing activities of IRP1 (Pantopoulos & Hentze, 1995a , b
; Constable et al., 1992
). The conversion of active bacterial aconitases to the inactive [3Fe4S] or apo-enzyme forms could likewise provide a comparable stress-responsive switch for stabilizing acn and other relevant transcripts in vivo, especially as the aconitase activity of E. coli is known to be sensitive to oxidative stress (Gardner & Fridovich, 1992
; Gardner et al., 1997
). Accordingly, the effect of MV-induced oxidative stress on aconitase activity and Acn protein synthesis was investigated in growing cultures of E. coli. Strain DJ901 (
soxR) was chosen because it allowed translation to be studied during oxidative stress without interference from SoxRS-dependent activation of acnA transcription (Gruer & Guest, 1994
; Cunningham et al., 1997
). In a typical experiment the total aconitase activity decreased by 60% whilst the AcnA and AcnB proteins increased three- to fourfold in response to added MV (Fig. 5
). Similar reciprocal effects were observed with H2O2 (0·5 mM) and manganous ions (50 µM). These findings are therefore consistent with the view that inactivated aconitases enhance the synthesis of Acn proteins in vivo, presumably by stabilizing the corresponding mRNAs.
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DISCUSSION |
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Early attempts to detect AcnA binding to mammalian IREs by gel retardation were unsuccessful (R. D. Klausner & J. R. Guest, unpublished) and the same was found with MICP, the AcnA of Legionella pneumophila (Mengaud & Horwitz, 1993 ). Three IRE-like sequences of unknown significance have been found in E. coli (Dandekar et al., 1998
): one is in the frdB coding region; another is in a large multi-component stemloop and coding region located upstream of the fepB gene; and the third is in the 5'UTR of the hemA gene. The transcriptiontranslation activity of a hemAlacZ fusion of uncertain origin (kindly provided by R. P. Gunsalus) was unaffected by inactivating the acnA and acnB genes (M. A. Quail & J. R. Guest, unpublished).
The binding affinities of apo-AcnA and apo-AcnB for the acn 3'UTRs (KD8·0 and 1·3 µM, respectively) are much lower than have been reported for IRP1 and IRE (KD=550 pM; Butt et al., 1996
). Nevertheless, relative to the intracellular concentrations of AcnA and AcnB (6 and 12 µM maximum, respectively), the apo-protein binding affinities are entirely compatible with the operation of two sensitive physiologically poised switches. Higher affinities might indeed be deleterious to the growth of E. coli. In view of its great instability, AcnB could provide a particularly sensitive mechanism for stress-induced apo-enzyme formation and mRNA binding/stabilization. It is possible that the observed mRNA-binding affinities are underestimated because the formation of stemloops that might be needed for optimal apo-Acn binding could have been limited by the use of denatured and rapidly cooled RNA in the gel retardation experiments. It is also relevant that longer 3'UTRs are needed for retardation than for detecting translational enhancement in vitro. Thus, the shorter subfragments, A4 and B3, were not retarded (Fig. 2
) even though they contain sufficient of the 3'UTR to enhance AcnA and AcnB synthesis from DNA-derived transcripts in transcriptiontranslation tests (Fig. 4
). This could mean that the 3'UTRs contain several apo-Acn-binding sites, not all of which are located in the minimal fragments, or that secondary structural elements form more readily in longer RNA molecules.
Attempts to identify potential binding sites for the Acn apo-protein by comparing the sequences and secondary structural features of the two acn 3'UTRs revealed two paired motifs, AAACACAAUGC and CAUUUU, that flank putative stemloops A and B
in each 3'UTR (Fig. 8
). Their locations correlate precisely with the minimal requirements for detecting translational enhancement, 169>A>71 and 80>B>0 bp, for the respective acnA and acnB 3'UTRs (Fig. 4
). There is also a sequence related to the first of these motifs downstream of B
(Fig. 8
), which might contribute to a second binding site and simultaneously explain why a longer 3'UTR (279 bp) improves translational enhancement with acnB. If significant, these motifs should be regarded as common features of potentially different AcnA- and AcnB-binding sites because there is no evidence that the two apo-proteins bind to identical sites even though they exhibit overlapping regulatory activities. These sites are clearly different from IREs, where sequence conservation is largely confined to the loops of the stemloops recognized by IRPs. Searching the E. coli genome for pairs of motifs analogous to those flanking the A2 and B2 stemloops, but with variable hyphenation, revealed no identities. Sequences differing at only one position exist but there were no preferred locations or functional relationships among the corresponding genes.
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The overlapping (cross-reacting) feature of the proposed AcnA/B autoregulatory system probably ensures that despite variations in relative amounts of the two acn transcripts (and their products) in different growth phases, Acn synthesis will increase in response to oxidative stress. However, in view of the sixfold higher affinity of apo-AcnB for acn 3'UTR fragments relative to apo-AcnA, it is envisaged that AcnB will play the major role in mounting a particularly sensitive response to oxidative stress in growing cultures by enhancing the synthesis of the stabler AcnA (as well as AcnB) for survival and recovery purposes. The proposed autoregulatory mechanisms also provide a very plausible explanation for previous observations that AcnA and AcnB amplification from pUC-derived plasmids greatly exceeds the amounts expected from copy number alone (i.e. because accumulating apo-Acn stabilizes the acn transcripts).
The present work has revealed a hitherto undetected mechanism for modulating bacterial gene expression at the translational level based on the redox-sensitivity of the aconitases. It offers a rapid initial response to oxidative stress that is activated before the longer-term SoxRS and OxyR transcriptional regulatory mechanisms take effect (Hidalgo & Demple, 1996 ). It is switched by the interconversion of catalytic and RNA-binding forms of aconitase just like IRP1-mediated post-transcriptional regulation. Indeed, it could be speculated that RNA binding is an inherent property of the aconitases leading to the evolution of the high-affinity IRP1 system of higher organisms, where regulating gene expression from specific relatively stable transcripts is more important than in bacteria. It is also interesting that an ironsulphur cluster serves as the sensory cofactor as in the SoxR- and FNR-mediated switches. The Acn system has so far been shown to autoregulate aconitase synthesis but it could perform a more general role in mounting the oxidative stress response, and possibly in regulating hostpathogen interactions and bacterial differentiation, where aconitases have been implicated. Future work will be aimed at characterizing the Acn regulatory system and the effects of stress factors (using a semi-synthetic reporter gene), defining the moleular basis of the AcnmRNA interactions, and identifying other Acn-regulated genes.
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ACKNOWLEDGEMENTS |
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Received 11 June 1999;
revised 27 July 1999;
accepted 2 August 1999.