The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK1
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK2
Author for correspondence: Jeffrey Green. Tel: +44 114 222 4403. Fax: +44 114 272 8697. e-mail: jeff.green{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: acid stress, radical protein, pH homeostasis, pyruvate formate-lyase, formate
Abbreviations: dO2, dissolved oxygen tension; FNR, fumarate and nitrate reduction regulator; GST, glutathione S-transferase; PFL, pyruvate formate-lyase
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INTRODUCTION |
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The yfiD gene product has appeared in five proteomic screens. Firstly, as a polypeptide upregulated by expression of the heterologous oxygen-responsive FNR protein, HlyX, in anaerobically grown E. coli (Green & Baldwin, 1997 ). In the second analysis, YfiD was induced to high levels when the cytoplasm was acidified, i.e. during growth at pH 4·4, and upon the addition of propionate at pH 6 (Blankenhorn et al., 1999
). Interestingly, the effects of pH on gene expression are known to overlap with many other environmental factors, but particularly with oxygen availability (Slonczewski & Foster, 1996
). It has been noted that the acid-induced expression of amino acid decarboxylases is enhanced under anaerobic conditions (Meng & Bennett, 1992
; Neely et al., 1994
) and that expression of cytochrome o is repressed by acid stress (Cotter et al., 1990
). It was therefore suggested that there is a complex relationship between YfiD, pH and oxygen (Blankenhorn et al., 1999
). Thirdly, YfiD was decreased in gel-entrapped E. coli, and on the basis of its transient expression pattern it was suggested that YfiD is an early stationary phase protein (Perrot et al., 2000
). In the fourth analysis, enhanced yfiD expression was noted in strains harbouring heterologous polyhydroxyalkanoate biosynthesis genes (Han et al., 2001
). It was again suggested that YfiD was present under these particular conditions because the cytoplasm becomes more acidic. Finally, YfiD was detected as a phosphorylated protein in L-forms of E. coli (Freestone et al., 1998
). Thus, although the exact physiological role of YfiD has not yet been assigned, this protein has emerged from a range of studies related to anaerobic and acidic growth. Therefore, because yfiD expression is regulated by FNR and the YfiD gene product is acid-inducible, we have investigated the effects of anaerobiosis, pH and growth phase on yfiD expression and activation of the YfiD protein. We find that the major regulator of yfiD expression is FNR, but that yfiD expression is induced at the level of transcription during growth under acidic conditions. Furthermore, the activation of YfiD by PFL activase is enhanced by growth at low pH in the presence of propionate or benzoate. In addition, accumulation of lactate, succinate, pyruvate and formate is greater for the yfiD mutant strain than for the parent, consistent with a role for YfiD in reducing exposure to acidic metabolic end products and in directing carbon flux.
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METHODS |
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Protein purification.
YfiD was initially purified as a glutathione S-transferase (GST)YfiD fusion protein amplified in aerobically grown E. coli BL21(pGS1011). Cultures (500 ml) were grown at 37 °C with vigorous shaking in 2xYT medium (Sambrook et al., 1989 ) for 3 h, at which point the expression of the gstyfiD fusion was initiated by addition of IPTG to a final concentration of 30 mg l-1. Incubation was continued for a further 3 h before the cultures were harvested by centrifugation. Clarified French pressure cell extracts (up to 150 mg total protein, in 10 mM Tris/HCl, pH 8·0 containing 10 mM benzamidine and 0·1 mM PMSF) were applied to a column (50x15 mm) of glutathioneagarose (Sigma) equilibrated with 50 mM Tris/HCl, pH 6·8 containing 0·5 mM DTT. The column was washed with 10 vols of the same buffer before eluting the GSTYfiD fusion with 50 mM Tris/HCl, pH 8·0 containing 10 mM reduced glutathione. Aliquots of the fusion protein were dialysed to remove glutathione and the pure YfiD protein was obtained by cleavage of the desalted GSTYfiD fusion with thrombin (14 U) for 2 h at 37 °C, followed by chromatography on glutathioneSepharose to remove the released GST. The product contains an extra 15 aa, which are derived from the thrombin-sensitive linker, whose presence was confirmed by the total amino acid analysis and N-terminal sequencing. Protein concentrations were estimated with the Bio-Rad protein reagent.
Characterization of YfiD.
Total metal ion contents and electrospray mass spectrometry measurements were done with redissolved freeze-dried samples of isolated YfiD that had been dialysed against several changes of 1 mM ammonium acetate, pH 6·0. The native molecular mass of YfiD was also estimated by gel filtration on a Superdex 200 HR 10/30 column (Amersham-Pharmacia) equilibrated with 50 mM sodium phosphate buffer, pH 7·0 containing 0·15 M sodium chloride, and calibrated with a 650066000 molecular weight marker kit for gel filtration (Sigma). N-terminal amino acid sequencing was achieved by Edman degradation using an Applied Biosystems protein sequencer. Standard technologies were used for Western blotting. Polyclonal YfiD anti-sera were raised in rabbits (dwarf lop) and used at a final dilution of 1:20000. Detection of cross-reacting material was achieved using the Amersham-Pharmacia ECL detection system with horseradish peroxidase conjugated secondary antibodies, or by nitro blue tetrazolium/X-phosphate for alkaline phosphatase conjugated secondary antibodies, and was quantified using ImageMaster software (Amersham-Pharmacia).
Continuous culture.
Chemostat cultures were grown in LH Fermentation chemostat vessels (either 1 l nominal capacity, 750 ml working volume or 2 l nominal capacity, 1130 ml working volume) maintained at 37 °C. The minimal medium had the following composition (g l-1): (NH4)2SO4, 2·0; K2HPO4, 1·0; NaH2PO4, 1·0; MgSO4.7H2O, 0·2; supplemented with trace element solution (Vishniac & Santer, 1957 ), 2 ml; and glucose, 30 mM. The pH was adjusted to 7·0±0·2 by automatic titration with sterile 2 M KOH. Dissolved oxygen levels were monitored with galvanic oxygen electrodes. For aerobic cultures the culture agitation speed (400500 r.p.m.) and air flow were adjusted to maintain a dissolved oxygen tension (dO2) >50% air saturation or <5% for microaerobic cultures. For oxygen-starved cultures, the air supply was replaced by oxygen-free nitrogen. In some experiments air and nitrogen mixtures were used to gas the cultures. Inocula were grown as batch cultures overnight in the continuous culture medium.
Glucose in feed medium and culture filtrates was estimated enzymically with glucose oxidase (Bergmeyer & Bernt, 1968 ). Low molecular mass overmetabolites in culture filtrates were monitored by HPLC using a Shodex Ion pak KC-811 column (8x300 mm) at 50 °C. Samples were eluted with 0·1% (v/v) phosphoric acid at a flow rate of 2·0 ml min-1 through detectors for UV (210 nm) and refractive index (Waters 490E and Waters 410, respectively) coupled to a data module (Millennium 2010). Peak areas were proportional to concentrations, which were determined using standard solutions of each organic acid. Acetate, formate and ethanol were also estimated using enzyme-based Boehringer Mannheim kits, which also served to confirm these peak assignments in the HPLC elution profiles; the estimates obtained by this method were similar to those obtained by calibrated HPLC.
To investigate whether the yfiD null mutant is physiologically disadvantaged compared to the parent, a chemostat containing carbon-limited medium was inoculated with approximately equal numbers of W3110 and JRG4033 bacteria by combining equivalent overnight cultures of these strains. The mixed culture was allowed to grow as a batch culture in the fermentation vessel for 8 h before switching to continuous growth with a dilution rate of 0·2 h-1. Samples (
5 ml) were taken immediately after switching to continuous growth and subsequently every 510 generations. The proportion of each strain in the culture vessel was determined by plating serial dilutions (in triplicate) of the culture on carbon-limited agar plates with and without kanamycin (30 mg l-1). The plates were incubated for 40 h at 37 °C before counting the number of c.f.u. recovered. The numbers were averaged and the percentage of JRG4033 (kanr bacteria) was calculated. The competition experiments were performed over 50 generations under aerobic (50% dO2) and microaerobic (5% dO2) conditions.
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RESULTS |
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It has long been recognized that there are overlaps in bacterial responses to anaerobiosis and acid stress at the level of gene regulation (Cotter et al., 1990 ; Meng & Bennett, 1992
; Neely et al., 1994
; Slonczewski & Foster, 1996
; Blankenhorn et al., 1999
). Therefore, we considered whether FNR and/or ArcAB, the major oxygen-sensing systems of E. coli contributed to the acid enhancement of yfiD expression. This could be achieved by enhancement of the activities of either, or both, regulatory systems at lower intracellular pH values. The data presented in Table 2
and Fig. 1
confirmed that FNR is responsible for the anaerobic induction of yfiD expression with dramatically reduced anaerobic yfiD::lac expression reported for the fnr mutant strain (200 Miller U at pH 7·2) compared to the parent (2990 Miller U at pH 7·2). As expected from the data in Table 1
, lowering the pH of the cultures increased anaerobic yfiD::lac expression for the parent (from 2990 Miller U at pH 7·2 to 4210 Miller U at pH 6·3) but there was only a small increase for anaerobic cultures of the fnr mutant (from 200 Miller U at pH 7·2 to 230 Miller U at pH 6·3). To test the possibility that FNR may be directly involved in the observed acid response, expression from the simple FNR-regulated synthetic promoter FF-41.5 (Wing et al., 1995
) was investigated at pH 7·2 and 6·3. The experiments revealed that although FNR-dependent expression from this promoter was subject to a small but reproducible enhancement after growth at low pH, it is unlikely that FNR activity is modulated by intracellular pH (Table 2
).
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Further analysis of the yfiD promoter revealed the absence of consensus sites for regulators such as PhoP and OmpR that are known to be involved in the response of E. coli to acid. However, two overlapping putative ArcA sites (7/10 bases matching the consensus centred at -97, AGTTgATgtA, and -94, TGTaAAacAA; non-consensus bases in lower case) were detected within the far upstream (-93·5) FNR site that is responsible for the anaerobic downregulation of yfiD expression (Green et al., 1998 ), suggesting that ArcA might prevent FNR binding at this location and thereby increase yfiD expression. Therefore, the effects of an arcA mutation on yfiD::lac expression were tested (Table 2
). Anaerobic expression of yfiD was 22·7-fold lower in an arcA mutant strain (2540 Miller U compared to 6810 Miller U for the same strain expressing arcA from the plasmid pRB38 at pH 7·2, and 4470 Miller U compared to 9210 Miller U at pH 6·3), indicating that ArcA has a positive effect on yfiD expression. The simplest explanation for this data, based on our knowledge of FNR-mediated regulation of yfiD expression (Green et al., 1998
; Green & Marshall, 1999
; Marshall et al., 2001
), is that ArcA relieves the repression mediated by the far upstream FNR dimer by competing for occupation of the -93 region of the yfiD promoter. Expression of the solely FNR-regulated model promoter FF-41.5 (Wing et al., 1995
) was tested in the arcA background to determine whether there was any effect of removing ArcA on a simple FNR-driven promoter. Deletion of arcA had no significant effect on FNR-mediated expression from FF-41.5 at both pH 7·2 and pH 6·3, and thus the ArcA-mediated enhancement of yfiD expression (Table 2
) is probably mediated by ArcA competing with FNR for occupancy of the -93 region of the yfiD promoter, where FNR can act negatively and ArcA positively, and that the positive effects of ArcA are magnified by the presence of arcA in multiple copies. However, this mechanism is not directly responsible for the anaerobic acid induction of yfiD expression because the yfiD promoter remains pH-responsive in the arcA background (2540 Miller U at pH 7·2, compared to 4470 Miller U at pH 6·3) (Table 2
).
Analysis of the yfiD promoter region revealed the presence of a near consensus (cAaTGGTTTTACCAATT, 12/14, non-consensus bases in lower case) PdhR binding site (Quail & Guest, 1995 ) centred at +17 relative to the transcript start. The PdhR protein is responsible for repressing the expression of the pyruvate dehydrogenase complex, and thus plays a key role in the regulation of the central metabolic pathways in E. coli. PdhR senses the status of the intracellular pyruvate pool by binding the co-effector, pyruvate, and because the PdhRpyruvate complex cannot bind target DNA, expression of the pdhRaceEFlpd operon, encoding pyruvate dehydrogenase, is derepressed when pyruvate is available (Quail & Guest, 1995
). The expression of the yfiD::lac reporter was tested in JRG2547, a strain that carries the pdhRc mutation that prevents PdhR binding to target DNA (Quail & Guest, 1995
). The data show that, as might be predicted from the position of the PdhR box, PdhR acts as a weak anaerobic repressor of yfiD expression (2990 Miller U at pH 7·2 for the parent compared to 3740 Miller U for the pdhRc mutant). Furthermore, because acidic growth conditions still enhance yfiD expression in a pdhRc background (3740 Miller U at pH 7·2 compared to 4950 Miller U at pH 6·3), it would appear that PdhR does not have a major role in acid-induced yfiD expression (Table 2
).
A yfiD null mutant accumulates increased levels of acidic metabolic end products
To attempt to define a role for the YfiD protein, a yfiD null mutant strain (JRG4033) was created by internal deletion and insertion of a kanamycin resistance cassette into the yfiD gene of E. coli W3110. After Western blotting to confirm that JRG4033 was unable to produce YfiD protein (Fig. 1), a range of phenotypic tests were applied to the mutant, including: monitoring growth and viability of cultures grown aerobically and anaerobically on a variety of carbon sources; sensitivity to UV irradiation, ethanol and freezethaw cycling; and morphological differences under light and electron microscopy. However no obvious phenotype was indicated by these tests. To investigate whether YfiD plays a role in resisting acid stress, the responses of the parent (W3110) and the yfiD mutant (JRG4033) to acid shock were monitored. Under aerobic and anaerobic conditions the parent and mutant responded similarly in viability tests to acid shock at pH 2·5 (inorganic acid) or at pH 4·5 (weak organic acid), after growth at pH 7·2 or pH 5·7 (not shown), indicating no fundamental role for YfiD in resisting, or adapting to, acid stress. Therefore, it was concluded that the role of YfiD must be more subtle than could be revealed by the simple tests employed.
Because the tests described above failed to reveal a phenotype, and because yfiD expression is maximal under microaerobic conditions (Marshall et al., 2001 ), the properties of the yfiD mutant (JRG4033) and parent (W3110) strains were assessed in aerobic (50% dO2) and microaerobic (5% dO2) continuous culture competition experiments. When grown aerobically, the proportions of parent and mutant in the culture were essentially constant over 50 generations. However, when cultured microaerobically (<5% dO2) the proportion of the yfiD null mutant, as estimated by the relative number of kanamycin-resistant (yfiD null mutant) c.f.u., was reduced from 49% to 29%, and from 45% to 23%, in two independent experiments, within 35 generations. These data indicate that the yfiD mutant strain has a microaerobic growth defect. To investigate this further, the properties of the mutant (JRG4033) and parent (W3110) were compared in chemostat cultures under both aerobic and oxygen-starved conditions. The measured growth parameters (µmax and Ymax, max growth rate and max growth yield respectively) indicated that the disruption of the yfiD gene did not have a profound effect on the growth of E. coli under oxygen-starved conditions, confirming the observations made in batch culture. However, analysis of the culture medium of the yfiD mutant strain showed that it contained elevated levels of lactate, succinate, pyruvate and formate compared to the parental strain (Table 3
). The concentrations of other metabolic end products (carbon dioxide, acetate and ethanol) were similar for both strains. During growth in the presence of oxygen there was no significant accumulation of any of the overmetabolites by either strain (not shown). These observations indicate that, directly or indirectly, YfiD acts to prevent the accumulation of lactate, succinate, pyruvate and formate during oxygen-starved growth, either by routing carbon flux away from these products or by removing them once they have formed. Such a role is in accord with the anaerobic- and acid-inducibility of yfiD expression, and also with the roles of other acid-inducible genes whose products appear to remove, or to prevent the accumulation of, acidic compounds.
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Expression of yfiD is enhanced in stationary phase
Previous studies indicated that yfiD is expressed throughout the anaerobic growth cycle but that expression increased a further 1·7-fold in stationary phase cultures compared to exponential phase (Green et al., 1998 ). A more thorough analysis of the growth phase-dependency of yfiD::lac expression was undertaken following the suggestion that YfiD is an early stationary phase protein (Perrot et al., 2000
). The data revealed that in cultures buffered at pH 7·2, aerobic expression of yfiD::lac was low throughout the growth cycle, but increased as growth proceeded (Fig. 3
). During the anaerobic growth cycle of similarly buffered cultures, yfiD::lac expression was much higher than observed aerobically, but followed the same pattern, with maximum ß-galactosidase activity being observed in stationary phase. Accordingly, under these conditions, YfiD protein was at the limits of detection by Western blotting of whole cell samples obtained at intervals during aerobic growth (<1000 molecules per cell), but accumulated in anaerobically grown cultures reaching a concentration of approximately 100000 molecules per cell (or 3·4% of total protein) after 7 h growth (not shown). This level of YfiD protein was maintained even after 48 h, indicating that YfiD is not an early stationary phase protein under these growth conditions, but that it accumulates throughout the growth cycle and is present for at least 48 h after growth has ceased.
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DISCUSSION |
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The overlapping pattern of regulation of yfiD and pflB has acquired new significance because during the re viewing of this work it has been shown that YfiD can act as a spare part by providing an active glycyl radical domain to restore activity to oxygenolytically cleaved PFL (Wagner et al., 2001 ). PFL cleaves pyruvate to form acetyl-CoA (which is subsequently converted to acetate) and formate (which is converted to carbon dioxide and hydrogen) during anaerobic growth. Thus, it is suggested that YfiD may act to maintain/restore PFL activity to bacteria that have been exposed to oxygen (Wagner et al., 2001
). Such a role is consistent with FNR-mediated regulation tuned for maximum yfiD expression in microaerobic conditions (Marshall et al., 2001
), and with PdhR-mediated repression of yfiD expression in response to low intracellular pyruvate. Moreover, the chemostat studies with the yfiD mutant provide further support for the spare part hypothesis, which predicts that in the absence of YfiD under microaerobic conditions, carbon flux through PFL would be reduced due to an inability to repair oxygenolytically cleaved PFL. Reduced flux through PFL could lead to increased excretion of pyruvate, succinate and lactate, but not acetate, ethanol or formate. The data presented here indicate that a lesion in yfiD leads to excretion of increased levels of pyruvate (16-fold greater than for the parent strain), succinate (>15-fold higher) and lactate (>140-fold higher) whereas the levels of ethanol and acetate were similar for both the parent and yfiD mutant strains (Table 3
). All these features are consistent with YfiD acting to maintain high levels of PFL activity during microaerobic growth. The sevenfold increase in formate excretion for the yfiD mutant cultures does not at first sight appear to be consistent with this role. However, because the growth medium contains limited amounts of oxygen and no other exogenous electron acceptors, it could be that the reduced flux through PFL, caused by the inability to rescue the activity of oxygen-damaged PFL because of the absence of YfiD, has not allowed sufficient formate to accumulate to activate the FhlA-regulated formate hydrogen-lyase system in the mutant cultures. Previous studies have shown that maximal anaerobic expression of hycB (a component of formate hydrogen-lyase) at pH 7·0 in vivo requires the presence of 30 mM formate in the culture medium (Rossmann et al., 1991
). Moreover, 10 mM formate has been shown to be necessary for maximum activity of FhlA in vitro (Hopper et al., 1994
). Although these studies are not directly comparable to those described here, they do indicate that substantial formate accumulation is required for the optimal induction of formate hydrogen-lyase. Consequently, if formate hydrogen-lyase is not induced in the yfiD mutant strain, due to subinductive intracellular formate concentrations resulting from reduced flux through PFL, then formate would not be disproportionated to carbon dioxide and water. This could lead to the detection of increased levels of formate in the yfiD mutant cultures compared to the parental cultures, where, because the carbon flux through PFL is uninhibited, sufficient formate is produced to induce a functional formate hydrogen-lyase. Other possibilities that cannot be discounted are that YfiD may contribute directly or indirectly to the metabolism of formate, perhaps by reversing the PFL reaction, or that the formate accumulating in the yfiD mutant cultures arises from a source other than PFL. Further work will be needed to resolve these questions.
Consistent with the proposal that YfiD acts as a radical domain for damaged PFL and possibly other radical proteins in E. coli, the YfiD radical shares many features with that of PFL. The location of the YfiD radical was presumed to be G102 by analogy with PFL (Wagner et al., 1992 ); and this has recently been shown to be the case (Wagner et al., 2001
). Here it is shown that the YfiD radical is inserted by the PFL activase, PflA. Therefore, yfiD and pflB not only share similar regulation at the transcriptional level, but the activation, and thus the activities, of the two proteins are also linked. In PFL the glycine radical acts as a radical store when the protein is in the resting state (Becker et al., 1999
) and it would now seem that a similar function is fulfilled by G102 in YfiD (Wagner et al., 2001
). The observation that the radical content of YfiD is increased at low pH suggests that activated PFL is more easily oxygenolytically cleaved under such conditions and consequently more YfiD is activated to restore activity to the damaged PFL protein. Whether the YfiD protein can, under certain conditions, have activity per se or whether its function is only to repair oxidatively damaged PFL is as yet unknown. The reaction mechanism of PFL requires an amino acid triad consisting of G734, C418 and C419. In the resting state the radical resides at G734 but during initiation of the PFL reaction it is transferred to C419 and then to C418 (Becker et al., 1999
). This sequence can be reversed to return the protein to the resting state, allowing multiple turnovers without regeneration of the glycyl radical. In contrast to PFL, the YfiD protein has only one cysteine residue (C31) and thus if it has independent activity it must use a different catalytic mechanism to that of PFL. Other glycyl radical enzymes are known that lack the adjacent cysteine motif of PFL (Sawers & Watson, 1998
; Selmer & Andrei, 2001
). The failure to observe increased oxygenolytic cleavage of YfiD from cultures unable to express the PFL deactivase AdhE (a multifunctional alcohol dehydrogenase), which acts to quench the PFL radical and thereby prevent oxidative cleavage occurring is consistent with the proposed role of YfiD, because YfiD would need to be activated under conditions that could damage PFL. Thus, the work reported here describes strong links between the regulation of expression and activity of YfiD and PFL, in accord with the proposed role of YfiD as a radical-containing module that can be plugged into oxidatively damaged PFL to restore activity to this key enzyme.
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ACKNOWLEDGEMENTS |
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Received 7 September 2001;
revised 20 November 2001;
accepted 27 November 2001.
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