In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states

David P. Dibden and Jeffrey Green

Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK

Correspondence
Jeffrey Green
jeff.green{at}sheffield.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
FNR proteins are transcription regulators that sense changes in oxygen availability via assembly–disassembly of [4Fe–4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe–4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron–sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions, ~45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR- and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The FNR protein is an oxygen-responsive transcription regulator that controls the expression of more than 100 transcriptional units in Escherichia coli, and coordinates the transition from aerobic to anaerobic growth (Becker et al., 1996; Covert et al., 2004; Gonzalez et al., 2003; Guest et al., 1996; Kang et al., 2005; Salmon et al., 2003; Sawers, 1999; Unden & Bongaerts, 1997). The intracellular concentration of FNR is similar under both aerobic and anaerobic conditions (Sutton et al., 2004a; Unden & Duchene, 1987), but FNR is activated under anaerobic conditions by the acquisition of [4Fe–4S] clusters that promote the formation of homodimers, and enhance DNA binding at sites resembling the consensus sequence TTGAT-N4-ATCAA (Bauer et al., 1999; Eiglmeier et al., 1989; Green et al., 1996, 2001; Green & Paget, 2004; Khoroshilova et al., 1995, 1997; Kiley & Beinert, 1999, 2003; Lazazzera et al., 1993, 1996; Moore & Kiley, 2001; Patschkowski et al., 2000; Popescu et al., 1998). Four essential cysteine residues (Cys20, 23, 29 and 122) ligate the FNR [4Fe–4S] cluster, which is incorporated in a process involving the isc operon (Schwartz et al., 2000). Accordingly, deletion of the iscS gene (a cysteine desulphurase that provides the sulphur for iron–sulphur cluster assembly) impairs FNR activity in vivo (Schwartz et al., 2000). Furthermore, comparison of the effects on FNR, which requires an iron–sulphur cluster for activity, and those on FNR*, an FNR variant that is active in the absence of an iron–sulphur cluster, suggests that the isc operon plays a major role in the assembly of the FNR iron–sulphur cluster (Schwartz et al., 2000).

The [4Fe–4S] form of FNR acts as both a positive and a negative regulator of gene expression, activating transcription by recruiting RNA polymerase, or repressing transcription by inhibiting the formation of productive promoter–RNA polymerase interactions (Barnard et al., 2004; Bell & Busby, 1994; Blake et al., 2003; Browning et al., 2003; Green & Marshall, 1999; Green et al., 1998; Lamberg & Kiley, 2000; Lamberg et al., 2002; Li et al., 1998; Lonetto et al., 1998; Marshall et al., 2001; Meng et al., 1997; Williams et al., 1997; Wing et al., 2000). Exposure of E. coli to air is sensed by the disassembly of the FNR iron–sulphur clusters. The [4Fe–4S] clusters are first converted to [2Fe–2S] clusters (Crack et al., 2004; Jordan et al., 1997; Khoroshilova et al., 1997; Sutton et al., 2004a, b). This inactivates FNR by promoting the formation of FNR monomers, and inhibition of DNA binding (Lazazzera et al., 1996). When oxygen persists in the environment, the [2Fe–2S] clusters disassemble, yielding apo-FNR (Green et al., 1991; Achebach et al., 2005) in a process that can be driven by superoxide, a by-product of aerobic metabolism (Sutton et al., 2004b). Consequently, FNR-activated genes are switched off, and FNR-repressed genes are switched on under aerobic conditions.

Studies in vitro have shown that under anaerobic conditions iron–sulphur clusters can be incorporated into the isolated apo-FNR protein, and the reconstituted protein can then bind to DNA with high affinity, and regulate transcription from target promoters (Green et al., 1996; Jordan et al., 1997; Khoroshilova et al., 1995). Furthermore, in vivo studies of the expression of an FNR-activated gene, and the reactivity of the cysteine thiol groups (Cys20, 23, 29 and 122) of FNR that ligate the iron–sulphur clusters, suggest that inactive apo-FNR and active iron–sulphur-containing FNR can be interconverted when cultures are shifted between aerobic and anaerobic conditions (Engel et al., 1991; Six et al., 1996). Thus, under aerobic conditions, the cysteinyl residues that are used to ligate both the [4Fe–4S] and [2Fe–2S] clusters of FNR are reactive, and can be carboxymethylated, but when cultures are shifted to anaerobic conditions, the cysteinyl residues are protected (Engel et al., 1991). Moreover, it has recently been suggested that aerobically grown E. coli cells contain significant amounts of apo-FNR (Achebach et al., 2005). The simplest explanation for these observations is that a significant amount of FNR is present in the apo form under aerobic conditions, and, upon transfer to anaerobic conditions, apo-FNR acquires iron–sulphur clusters. However, it has been suggested that rather than being available to receive an iron–sulphur cluster, apo-FNR protein is mostly degraded, and that iron–sulphur clusters are mostly incorporated into newly synthesized FNR (Patschkowski et al., 2000). Here, evidence is presented to support the view that FNR is cycled between active and inactive states in the absence of de novo FNR synthesis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The bacterial strains used were: E. coli MC1000 (lac), and the isogenic fnr mutant JRG1728 (Spiro & Guest, 1987a); E. coli MG1655, and the isogenic clpX mutant. The plasmids used were: pJLA502 (ApR), an expression vector with a temperature-inducible promoter (Schauder et al., 1987); pGS199 (ApR), a pJLA502-based fnr expression plasmid (Spiro & Guest, 1987b); and pFF-41.5 (TetR), a low-copy-number plasmid with a class II FNR-activated promoter fused to lacZ (Wing et al., 1995). Standard methods were used to transform bacteria, and isolate DNA (Sambrook & Russell, 2001). Bacterial cultures were grown in Lennox broth (Lennox, 1955) containing tryptone (10 g l–1), yeast extract (5 g l–1), NaCl (5 g l–1), and supplemented with antibiotics as indicated: tetracycline (35 mg l–1), ampicillin (100 mg l–1), kanamycin (50 mg l–1). Cultures were grown from colonies on agar plates at 37 °C, either aerobically in conical flasks (10–20 ml in a 250 ml flask, shaking at 250 r.p.m.), or anaerobically in sealed bottles (7–25 ml, without shaking). The MIC values for chloramphenicol (to inhibit protein synthesis) and rifampicin (to inhibit transcription) were determined by measuring growth of aerobic cultures in the presence of increasing concentrations of the antibiotics. The MICs were 10 µg ml–1 for chloramphenicol, and 6 µg ml–1 for rifampicin.

lacZ reporter experiments.
E. coli JRG1728 (fnr), containing the reporter plasmid FF-41.5, was transformed with either pJLA502 (vector) or pGS199 (encoding FNR). Three independent cultures (20 ml) of both strains were grown under aerobic conditions at 25 °C for 5·5 h, before increasing the temperature to 42 °C for 30 min to induce FNR synthesis. The cultures were then returned to 25 °C for 1 h to switch off further fnr transcription. At this stage, one group of cultures was transferred to an anaerobic workstation (Don Whitley MK3), and poured into universal vials, which were sealed and placed in anaerobic jars (Oxoid). These anaerobic cultures were then incubated at 25 °C for 3·5 h. A second group of cultures was maintained under aerobic conditions. At each stage, samples of the cultures (2–3 ml) were taken for the measurement of {beta}-galactosidase activity in triplicate (Miller, 1972), and to estimate the amounts of FNR in the cytoplasm by using Western blotting. For immunoblotting, crude cell extracts were prepared by alkaline lysis, and, after matching the protein contents of the samples, polypeptides were separated by SDS-PAGE (10 % polyacrylamide). The polypeptides were then transferred to nitrocellulose membranes for detection of FNR using a polyclonal antibody (1 : 10 000 working dilution) and the ECL Plus system (Amersham). The relative amount of FNR in each sample was determined using ImageMaster software (Amersham).

Addition of 14C-labelled amino acids (0·185 MBq; Amersham) to post-induction cultures of JRG1728 (fnr) containing pGS199 was used to determine whether the shift to 25 °C (non-inducing condition) from 42 °C (inducing condition) halts FNR synthesis. FNR and the control protein aconitase were collected from cell-free extracts (0·8 ml) using anti-FNR and anti-aconitase sera (1 h incubation at 20 °C), followed by trapping of the antibody–protein complexes with Protein A Sepharose (0·1 ml). Aliquots were separated on SDS-polyacrylamide gels for autoradiography (as described below) and Western blotting (as described above; the aconitase antibody was used at a 1 : 10 000 working dilution).

Determination of protein and transcript stabilities.
Cultures of E. coli MC1000, MG1655 and MG1655 clpX were grown to mid-exponential phase under aerobic and anaerobic conditions, at which point chloramphenicol was added to a final concentration of 30 µg ml–1, which is threefold greater than the MIC. Aliquots (2 ml) were removed from the cultures at various time points after the addition of the antibiotic. After normalizing the protein contents of crude cell lysates, the polypeptides were separated by SDS-PAGE (10 % polyacrylamide), and transferred to a nitrocellulose membrane for detection of FNR, as described above.

To determine the stabilities of the ndh and dmsA transcripts, cultures of E. coli MC1000 were grown to mid-exponential phase (OD600~0·4–0·6), at which point transcription was inhibited by addition of rifampicin to a final concentration of 250 µg ml–1. At various time points after exposure to rifampicin, aliquots (1 ml) of the cultures were removed, and mixed with RNAprotect (Qiagen) to inhibit RNA degradation. Total RNA was then isolated using Qiagen RNeasy Mini-prep kits, according the manufacturer's instructions. Any contaminating DNA was degraded by incubation with 10 units RNase-free DNase I (Roche) for 30 min at 37 °C. The DNase I was then inactivated by incubation at 70 °C for 5 min. Aliquots (2 µl) of the DNA-free RNA samples were mixed with 2 µl 200 mM MOPS (pH 7·0), 4 µl 37 % formaldehyde solution, 10 µl formamide, and 1 µl ethidium bromide (200 µg ml–1). The mixtures were incubated at 55 °C for 1 h, and then chilled on ice for 10 min, before visualization on 1·5 % (w/v) denaturing (formaldehyde) agarose gels.

To determine the relative abundance of the dmsA and ndh transcripts, the total RNA samples were used as the templates in RT-PCR reactions. These reactions contained equal amounts of template (~2 µg RNA) and 25 units reverse transcriptase (ABgene), 5 µl Reddymix (ABgene), 2·5 µM forward primer, and 2·5 µM reverse primer, in a total volume of 10 µl. The primers used were: DPD007 (5'-GAGTAGTCGCCGTAATGGT-3') and DPD007a (5'-ATGCGGTATTGGCTGC-3'), which yield a 600 bp product for dmsA; and DPD008 (5'-GGCCTGACCAACGAAG-3') and DPD008a (5'-CCAGCTGGTGTGCAGC-3'), which yield a 400 bp product for ndh. The RT-PCR program consisted of the following steps: 1 cycle of 30 min at 47 °C, and 2 min at 95 °C, followed by 30 cycles of 95 °C for 20 s, 58 °C for 1 min, and 72 °C for 5 min. A final step of 5 min at 72 °C was added to the end of the program. The resulting cDNAs were visualized after separation on 1·8 % (w/v) agarose gels buffered with TBE (90 mM Tris, 90 mM borate, 50 mM EDTA), and staining with ethidium bromide (200 µg ml–1). Control reactions in which Reverse Transcriptase was omitted were used to ensure that the RNA samples were DNA free. The relative abundance of the cDNAs was determined using ImageMaster software (Amersham).

Determination of transcript abundance in the presence and absence of oxygen.
To ensure that chloramphenicol (30 µg ml–1) was sufficient to inhibit protein synthesis, cultures of E. coli MC1000 were incubated at 37 °C to mid-exponential phase, and chloramphenicol was added to half of the samples. Incubation was then continued for 30 min, before 14C-labelled amino acids (0·185 MBq; Amersham) were added. All cultures were then incubated at 37 °C for a further 1 h, before collecting the bacteria by centrifugation. Crude cell lysates were separated by SDS-PAGE using a 10 % (w/v) gel. After fixing in 2-propanol : water : acetic acid (25 : 65 : 10) for 30 min, and soaking in Amplify fluorographic reagent (Amersham) for a further 30 min, the gel was dried and exposed to Hyperfilm MP (Amersham) at –70 °C.

Cultures of E. coli MC1000 were grown at 37 °C under aerobic conditions to mid-exponential growth phase. Chloramphenicol was then added to a final concentration of 30 µg ml–1, and the incubation was continued for a further 30 min. At this point, samples (1 ml) were taken for total RNA isolation, and the cultures were then made anaerobic in a Don Whitley MK3 anaerobic workstation to transfer the cultures to sealed universal vials, which were incubated at 37 °C for 1 h in anaerobic jars (Oxoid). Further samples (1 ml) were taken for RNA isolation. The remaining culture was then transferred back into sterile 250 ml conical flasks for a final 1 h incubation at 37 °C, before the last set of samples (1 ml) for RNA isolation were obtained. Each aliquot of culture (1 ml) was rapidly mixed with 2 ml RNAprotect (Qiagen) to inhibit RNA degradation, and total RNA was isolated using a Qiagen RNeasy Mini-prep kit. After treatment with DNase I (see above), the quality of the resulting RNA samples was determined by electrophoresis, as described above. The mRNA transcripts for ndh and dmsA were amplified using RT-PCR, as described above. A control transcript, tatE, which is unresponsive to oxygen availability (Jack et al., 2001), was used to standardize the reactions, and was amplified using primers DPD005 (5'-CACTCAAAATGAGAGAGCTTTTC-3') and DPD005a (5'-GGTGAGATTAGTATTACCAAACTG-3') to yield a 200 bp fragment of tatE. The resulting cDNAs were visualized after separation by electrophoresis on 1·8 % (w/v) agarose TBE gels, as described above.

The effect of the ClpXP protease on FNR-mediated regulation of dmsA and ndh transcription was investigated by isolating total RNA from anaerobic cultures of MG1655 and MG1655 clpX, and estimating the abundance of the transcripts by RT-PCR, as described above. Once again, the tatE transcript served as a control.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The starting point for this work was the suggestion that after exposure of the E. coli FNR protein to oxygen, and consequent degradation of the [4Fe–4S] clusters, the resulting monomeric apoprotein is mostly degraded rather than recycled (Patschkowski et al., 2000). Earlier studies, in which the expression of an FNR-activated gene (frd, encoding fumarate reductase), and the reactivity of the cysteine thiol groups of FNR, were determined upon shifting cultures between aerobic and anaerobic conditions in the absence of de novo FNR synthesis, suggested that apo-FNR is recycled (Engel et al., 1991). Therefore, experiments were formulated to test the hypothesis that apo-FNR is recycled in vivo.

FNR synthesized under aerobic conditions is activated upon switching cultures to anaerobic conditions
The strategy adopted was to control FNR synthesis by using the thermo-inducible FNR expression plasmid pGS199 in the fnr lac double-mutant JRG1728. A model FNR-dependent promoter FF-41.5 fused to lacZ in the low-copy-number plasmid pFF-41.5 (Wing et al., 1995) was chosen as a reporter of FNR activity, because many natural FNR-regulated promoters are also dependent on other transcription factors for activation. Thus, JRG5438 carries the lacZ reporter pFF-41.5 and the FNR expression plasmid pGS199. An equivalent strain JRG5437 carrying the vector pJLA502 in place of pGS199 was used as a control. After pre-culturing under aerobic conditions at 25 °C, FNR synthesis was then induced by incubation at 42 °C, before returning the cultures to 25 °C to halt further FNR synthesis. Finally, the cultures were either transferred to anaerobic conditions at 25 °C, or held under aerobic conditions at 25 °C. At each stage, aliquots were removed to determine whether FNR was present in the bacteria, and the consequent effect on {beta}-galactosidase activity. This showed that before FNR synthesis was induced, expression from the FNR-dependent FF-41.5 promoter was low in both the test and control cultures (Fig. 1a, bars labelled 1). After induction of FNR synthesis, expression of the reporter gene was enhanced even under aerobic conditions (Fig. 1a, bars labelled 2 and 3). This apparent aerobic FNR activity is probably caused by the increase in the intracellular concentration of FNR (approx. three- to fivefold greater than that observed for chromosomally encoded FNR, as estimated by immunoblotting) after induction. Addition of 14C-labelled amino acids to post-induction cultures, and analysis of the radiolabelled cell-free extracts, revealed that although polypeptides with molecular masses corresponding to FNR and the control protein aconitase B were detected in Western blots of immunoprecipitates obtained using anti-FNR and anti-aconitase B sera, only the higher-molecular-mass species (aconitase B) was detected in the corresponding autoradiographs. The absence of FNR on the autoradiographs suggests, but does not prove, that FNR protein is not synthesized when cultures are shifted back to the non-permissive condition (25 °C) after induction of FNR synthesis at 42 °C (not shown). Despite the apparent absence of de novo FNR synthesis, upon transfer to anaerobic conditions {beta}-galactosidase activity was enhanced by approximately fourfold compared with cultures maintained under aerobic conditions (Fig. 1a, bars labelled 4). This response is consistent with the hypothesis that a portion of the FNR protein synthesized during the aerobic 42 °C phase, which was incapable of activating transcription (apo-FNR or [2Fe–2S] FNR), is converted into a transcriptionally competent form when the culture is transferred to anaerobic conditions.



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Fig. 1. Activation of an FNR-dependent model promoter upon transfer to anaerobic conditions in the absence of de novo FNR synthesis. The expression of fnr in JRG5438 was controlled by the thermo-inducible (42 °C) promoter of pGS199, and FNR activity was reported by activation of the FNR-dependent FF-41.5 promoter fused to the reporter gene lacZ. The control strain JRG5437 contained the vector (pJLA502) in place of pGS199. (a) Cultures of JRG5437 (FNR; black bars) and JRG5438 (FNR+; open and grey bars) were grown under aerobic conditions at 25 °C for 5·5 h. Aliquots were taken for {beta}-galactosidase assays (1). FNR synthesis was then induced by incubation at 42 °C for 30 min under aerobic conditions. Further samples to monitor FNR activity were taken (2). FNR synthesis was then halted by incubation at 25 °C for 1 h (3). Finally, the cultures were either maintained under aerobic conditions (4, grey bar) ortransferred to anaerobic conditions (4, open and black bars) for 3·5 h at 25 °C, before further samples were taken for {beta}-galactosidase assays. {beta}-Galactosidase activity was measured according to Miller (1972), using triplicate assays of triplicate cultures. The mean values and standard deviations are shown. (b) Representative anti-FNR immunoblots of bacterial lysates obtained from the samples indicated above. Before immunoblotting, the lysates were separated on denaturing polyacrylamide gels, and stained with Coomassie blue to allow equalization of the amounts of protein applied to each lane. Samples in lanes 1–4 are as described above for 1–4 in (a). (c) The relative amounts of FNR present at the various stages of the experiment, as determined using the ImageMaster software package with the immunoblots shown in (b). The amount of FNR present in lane 2 of each blot in (b) was set at 100 %.

 
Determination of the relative amounts of FNR present at each stage of the experiment showed that FNR synthesis was initiated upon transfer to the higher temperature [Fig. 1b (lane 2) and c (bar 2)]. After the cultures were returned to 25 °C (to inhibit further FNR synthesis) for 1 h, the cellular content of FNR protein was lowered by ~35 % [Fig. 1b (lane 3) and c (bar 3)]. Upon transfer to anaerobic conditions for 3·5 h, the cellular content of FNR was maintained, whereas under aerobic conditions, the relative amount of FNR decreased to ~10 % of the original amount [Fig. 1b (lane 4) and c (bar 4)]. This suggests that FNR protein turnover might be greater under aerobic compared with anaerobic conditions.

Stability of the E. coli FNR protein in vivo
It has been shown that the E. coli FNR protein can be activated in vitro by reconstituting the [4Fe–4S] clusters into apo-FNR under anaerobic conditions (Green et al., 1996; Jordan et al., 1997; Khoroshilova et al., 1995). This suggests that if apo-FNR is not quickly degraded under aerobic conditions in vivo then it could be recycled. Therefore, to further investigate the stability of the FNR protein in vivo under aerobic conditions, immunoblotting with anti-FNR serum was used to probe a series of samples taken from cultures treated with chloramphenicol to inhibit protein synthesis. Inhibition of protein synthesis was confirmed by the inability to detect radiolabelled polypeptides on autoradiographs of cell-free extracts obtained from cultures to which 14C-labelled amino acids had been added 30 min after chloramphenicol treatment. Labelled polypeptides were readily detected in equivalent cultures that had not been treated with chloramphenicol (not shown). The half-life of the FNR protein under aerobic conditions was estimated to be ~45 min (Fig. 2a). It has been reported that FNR is a substrate for the ClpXP protease (E. L. Mettert and P. J. Kiley, personal communication reported in Flynn et al., 2003), and thus FNR degradation in a clpX mutant was investigated. This revealed that, under aerobic conditions, FNR half-life in the clpX mutant was increased to ~80 min, suggesting that the ClpXP system contributes to, but is not the only agent mediating, the degradation of FNR under aerobic conditions (Fig. 2a). Measurements of the rate of [Fe–S] cluster incorporation into apo-FNR in vitro indicate a reconstitution rate of 0·02–0·09 nmol FNR min–1, equivalent to 1012–1013 molecules of FNR reconstituted per minute (Achebach et al., 2004). As the intracellular concentration of FNR is ~3000–4000 molecules per E. coli cell (Sutton et al., 2004a; Unden & Duchene, 1987), it would appear that FNR is sufficiently long lived in vivo under aerobic conditions to offer the possibility that it can be cycled between active and inactive forms.



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Fig. 2. Effect of a clpX mutation on FNR degradation and activity. (a) Quantification of immunoblots of FNR in crude lysates of cultures of MG1655 ({blacksquare}) and the MG1655 clpX mutant ({blacktriangleup}) maintained under aerobic conditions in the presence of chloramphenicol. Cultures were grown to mid-exponential phase, and then chloramphenicol (30 µg ml–1) was added, and aliquots were removed for immunoblotting with anti-FNR serum at the indicated times. The signals obtained were quantified using the ImageMaster software package. (b) RT-PCR products obtained from using total RNA from anaerobic cultures of MG1655 (lane 1), and MG1655 clpX (lane 2) as the template, and dmsA, ndh and tatE transcript specific primers.

 
Similar experiments carried out under anaerobic conditions suggested that the FNR protein is more stable than under aerobic conditions. In contrast to the aerobic experiments, for both the wild-type and the clpX mutant, 20–30 % of FNR protein was still detectable after 24 h (not shown).

Measurement of the relative amounts of an FNR-activated transcript (dmsA; Lamberg & Kiley, 2000) and an FNR-repressed transcript (ndh; Green & Guest, 1994; Meng et al., 1997; Spiro et al., 1989) in anaerobic cultures of MG1655 and MG1655 clpX revealed that the dmsA transcript was ~2·5-fold more abundant, and the ndh transcript was ~1·5-fold less abundant in the clpX mutant (Fig. 2b). The abundance of the tatE transcript, which does not respond to either FNR or oxygen availability (Jack et al., 2001), was the same in both strains (Fig. 2b). These observations are consistent with the enhanced stability of FNR in the clpX mutant.

In the absence of de novo FNR synthesis, the protein can be switched between active and inactive forms in vivo in response to oxygen availability
The experiments described above supported the hypothesis that FNR can be cycled between active and inactive states. However, the lac reporter experiments, and the experiments reported by Engel et al. (1991) using the FNR-activated frdA promoter, used overproduced FNR synthesized from pGS199. Therefore, the effects of oxygen availability on chromosomally encoded FNR activity were determined by measuring the abundance of the dmsA, ndh and tatE transcripts. Firstly, the half-lives of the dmsA and ndh transcripts had to be determined. This was achieved by estimating the relative amounts of each transcript using RT-PCR at fixed time points after inhibiting transcription with rifampicin. The half-lives of both transcripts were in the range 2–5 min, which is typical of E. coli mRNAs (not shown). The effects of oxygen availability on FNR-mediated regulation of dmsA and ndh transcription were then investigated by preparing total RNA from cultures treated with chloramphenicol to inhibit protein synthesis. The RNA samples served as the templates in RT-PCR assays, and the amplified cDNA was analysed by agarose gel electrophoresis. Under aerobic conditions, the abundance of the dmsA transcript was low (Fig. 3, upper panel, lane 1). However, upon transfer of the culture to anaerobic conditions, even in the absence of de novo FNR protein synthesis, the abundance of the dmsA transcript was enhanced (Fig. 3, upper panel, lane 2). Moreover, when air was reintroduced to the cultures, the amount of the dmsA transcript decreased (Fig. 3, upper panel, lane 3). The opposite pattern was observed with the FNR-repressed transcript ndh. In this case the abundance of the ndh transcript was greatest when cultures were exposed to air (Fig. 3, middle panel, lanes 1 and 3), and lowest under anaerobic conditions (Fig. 3, middle panel, lane 2). As expected (Jack et al., 2001), the tatE transcript did not respond to the changes in oxygen availability (Fig. 3, lower panel). These data suggest that de novo FNR protein synthesis is not necessary for FNR activation, and that FNR is cycled between active and inactive states in vivo.



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Fig. 3. In vivo activation and repression of FNR-regulated genes upon transfer from aerobic to anaerobic conditions in the absence of de novo FNR synthesis. Cultures of E. coli MC1000 were grown aerobically to mid-exponential phase, and then treated with chloramphenicol (30 µg ml–1) to inhibit protein synthesis. After a further 30 min incubation, aliquots (lane 1) were removed, and the cultures were transferred to anaerobic conditions. After 1 h, further samples (lane 2) were taken, and the cultures were then transferred to aerobic conditions, and, after another 1 h incubation, the final samples were taken (lane 3). The panels show the cDNA products generated by RT-PCR of the FNR-activated dmsA transcript, the FNR-repressed ndh transcript, and the FNR- and oxygen-independent tatE transcript using total RNA prepared from the aerobic (O2) and anaerobic (N2) samples described above.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial FNR proteins are transcription factors that coordinate the transition between aerobic and anaerobic metabolic modes (Guest et al., 1996; Sawers, 1999). They use an iron–sulphur-cluster-based switch to respond to changes in oxygen availability (see reviews by Green & Paget, 2004; Kiley & Beinert, 1999, 2003). The intracellular concentration of E. coli FNR is similar under aerobic and anaerobic conditions (Sutton et al., 2004a; Unden & Duchene, 1987), and studies with an FNR variant, FNR-L28H, which possesses a more stable iron–sulphur cluster suggest that [4Fe–4S] clusters are inserted into apo-FNR under both aerobic and anaerobic conditions (Bates et al., 2000). When the [4Fe–4S] form of FNR is exposed to oxygen in vitro or in vivo, it is first converted to a [2Fe–2S] form, and then to an apo-form (Crack et al., 2004; Engel et al., 1991; Green & Paget, 2004; Kiley & Beinert, 1999, 2003; Popescu et al., 1998; Sutton et al., 2004a, b). Thus, FNR protein isolated from anaerobic cultures is in the transcriptionally active [4Fe–4S] form (Lazazzera et al., 1996), whereas FNR isolated from aerobic cultures is in the inactive apo-form (Green et al., 1991). Early studies suggested that FNR could be recycled by being interconverted between inactive and active forms (Engel et al., 1991). More recently, it has been suggested that the ClpXP protease degrades FNR under aerobic conditions (E. L. Mettert and P. J. Kiley, personal communication reported in Flynn et al., 2003), and that de novo FNR synthesis might be necessary for the incorporation of iron–sulphur clusters (Patschkowski et al., 2000). The experiments described here show that FNR is more readily degraded in vivo under aerobic conditions compared with anaerobic conditions, and that ClpXP contributes significantly to this degradation, but makes a less pronounced contribution under anaerobic conditions. Nevertheless, it would appear that the FNR protein is sufficiently long lived (half-life ~45 min, under aerobic conditions) to allow cycling between active and inactive forms in vivo in the absence of de novo protein synthesis. At this point, it is not possible to exclude interconversion of [4Fe–4S] to [2Fe–2S] forms (Popescu et al., 1998). However, the simplest explanation for the data presented here and elsewhere is that in E. coli, [4Fe–4S] clusters are continually incorporated into apo-FNR, but, in the presence of oxygen, these are disassembled forming the transcriptionally inactive [2Fe–2S] form of the protein, which in turn is converted to apo-FNR by prolonged exposure to oxygen and endogenously generated reactive oxygen species (Fig. 4; Achebach et al., 2005; Green & Paget, 2004; Kiley & Beinert, 1999, 2003; Sutton et al., 2004a, b). The apparent enhanced degradation of FNR under aerobic conditions might indicate that the FNR dimer is more stable than the monomer in vivo. It has been suggested that FNR possesses both N- and C-terminal sequences that facilitate recognition by the ClpXP protease (Flynn et al., 2003). Clearly the direct involvement of the N-terminal region of FNR in the ligation of the sensory iron–sulphur cluster might cause one of the ClpXP recognition sequences to be masked upon iron–sulphur cluster incorporation, and thereby contribute to the enhanced stability of FNR under anaerobic conditions.



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Fig. 4. A simplified scheme illustrating the cycling of FNR between active and inactive states. FNR protein is synthesized under aerobic and anaerobic conditions and [4Fe–4S] clusters ({square}) are incorporated, promoting the formation of transcriptionally active homodimers. In the presence of oxygen, these [4Fe–4S] clusters are rapidly disassembled generating inactive FNR with [2Fe–2S] clusters ({lozenge}). The [2Fe–2S] clusters are further degraded to yield apo-FNR protein, which can then reacquire [4Fe–4S] clusters, and continue around the cycle again. In addition, some [2Fe–2S] containing FNR may be converted to the [4Fe–4S] form. To maintain the intracellular concentration of FNR, fnr expression is autoregulated (Spiro & Guest, 1987b), and monomeric apoprotein is more prone to degradation (mediated in part by the ClpXP protease) than the [4Fe–4S]-containing dimer. The width of the arrow is intended to suggest the relative rate of the reaction.

 
In conclusion, the data presented here and elsewhere suggest that FNR is continually cycling between the inactive and active states. In the absence of oxygen, the cycle is interrupted at the [4Fe–4S] to [2Fe–2S] conversion, and consequently the transcriptionally active [4Fe–4S] form accumulates, and FNR-mediated control of gene expression is exerted. This scheme (Fig. 4) accounts for the isolation of apo-FNR from aerobic cultures of E. coli (Green et al., 1991; Achebach et al., 2005), the oxygen-dependent changes in the reactivities of the cysteine thiols that act as ligands for the FNR iron–sulphur cluster (Engel et al., 1991), and the ability to incorporate functional [4Fe–4S] clusters into apo-FNR in vitro (Green et al., 1996; Jordan et al., 1997; Khoroshilova et al., 1995). Thus, it seems likely that FNR continually samples its environment by cycling through inactive and active states, providing an economical and rapidly responding mechanism to monitor changes in environmental oxygen.


   ACKNOWLEDGEMENTS
 
We thank the Biotechnology and Biological Sciences Research Council (UK), and the Medical Research Council (UK), for supporting this work, and F. R. Blattner for providing the MG1655 clpX mutant.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Achebach, S., Tran, Q. H., Vlamis-Gardikas, A., Mullner, M., Holmgren, A. & Unden, G. (2004). Stimulation of Fe–S cluster insertion into apo-FNR by Escherichia coli glutaredoxins 1, 2 and 3 in vitro. FEBS Lett 565, 203–206.[CrossRef][Medline]

Achebach, S., Selmer, T. & Unden, G. (2005). Properties and significance of apoFNR as a second form of air-inactivated [4Fe–4S] FNR of Escherichia coli. FEBS J 272, 4260–4269.[Abstract/Free Full Text]

Barnard, A. M. L., Green, J. & Busby, S. J. W. (2004). Transcription regulation by tandem-bound FNR at Escherichia coli promoters. J Bacteriol 185, 5993–6004.[CrossRef]

Bates, D. M., Popescu, C. V., Khoroshilova, N., Vogt, K., Beinert, H., Munck, E. & Kiley, P. J. (2000). Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe–4S]2+ cluster to oxygen. J Biol Chem 275, 6234–6240.[Abstract/Free Full Text]

Bauer, C., Elsen, S. & Bird, T. H. (1999). Mechanisms for redox control of gene expression. Annu Rev Microbiol 53, 495–523.[CrossRef][Medline]

Becker, S., Holighaus, G., Gabrielczyk, T. & Unden, G. (1996). O2 as the regulatory signal for FNR-dependent gene expression in Escherichia coli. J Bacteriol 178, 4515–4521.[Abstract/Free Full Text]

Bell, A. & Busby, S. J. W. (1994). Location and orientation of an activating region in the Escherichia coli transcription factor, FNR. Mol Microbiol 11, 383–390.[Medline]

Blake, T., Barnard, A., Busby, S. J. W. & Green, J. (2003). Transcription activation by FNR: evidence for a functional activating region 2. J Bacteriol 184, 5855–5861.[CrossRef]

Browning, D., Lee, D., Green, J. & Busby, S. (2003). Secrets of transcription initiation taught by the Escherichia coli FNR protein. In Signals, Switches, Regulons and Cascades: Control of Bacterial Gene Expression, pp. 127–142. Edited by D. A. Hodgson & C. M. Thomas. Cambridge: Cambridge University Press.

Covert, M. W., Knight, E. M., Reed, J. L., Herrgard, M. J. & Palsson, B. O. (2004). Integrating high-throughput and computational data elucidates bacterial networks. Nature 429, 92–96.[CrossRef][Medline]

Crack, J., Green, J. & Thomson, A. J. (2004). Mechanism of oxygen sensing by the bacterial transcription factor fumarate–nitrate reduction (FNR). J Biol Chem 279, 9278–9286.[Abstract/Free Full Text]

Eiglmeier, K., Honore, N., Iuchi, S., Lin, E. C. C. & Cole, S. T. (1989). Molecular genetic analysis of FNR-dependent promoters. Mol Microbiol 33, 869–878.

Engel, P., Trageser, M. & Unden, G. (1991). Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability. Arch Microbiol 156, 463–470.[Medline]

Flynn, J. M., Neher, S. B., Kim, Y. I., Sauer, R. T. & Baker, T. A. (2003). Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol Cell 11, 671–683.[CrossRef][Medline]

Gonzalez, R., Tao, H., Purvis, J. E., York, S. W., Shanmugam, K. T. & Ingram, L. O. (2003). Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol Prog 19, 612–623.[CrossRef][Medline]

Green, J. & Guest, J. R. (1994). Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. Mol Microbiol 12, 433–444.[Medline]

Green, J. & Marshall, F. A. (1999). Identification of a surface of FNR overlapping AR1 that is required for repression of gene expression. J Biol Chem 274, 10244–10248.[Abstract/Free Full Text]

Green, J. & Paget, M. S. (2004). Bacterial redox sensors. Nat Rev Microbiol 2, 954–966.[CrossRef][Medline]

Green, J., Trageser, M., Six, S., Unden, G. & Guest, J. R. (1991). Characterization of the FNR protein of Escherichia coli, an iron binding transcriptional regulator. Proc Biol Soc 244, 137–144.

Green, J., Bennett, B., Jordan, P., Ralph, E. T., Thomson, A. J. & Guest, J. R. (1996). Reconstitution of the [4Fe–4S] cluster in FNR and demonstration of the aerobic–anaerobic transcription switch in vitro. Biochem J 316, 887–892.[Medline]

Green, J., Baldwin, M. L. & Richardson, J. (1998). Downregulation of Escherichia coli yfiD expression by FNR occupying a site at –93·5 involves the AR1-containing face of FNR. Mol Microbiol 29, 1113–1123.[CrossRef][Medline]

Green, J., Scott, C. & Guest, J. R. (2001). Functional versatility in the CRP-FNR superfamily of transcription factors: FNR and FLP. Adv Microb Physiol 44, 1–34.[Medline]

Guest, J. R., Green, J., Irvine, A. S. & Spiro, S. (1996). The FNR modulon and FNR-regulated gene expression. In Regulation of Gene Expression in Escherichia coli, pp. 317–342. Edited by E. C. C. Lin & A. S. Lynch. Austin, TX: R. G. Landes & Co.

Jack, R. L., Sargent, F., Berks, B. C., Sawers, G. & Palmer, T. (2001). Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth. J Bacteriol 183, 1801–1804.[Abstract/Free Full Text]

Jordan, P. A., Thomson, A. J., Ralph, E. T., Guest, J. R. & Green, J. (1997). FNR is a direct oxygen sensor having a biphasic response curve. FEBS Lett 416, 349–352.[CrossRef][Medline]

Kang, Y., Weber, K. D., Qiu, Y., Kiley, P. J. & Blattner, F. R. (2005). Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function. J Bacteriol 187, 1135–1160.[Abstract/Free Full Text]

Khoroshilova, N., Beinert, H. & Kiley, P. J. (1995). Association of a polynuclear iron–sulfur center with a mutant FNR protein enhances DNA-binding. Proc Natl Acad Sci U S A 92, 2499–2505.[Abstract/Free Full Text]

Khoroshilova, N., Popescu, C., Munck, E., Beinert, H. & Kiley, P. J. (1997). Iron–sulphur disassembly in the FNR protein of Escherichia coli by O2: [4Fe–4S] to [2Fe–2S] conversion with loss of biological activity. Proc Natl Acad Sci U S A 94, 6087–6092.[Abstract/Free Full Text]

Kiley, P. J. & Beinert, H. (1999). Oxygen sensing by the global regulator FNR: the role of the iron-sulfur cluster. FEMS Microbiol Rev 22, 341–352.[CrossRef]

Kiley, P. J. & Beinert, H. (2003). The role of Fe–S proteins in sensing and regulation in bacteria. Curr Opin Microbiol 6, 181–185.[CrossRef][Medline]

Lamberg, K. E. & Kiley, P. J. (2000). FNR-dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR-activating regions 1 and 3. Mol Microbiol 38, 817–827.[CrossRef][Medline]

Lamberg, K. E., Luther, C., Weber, K. D. & Kiley, P. J. (2002). Characterization of activating region 3 from Escherichia coli FNR. J Mol Biol 315, 275–283.[CrossRef][Medline]

Lazazzera, B. A., Bates, D. & Kiley, P. J. (1993). The activity of the Escherichia coli transcription factor FNR is regulated by a change in oligomeric state. Genes Dev 7, 1993–2005.[Abstract]

Lazazzera, B. A., Beinert, H., Khoroshilova, N., Kennedy, M. C. & Kiley, P. J. (1996). DNA-binding and dimerization of the Fe–S containing FNR protein Escherichia coli are regulated by oxygen. J Biol Chem 271, 2762–2768.[Abstract/Free Full Text]

Lennox, E. S. (1955). Transduction of linked genetic characters of host by bacteriophage P1. Virology 1, 190–206.[CrossRef][Medline]

Li, B., Wing, H., Lee, D., Wu, H. & Busby, S. (1998). Transcription activation by Escherichia coli FNR protein: similarities to and differences from, the CRP paradigm. Nucleic Acids Res 26, 2075–2081.[Abstract/Free Full Text]

Lonetto, M. A., Rhodius, V., Lamberg, K., Kiley, P., Busby, S. & Gross, C. (1998). Identification of a contact site for different transcription activators in region 4 of the Escherichia coli RNA polymerase {sigma}70 subunit. J Mol Biol 284, 1353–1365.[CrossRef][Medline]

Marshall, F. A., Messenger, S. L., Wyborn, N. R., Guest, J. R., Wing, H., Busby, S. J. W. & Green, J. (2001). A novel promoter architecture for microaerobic activation by the anaerobic transcription factor FNR. Mol Microbiol 39, 747–753.[CrossRef][Medline]

Meng, W., Green, J. & Guest, J. R. (1997). FNR-dependent repression of ndh gene expression requires two upstream FNR-binding sites. Microbiology 143, 1521–1532.[Medline]

Miller, J. H. (1972). Assay of {beta}-galactosidase. In Experiments in Molecular Genetics, pp. 352–355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Moore, L. J. & Kiley, P. J. (2001). Characterization of the dimerization domain in the FNR transcription factor. J Biol Chem 276, 45744–45750.[Abstract/Free Full Text]

Patschkowski, T., Bates, D. M. & Kiley, P. J. (2000). Oxidative stress. In Bacterial Stress Responses, pp. 61–78. Edited by G. Storz & R. Hengge-Aronis. Washington, DC: American Society for Microbiology.

Popescu, C. V., Bates, D. M., Beinert, H., Munck, E. & Kiley, P. J. (1998). Mössbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia coli. Proc Natl Acad Sci U S A 95, 13431–13435.[Abstract/Free Full Text]

Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W. & Gunsalus, R. P. (2003). Global gene expression profiling in Escherichia coli K12: the effects of oxygen availability and FNR. J Biol Chem 278, 29837–29855.[Abstract/Free Full Text]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sawers, G. (1999). The aerobic/anaerobic interface. Curr Opin Microbiol 2, 181–187.[CrossRef][Medline]

Schauder, B., Blocker, H., Frank, R. & McCarthy, J. E. G. (1987). Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region. Gene 52, 279–283.[CrossRef][Medline]

Schwartz, C. J., Djaman, O., Imlay, J. A. & Kiley, P. J. (2000). The cysteine desulfurase, IscS, has a major role in in vivo Fe–S cluster formation in Escherichia coli. Proc Natl Acad Sci U S A 97, 9009–9014.[Abstract/Free Full Text]

Six, S., Trageser, M., Kojro, E., Fahrenholz, F. & Unden, G. (1996). Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, an O2-responsive, Fe containing transcriptional regulator of Escherichia coli. J Inorg Biochem 62, 89–102.[CrossRef][Medline]

Spiro, S. & Guest, J. R. (1987a). Activation of the lac operon of Escherichia coli by a mutant FNR protein. Mol Microbiol 1, 53–58.[Medline]

Spiro, S. & Guest, J. R. (1987b). Regulation and over-expression of the fnr gene of Escherichia coli. J Gen Microbiol 133, 3279–3288.[Medline]

Spiro, S., Roberts, R. E. & Guest, J. R. (1989). FNR-dependent repression of the ndh gene of Escherichia coli and metal ion requirement for FNR-regulated gene expression. Mol Microbiol 3, 601–608.[Medline]

Sutton, V. R., Mettert, E. L., Beinert, H. & Kiley, P. J. (2004a). Kinetic analysis of the oxidative conversion of the [4Fe–4S]2+ cluster of FNR to a [2Fe–S]2+ cluster. J Bacteriol 186, 8018–8025.[Abstract/Free Full Text]

Sutton, V. R., Stubna, A., Patschowski, T., Munck, E., Beinert, H. & Kiley, P. J. (2004b). Superoxide destroys the [2Fe–2S]2+ cluster of FNR from Escherichia coli. Biochemistry 43, 791–798.[CrossRef][Medline]

Unden, G. & Bongaerts, J. (1997). Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation. Biochim Biophys Acta 1320, 217–234.[Medline]

Unden, G. & Duchene, A. (1987). On the role of cyclic AMP and the Fnr protein in Escherichia coli growing anaerobically. Arch Microbiol 147, 195–200.[CrossRef][Medline]

Williams, S. M., Savery, N. J., Busby, S. J. W. & Wing, H. J. (1997). Transcription activation at Class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase {alpha} subunit. Nucleic Acids Res 25, 4028–4034.[Abstract/Free Full Text]

Wing, H. J., Williams, S. M. & Busby, S. J. W. (1995). Spacing requirements for transcription activation by Escherichia coli FNR protein. J Bacteriol 177, 6704–6710.[Abstract/Free Full Text]

Wing, H. J., Green, J., Guest, J. R. & Busby, S. J. W. (2000). Role of activating region 1 of Escherichia coli FNR protein in transcription activation at Class II promoters. J Biol Chem 275, 29061–29065.[Abstract/Free Full Text]

Received 6 June 2005; revised 1 September 2005; accepted 12 September 2005.



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