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
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ABSTRACT |
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INTRODUCTION |
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The [4Fe4S] 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 promoterRNA 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 ironsulphur clusters. The [4Fe4S] clusters are first converted to [2Fe2S] 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 [2Fe2S] 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 ironsulphur 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 ironsulphur clusters, suggest that inactive apo-FNR and active ironsulphur-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 [4Fe4S] and [2Fe2S] 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 ironsulphur clusters. However, it has been suggested that rather than being available to receive an ironsulphur cluster, apo-FNR protein is mostly degraded, and that ironsulphur 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.
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METHODS |
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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 (23 ml) were taken for the measurement of -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 antibodyprotein 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 ml1, 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 (OD6000·40·6), at which point transcription was inhibited by addition of rifampicin to a final concentration of 250 µg ml1. 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 ml1). 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 ml1). 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 ml1) 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 ml1, 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.
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RESULTS |
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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
-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
-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 [2Fe2S] FNR), is converted into a transcriptionally competent form when the culture is transferred to anaerobic conditions.
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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 [4Fe4S] 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 [FeS] cluster incorporation into apo-FNR in vitro indicate a reconstitution rate of 0·020·09 nmol FNR min1, equivalent to 10121013 molecules of FNR reconstituted per minute (Achebach et al., 2004
). As the intracellular concentration of FNR is
30004000 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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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 25 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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DISCUSSION |
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ACKNOWLEDGEMENTS |
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Received 6 June 2005;
revised 1 September 2005;
accepted 12 September 2005.
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