Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-Universität Mainz, Becherweg 15, 55 099 Mainz, Germany
Correspondence
Gottfried Unden
unden{at}uni-mainz.de
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ABSTRACT |
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INTRODUCTION |
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The DcuSR two-component system of Escherichia coli consists of the sensory kinase DcuS and the regulator DcuR, and controls the expression of genes in response to extracellular C4-dicarboxylates like fumarate and succinate (Jones & Gunsalus, 1987; Zientz et al., 1998
; Golby et al., 1999
; Davies et al., 1999
; Janausch et al., 2002a
). The major target genes encode enzymes of aerobic or anaerobic C4-dicarboxylate metabolism, and include the structural gene dcuB for the fumarate/succinate antiporter DcuB, the frdABCD operon for fumarate reductase, and the dctA gene for the succinate uptake carrier DctA. In addition, expression of a number of other genes that are not related to C4-dicarboxylate metabolism is regulated by DcuSR (Oshima et al., 2002
).
To date, the sensor of the DcuSR system has mainly been studied, and the structure of the periplasmic domain containing the signal-binding domain has been determined (Janausch et al., 2002b; Pappalardo et al., 2003
). The sensor has been isolated and functionally reconstituted in proteoliposomes. After reconstitution, the sensor is functionally intact and responds to the presence of fumarate or succinate (Janausch et al., 2002b
). Reconstituted DcuS is able to transfer a phosphoryl group to the response regulator DcuR, which after phosphorylation gains the ability to bind to DNA (Janausch et al., 2002b
). However, the details of DNA binding by DcuR are not known, although they would be important for understanding the complete DcuSR system. For this reason, the phosphorylation of DcuR and the binding of DcuR to target DNA were studied. Studies on phosphoryl transfer indicated a specific interaction between DcuR and DcuS during the reaction.
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METHODS |
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DcuR-D56N was constructed by site-directed mutagenesis with the QuikChange mutagenesis kit (Stratagene), using plasmid pMW180 (Janausch et al., 2002b) as a template, and primers DcuR-D56N-Mut28-for (GAC CTG ATA TTG CTC AAT ATC TAT ATG) and DcuR-D56N-Mut28-rev (GCA TAT AGA TAT TGA GCA ATA TCA GGT C). In the resulting plasmid pMW267 (encoding DcuR-D56N), the presence of a mutation (D56N) was verified by DNA sequencing. Plasmid pMW267 was used for overproduction and isolation of DcuR-D56N, as described for DcuR.
DNA binding and gel retardation.
Prior to its use in gel retardation assays, His6-DcuR was phosphorylated by incubation with acetyl phosphate. In this reaction, 10 µg DcuR in 20 µl buffer (50 mM Tris/HCl, pH 7·0, 5 mM MgCl2, 1 mM dithiothreitol) were incubated for 60 min at 37 °C with 50 mM acetyl phosphate and then used immediately for DNA-binding studies. For estimation of the phosphorylation state, various response regulators (DcuR, ArcA, NarL) were subjected to isoelectric focussing. The procedure enabled the separation of phosphorylated and non-phosphorylated regulators (unpublished data). The regulators incubated with acetyl or carbamoylphosphate were phosphorylated generally to about 50 %, and maximally up to approximately 75 % of the total regulator protein. For experiments involving DcuR-P, it was assumed that 50 % of the DcuR protein was present as DcuR-P.
The DNA fragments for gel retardation were obtained by PCR. The intergenic region in front of dcuB was amplified by PCR with the oligonucleotide primers dcuB1EcoRI (GAT AGT GAA TTC CAT GTG) and dcuB2EcoRI (AAA CAA GAA TTC CAA TAA CG) from E. coli AN387 genomic DNA, and cloned via the EcoRI sites into pBlueskript KS (Alting-Mees & Short, 1989), resulting in plasmid pMW195 with the complete intergenic region of dcuB. The dcuB promoter fragment (585 bp) for gel retardation was amplified with the same primers from pMW195. The DNA fragment (463 bp) with the frdA promoter region was amplified by PCR with primers frdA1EcoRI (GAC GGA ATT CCG CCA TAA TCG C) and frdA2EcoRI (GCG CGG CAG GAA TTC CAG C) from pNU31 (Cole, 1982
). The DNA fragment (481 bp) with the dctA promoter region was obtained by PCR with primers dctAEcoRI (CGC TGG ATG AAT TCG GCA TGG G) and dctABamHI (CAG AGA GGG ATC CAT AGG GTC TCC) from pMW103 (Zientz et al., 1998
). The sdhC promoter region (674 bp) was prepared by PCR from a colony of E. coli AN387 with primers sdhC1EcoRI (GAA AGA GGG GAA TTC CTG GGT AC) and sdhC2EcoRI (CGC GAT GGA GAA TTC ACG CTA TC). In the sequences above, the underlined sequences represent restriction sites.
For gel retardation, the DNA fragments were amplified from the corresponding plasmids, digested with EcoRI or with EcoRI and BamHI, purified with a Qiaquick PCR Purification Kit (Qiagen) and labelled with [-33P]dATP on both strands. The labelling mixture contained 0·1 pmol DNA fragment, Klenow reaction buffer (MBI Fermentas), 0·25 mM dNTP mix (without labelled nucleotide), 8·1x105 Bq [
-33P]dATP and 5 U Klenow enzyme (exo fragment). Incubation was carried out for 25 min at 30 °C and then for 10 min at 75 °C.
Gel retardation assays were performed essentially as described previously (Drapal & Sawers, 1995; Wackwitz et al., 1999
). The phosphorylated DcuR protein was incubated with labelled DNA (5 nM) in binding buffer (50 mM Tris/HCl, pH 7·5, 5·5 %, w/v, glycerol, 0·1 mM EDTA, 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 5 µg sonicated calf thymus DNA) for 20 min at room temperature. After incubation, the reaction mix was applied to a non-denaturing polyacrylamide gel (5 %) buffered with Tris/borate/EDTA (TBE) buffer (Sambrook et al., 1989
).
DNase I footprinting.
Plasmid pMW195, carrying the dcuB promoter, was restricted with HindIII and Cfr42I. The resulting 640 bp fragment, containing the complete dcuB-dcuR intergenic region, was restricted with HinfI, yielding a 421 bp DNA fragment. The dcuB promoter fragments of 640 and 421 bp were labelled on one strand with [-33P]dATP for 30 min at 30 °C, followed by 10 min at 75 °C. The reaction mixture contained 2 pmol DNA fragment, Klenow reaction buffer, 0·25 mM dNTP mix (without labelled nucleotide), 1·63x106 Bq [
-33P]dATP and 20 U Klenow enzyme (exo fragment). DNase I footprinting was performed essentially as described previously (Drapal & Sawers, 1995
). DcuR-P (see Methods, DNA binding and gel retardation) was incubated with labelled DNA (40 nM) for 30 min at room temperature in 50 µl footprinting buffer (50 mM Tris/HCl, pH 7·0, 10 %, w/v, glycerol, 0·1 mM EDTA, 10 mM MgCl2, 50 mM KCl, 0·1 mM dithiothreitol). The footprinting reaction and electrophoresis were performed as described by Drapal & Sawers (1995)
. As a size marker, plasmid DNA including the dcuB promoter, sequenced by the chain-termination method (T7-Sequencing kit, Pharmacia), was used.
Phosphorylation state of reconstituted DcuS in proteoliposomes.
Detergent-solubilized DcuS was incorporated into liposomes by detergent removal using Bio-Beads, as described previously (Janausch et al., 2002b). An 80 µl aliquot of the proteoliposome suspension with reconstituted DcuS (27 µg DcuS, 530 µg E. coli phospholipids) was mixed with (final concentrations) 10 mM MgCl2, 1 mM dithiothreitol and 20 mM fumarate, and subjected to three cycles of rapid freezing in liquid nitrogen, with slow thawing at 20 °C. After the final thawing, the proteoliposomes were kept for 1 h at 20 °C. Then, 2·5 µl [
-33P]ATP (110 TBq mmol1) was added to a final concentration of 0·1 µM ATP, and the reaction mixture incubated for 60 min at 20 °C. To test the effect of DcuR and DcuR-D56N on the phosporylation of DcuS in proteoliposomes, DcuR and DcuR-D56N were included during phosphorylation. At the indicated times, samples (10 µl) were withdrawn, mixed with 10 µl SDS loading buffer, and subjected to SDS-PAGE (Laemmli, 1970
). After electrophoresis, the gels were exposed to a phosphorimaging screen (Fuji BAS-MP2040) for identification of radioactive bands in the phosphorimager (Fuji BAS 1500).
Other methods.
Protein concentration was measured using the method of Bradford (1976).
-Galactosidase activity was determined according to Miller (1992)
. The method of Laemmli (1970)
was used for SDS-PAGE.
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RESULTS |
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If DcuR-D56N instead of DcuR was present initially, then the level of DcuS phosphorylation was significantly higher, as expected, due to lack of dephosphorylation by DcuR-D56N. Addition of wild-type DcuR in the second phase of the experiment resulted in a decrease in DcuS phosphorylation by about 25 %. The residual phosphorylation of DcuS, therefore, was higher than in the previous experiment (Fig. 5a), when only DcuR was included.
Each of the experiments therefore indicates that DcuR-D56N is still able to interact with DcuS and to compete with DcuR for binding and interaction. Thus, inactivation of the phosphorylation site in DcuR-D56N inhibits only phosphorylation of DcuR and DNA binding, but not interaction with DcuS.
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DISCUSSION |
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The formation of two retardation complexes (complexes I and II) could have been due to the presence of two binding sites with high and low affinity. For example, the presence of more than one binding site has been shown for the response regulators ArcA-P and NarL-P at the pfl, aldA, sdhC and narG promoters (Walker & DeMoss, 1994; Drapal & Sawers, 1995
; Shen & Gunsalus, 1997
; Pellicer et al., 1999
). The sdhC promoter contains four independent ArcA-binding sites with differing affinities for ArcA-P (Shen & Gunsalus, 1997
).
The formation of two retardation complexes could also be due to binding of DcuR in different oligomeric states. Thus, the ArcA-P binding sites in front of the pfl and sdhC promoters expand with increasing ArcA-P concentrations, since ArcA-P occupies a larger segment on the DNA (Drapal & Sawers, 1995; Lynch & Lin, 1996
). The dcuB promoter, on the other hand, contains only one DcuR-binding site, and the size of the protected site remains constant with increasing DcuR concentration. Therefore, the basis of complex II formation in the gel retardation assay is not clear. It cannot be ruled out, however, that complex II is a complex of DNA with non-phosphorylated DcuR. The finding that an equivalent of complex II is formed with all DcuR-specific promoters supports this assumption. In addition, the mobilities and app. KD values for complex II and the DcuR/DNA complex are similar.
DcuR-P binds at a relatively large site of 4252 nt at the dcuB promoter (Fig. 6). The protected area is similar in size to sequences of 5094 bp protected by the response regulator ArcA-P (Tardat & Touati, 1993
; Drapal & Sawers, 1995
; Lynch & Lin, 1996
; Cotter et al., 1997
). The protected sequence is AT rich, but there is only a small palindromic sequence (AGTTAA TTAACT) close to the 3' end of the protected sequence. The protected site is centred around 376 bp upstream of the transcriptional start site of dcuB (Golby et al., 1998
). The dcuB promoter region contains, in addition, predicted cyclic AMP receptor protein (CRP), FNR and NarL binding sites (Golby et al., 1998
), which are all located more than 270 bp downstream of the DcuR site. Identification of DcuR-binding sites at other promoters will be required to derive the DcuR consensus sequence.
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After this paper had been accepted for publication, a study on a similar subject was published (Abo-Amer et al., 2004).
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ACKNOWLEDGEMENTS |
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Received 10 November 2003;
revised 19 December 2003;
accepted 23 December 2003.
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