1 Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, 33615 Bielefeld, Germany
2 Experimentelle Biophysik, Fakultät für Physik, Universität Bielefeld, 33615 Bielefeld, Germany
Correspondence
Anke Becker
Anke.Becker{at}genetik.uni-bielefeld.de
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ABSTRACT |
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B. B. and F. W. B. contributed equally to this work.
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INTRODUCTION |
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Bacterial exopolysaccharides (EPSs) are important for nodule infection. S. meliloti is able to synthesize two acidic EPSs, succinoglycan (EPS I) and galactoglucan (EPS II). Infection of Medicago sativa root nodules by S. meliloti depends on low-molecular-mass forms of EPS I or EPS II (Glazebrook & Walker, 1989; Gonzalez et al., 1996
; Wang et al., 1999
). EPS II is composed of alternating glucose and galactose residues which are decorated by acetyl and pyruvyl groups (Her et al., 1990
). The biosynthesis of EPS II is directed by the 30 kb exp gene cluster, containing 22 genes organized in four operons (Becker et al., 1997
; Rüberg et al., 1999
).
Under standard culture conditions in a complex medium, wild-type strain S. meliloti 2011 synthesizes EPS I and only traces of EPS II. The biosynthesis of EPS II is increased by phosphate-limiting conditions (Zhan et al., 1991) or a mutation in either of the regulatory genes mucR (Keller et al., 1995
; Zhan et al., 1989
) and expR (Glazebrook & Walker, 1989
; Pellock et al., 2002
), which are unlinked to the exp gene cluster. Extra copies of the regulatory gene expG located in the exp gene cluster (Astete & Leigh, 1996
; Becker et al., 1997
; Rüberg et al., 1999
) stimulate transcription of the expA, expD and expE operons (Rüberg et al., 1999
). Under phosphate-limiting conditions the enhanced transcription of these operons requires expG, implying that ExpG acts as a transcriptional activator of exp gene expression (Astete & Leigh, 1996
; Rüberg et al., 1999
).
ExpG was grouped into the MarR family of regulatory proteins (Becker et al., 1997). Like many other transcriptional regulators MarR-type regulators bind DNA through a helixturnhelix (HTH) motif (Cohen et al., 1993
; Sulavik et al., 1995
). An assortment of biological functions, e.g. the expression of resistance to multiple antibiotics, detergents and oxidative stress agents, organic solvents and pathogenic factors, is controlled by members of the MarR family (Alekshun & Levy, 1999
; Miller & Sulavik, 1996
). Most members act as repressors and only a few as activators (Egland & Harwood, 1999
; Komeda et al., 1996
; Oscarsson et al., 1996
). Recently, we showed that ExpG itself exerts positive regulation of exp gene expression by binding to the expA1, expG/expD1 and expE1 promoter regions in the exp gene cluster (Bartels et al., 2003
).
In this paper we describe three distinct DNA sequence elements of the ExpG binding sites and their contribution to the specific binding process. Association and dissociation kinetics were characterized by ensemble and single-molecule methods.
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METHODS |
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Proteins.
Expression, of recombinant ExpG(His)6 fusion protein was performed essentially as described previously (Bartels et al., 2003). Purification was carried out by Ni-NTA affinity chromatography (Qiagen). Purified fusion protein was concentrated using an Ultrafree 4 centrifugal concentrator (Millipore), resuspended in buffer (250 mM NaCl, 10 mM Tris, 1 mM DTT and 50 %, v/v, glycerol) and stored at 20 °C. The concentration of purified protein was determined by using the Bio-Rad Protein Assay (Bradford, 1976
).
DNA fragments.
DNA fragments I, II and III (see Fig. 2a) and competitor fragments for electrophoretic mobility shift assays (EMSAs) were generated by PCR as described previously (Bartels et al., 2003
). Following hybridization of the oligonucleotides (Fig. 2b
) and their respective antisense oligonucleotides (synthesized by Qiagen) as described by Bertram-Drogatz et al. (1998)
, the double-stranded hybridization products were inserted into pUC18 (Yanisch-Perron et al., 1985
). The resulting plasmids were used as templates for amplification of fragments KF-A1c, d, e, f, g and h, KF-Ge and KF-E1e by PCR (for fragment lengths, see Fig. 2b
) employing primers M13uni (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and M13rev (5'-AGCGGATAACAATTTCACACAGGA-3'). These plasmids were also used for amplification of DNA fragments KF-A1e, f, g and h for atomic force microscopy (AFM) force spectroscopy employing primers M13uni (see above) and 5'SH-labelled primer M13rev (see above). DNA fragments for AFM imaging were amplified by PCR with primer ExpG1 (5'-AAAACTCGAGAGTCGTGTCTTACCGGGTTG-3') and M13uni (see above) from pFPG3/41 (pUC18 carrying the 348 bp EcoRIHindIII fragment that comprises the intergenic region between expD1 and expG).
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HPLC gel-permeation chromatography of ExpG(His)6.
Size distribution of ExpG(His)6 was measured by gel-permeation chromatography on a TSK Gel G2000SW column (TosoHaas) with a flow rate of 0·5 ml min1 (eluent: 50 mM sodium phosphate buffer, pH 7·0). Calibration was performed with an LMW Gel Filtration Calibration Kit (Amersham Biosciences). Protein absorbance was measured at 280 nm.
AFM imaging.
DNA fragments for AFM imaging consisted of 158 bp of the expG promoter region and a non-binding sequence of 990 bp. DNA (0·2 ng µl1) and ExpG protein (0·2 ng µl1) were mixed in buffer solution (50 mM Tris, 25 mM NaCl, 4 mM NiCl2, pH 8·3) and left to incubate for 5 min before being brought to a freshly cleaved mica surface (Provac AG, Balzers, Liechtenstein) which was then immediately installed under the AFM liquid cell. The concentration of Ni2+ counter-ions was optimized to yield a flexible immobilization of the DNA to the mica surface by electrostatic attraction (Hansma & Laney, 1996). ProteinDNA complexes were investigated at 25 °C with a commercial AFM (Multimode, Veeco Instruments) in tapping mode, using oxide-sharpened Si3N4 cantilevers (Veeco Instruments) at a resonance frequency of about 28 kHz. Images were taken at a scan rate of 2 Hz, while the setpoint was kept at 0·2 V. Amplitude and phase were recorded simultaneously (from the signal in trace/retrace direction) to distinguish between DNA and protein (Lysetska et al., 2002
).
Sample surface and AFM tip modification.
For force spectroscopy measurements, sample surfaces and AFM tips were functionalized as described previously (Bartels et al., 2003). Briefly, Si3N4 cantilevers (Microlever; Thermomicroscopes, Sunnyvale, CA, USA) were first activated by dipping for 10 s in concentrated nitric acid and silanized in a solution of 2 % aminopropyltriethoxysilane (Sigma) in dry toluene for 2 h. After washing with toluene, the cantilevers were incubated with 1 mM N-hydroxysuccinimide-poly(ethylene glycol)-maleimide (Shearwater Polymers) in 0·1 M potassium phosphate buffer, pH 8·0, for 30 min at room temperature. After washing with phosphate buffer, the cantilevers were incubated overnight at 4 °C with 10 ng µl1 of the respective DNA target sequence (see above) bearing a thiol label in binding buffer solution (50 mM Tris/HCl, 100 mM NaCl, 0·1 mM NiCl2, pH 8·3). The cantilevers were washed with binding buffer and used for force spectroscopy experiments. Modified tips were usable for at least a week if stored at 4 °C.
Mica surfaces (Provac) were silanized with aminopropyltriethoxysilane in an exsiccator (Lyubchenko et al., 1993) and incubated with 4 µM ExpG(His)6 protein and 20 µM bis(sulfosuccinimidyl)suberate sodium salt (Sigma) in 0·1 M potassium phosphate buffer, pH 7·5, for 1 h at 4 °C. The sample was washed with binding buffer afterwards. Modified surfaces were stable for at least 2 days if stored at 4 °C.
Dynamic force spectroscopy.
Force spectroscopy measurements were performed with a commercial AFM head (Multimode; Veeco Instruments) at 25 °C. Acquisition of the cantilever deflection force signal and the vertical movement of the piezoelectric elements was controlled by a 16 bit AD/DA card (PCI-6052E; National Instruments) and a high-voltage amplifier (600H; NanoTechTools) via a home-built software based on Labview (National Instruments). The deflection signal was low-pass filtered (<6 kHz) and box-averaged by a factor of 10, giving a typical experimental dataset of 2000 points per forcedistance curve.
The spring constants of all AFM cantilevers were calibrated by the thermal fluctuation method (Hutter & Bechhoefer, 1993) with an absolute uncertainty of approximately 15 %. Spring constants of the cantilevers used ranged from 12 pN nm1 to 15 pN nm1.
For loading-rate-dependent measurements, the retract velocity of the piezo was varied while keeping the approach velocity constant. The measured forcedistance curves were analysed with a Matlab program (MathWorks) and corrected to display the actual molecular distances calculated from the z piezo extension. To obtain the loading rate, the retract velocity was then multiplied by the elasticity of the molecular system, which was determined from the slope of the corrected forcedistance curves on the last 20 data points before the unbinding events.
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RESULTS AND DISCUSSION |
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The topography of the binding site was investigated by AFM in buffer solution. The 158 bp promoter region of expG (Bartels et al., 2003) was extended by a 990 bp non-binding sequence at one end, resulting in a 390 nm DNA fragment suitable for AFM imaging. In accordance with this experimental setup, bound proteins were observed at only one end of a given DNA fragment, confirming the binding to the promoter region. Furthermore, AFM revealed a change in DNA conformation during the process of unbinding (Fig. 1
), with a different curvature of the promoter region. Such a structural change was a recurring motif and has been observed for at least eight different DNA fragmentprotein complexes. Proteins binding at other DNA sites or without structural transition have not been observed. Although the unbound and bound state was not directly observed in reverse order, it can be assumed that the DNA acquires its characteristic bend during the formation of the proteinDNA complex.
A conserved 21 bp region with a palindromic sequence which may constitute the binding site of ExpG was recently found in the promoter regions of expA1, expG, expD1 and expE1 (Bartels et al., 2003; Lloret et al., 2002
). In addition to this conserved sequence two further regions in the exp promoter fragments, box 1 and box 2, share similarities (Fig. 2b
). Eight different competitor fragments (Fig. 2b
) were designed to test the importance of these two boxes and the palindromic sequence for binding of ExpG(His)6. The double-stranded hybridization products from 28 bp to 80 bp (see Methods and Fig. 2b
) were not effective competitors in EMSA experiments. To exclude the possibility that the competitor fragments were too short for protein binding, although they may carry the specific binding-site sequence, these fragments were cloned into the pUC18 vector. Flanking sequences derived from the pUC18 vector added 102 bp to the specific sequences from the exp promoter regions so that the fragments measured from 130 bp to 182 bp (Fig. 2b
).
Competitor fragments KF-A1e, g and h, KF-Ge and KF-E1e contained the three motifs of the expA, expG/expD and expE promoter regions, respectively. Fragments KF-A1e (Fig. 3a), KF-Ge (Fig. 3b
) and KF-E1e (Fig. 3c
) with the wild-type sequence were effective competitors for the binding of ExpG(His)6 to DNA fragments I, II and III containing the expA, expG/expD and expE promoter regions, respectively. KF-A1d, containing only the conserved palindrome region (Fig. 2b
) did not compete out binding of ExpG(His)6 (Fig. 3a
). This was also the case for fragment KF-A1c, which included box 1, the palindrome region and 3 bp of box 2 (Figs 2b and 3a
), suggesting that at least the palindrome region and box 2 are required for binding of ExpG.
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ExpG, which contains a HTH-MarR motif at the C-terminus (residues 70-164), is a member of the MarR family, which belongs to a supergroup of eight regulator families sharing a conserved extended sequence including the classical HTH motif (Perez-Rueda & Collado-Vides, 2001). The HTH motif is one of the most common DNA-binding motifs in proteins that control transcription initiation (Sauer et al., 1982
). In repressor proteins the HTH binding motif is predominantly situated at the N-terminus, whereas activators mainly contain this motif at the C-terminus (Perez-Rueda & Collado-Vides, 2001
). This observation is in agreement with the C-terminal position of this motif in the transcriptional activator ExpG.
Binding kinetics of the ExpGDNA complexes
With this competition assay available, we aimed to determine the on- and off-rates of the ExpG(His)6DNA complexes of the expD/expG, expE1 and expA1 promoter fragments (see Methods). To determine the protein concentration for analysis of the binding kinetics we carried out EMSAs with increasing protein concentrations and DNA fragment II (Fig. 6), DNA fragment I and DNA fragment III (data not shown). Only at a protein concentration of 0·28 µg µl1 (1·2x105 M) was the electrophoretic mobility of the proteinDNA complex more strongly reduced in comparison to the lower protein concentrations. This may indicate the formation of a protein tetramerDNA complex compared to a protein dimerDNA complex that is probably formed at lower protein concentrations (Fig. 6
). The protein concentration used in the EMSAs to investigate the binding kinetics of ExpG and the different exp promoter fragments was in the range 6·5x104 µg µl1 to 0·013 µg µl1 (2·8x108 M to 5·6x107 M).
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ACKNOWLEDGEMENTS |
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Received 29 June 2004;
revised 5 October 2004;
accepted 7 October 2004.
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