Control of bacterial nitrate assimilation by stabilization of G-quadruplex DNA† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc06057a Click here for additional data file.

Ligand-specific control of nitrate assimilation in Paracoccus denitrificans by stabilization of DNA G-quadruplex in the promoter region of nas.


DNA AND LIGANDS
The nasT' oligonucleotides d-5'(GGG-AGC-GGG-ACG-GGG-GCC-GGG)-3' and nasT'FRET (5'-FAM-d[GGG-AGC-GGG-ACG-GGG-GCC-GGG]-TAMRA-3'; donor fluorophore FAM is 6-carboxyfluorescein; acceptor fluorophore TAMRA is 6carboxytetramethyl-rhodamine) were purchased from Eurogentec, HPLC purified and dissolved as a stock solution (1 mM and 100 µM respectively) in MilliQ water. Further dilutions were made into sodium cacodylate buffer (10 mM, pH 7.4) containing either KCl, NaCl, LiCl or NH4Cl, each at 100 mM final concentration, as required. Samples were thermally annealed in a heat block at 95°C for 5 mins and cooled slowly to ambient temperature overnight. TmPyP4 was purchased from Sigma Aldrich and ligand 1 was prepared as previously described. 1 The structures of the ligands used in this study are given below:

UV SPECTROSCOPY
DNA melting/annealing was performed using an Agilent Technologies Cary 60 UV-Vis absorbance spectrometer equipped with a Quantum Northwest TC1 thermal peltier controller. TDS profiles were recorded using a low volume quartz cuvette by monitoring the absorbance at 295 nm. Samples (200 μL) were prepared to final oligonucleotide concentrations of 1.25-10 μM in buffer containing 0-100 mM of KCl, NaCl, LiCl or NH4Cl. Each oligonucelotide solution containing the relevant cation was covered with a layer of silicone oil and stoppered prior to thermal cycling. Samples were held at 20°C for 5 mins then cycled to 95°C three times at a rate of 0.5°C/min, with a 5 mins hold at 20°C and 95°C. Data points were recorded every 1°C and profiles shown in Figure S1 are the average of three scans. Tm values were determined by the baseline method. 2 UV difference spectra were performed at a DNA concentration of 2.5 μM. Spectra were recorded over a wavelength range of 400-200 nm at 20°C and 80°C. The difference spectrum was calculated by subtraction of the folded spectrum (20°C) from the unfolded spectrum (80°C) and normalized so the maximum change in absorption was set to +1, as previously described. 3

CIRCULAR DICHROISM
CD spectra were recorded on a Jasco J-810 spectropolarimeter using a 1 mm path length quartz cuvette. nasT' (10 μM) was measured at 20°C, over 220-320 nm; scanning at 200 nm/min with a response time of 1 s, 0.5 nm pitch and 2 nm bandwidth. The CD spectra shown in Figure. 1B represent an average of three scans and are buffer subtracted and zero corrected at 320 nm. Ligand titrations were performed by pipetting small aliquots of 1 mM stock solutions into the sample and mixing thoroughly.

FLUORESCENCE SPECTROSCOPY
Fluorescence spectra were recorded on a Perkin-Elmer LS-55 fluorescence spectrometer. The nasT' oligonucleotide (400 μM) was dissolved in a buffer containing sodium cacodylate (10 mM, pH 7.4) and NaCl (100 mM) and annealed to form a working stock solution. The fluorescence emission spectrum of TmPyP4 at 1 μM in buffer containing 100 mM NaCl and 10 mM sodium cacodylate at pH 7.4 with excitation at 425 nm gives rise to a λem at 650 nm; emission spectra were recorded over a wavelength range of 435-800 nm at 20°C. By contrast, the fluorescence emission spectrum of 1 at 10 μM in buffer containing 100 mM NaCl and 10 mM sodium cacodylate at pH 7.4 with excitation at 470 nm gives rise to a λem at 560 nm; emission spectra were recorded over a wavelength range of 480-700 nm at 20°C.
Titrations were performed in a manner analogous to previous described, 4 a stock solution of ligand (either TmPyP4 or 1) in water was diluted into experimental buffer to which aliquots of pre-annealed nasT' oligonucleotide were added. Spectra were acquired immediately after addition and mixing; ligands were added until no further changes in fluorescence intensity was observed. Relative fluorescence (1-(F/F0)) was plotted against concentration of nasT' DNA to generate a hyperbolic binding curve ( Figure S2). This was fitted to an isotherm for two inequivalent binding sites: , where θ is the fraction of folded DNA, K1 and K2 are the association constants for ligand with the oligonucleotide, and [DNA] is the concentration of nasT' added to the reaction.

FÖRSTER RESONANCE ENERGY TRANSFER MELTING EXPERIMENTS
The ability of ligand TmPyP4 and 1 to affect the stability of nasT' DNA was assessed using a Förster resonance energy transfer (FRET) DNA melting based assay. The labelled oligonucleotide nasT'FRET was prepared as a 400 nM solution in buffer containing 10 mM sodium cacodylate (pH 7.4) with 100 mM NaCl and then thermally annealed. Strip-tubes (QIAgen) were prepared by aliquoting 10 μL of the annealed DNA, followed by 10 μL of the respective (2 x) ligand solutions. Fluorescence melting curves were determined in a QIAgen Rotor-Gene Q-series PCR machine, using a total reaction volume of 20 μL. Samples were held at 25°C for 5 minutes then ramped to 95°C, at increments of 1°C, holding the temperature at each step for 1 minute. Measurements were made with excitation at 470 nm and detection at 510 nm. Final analysis of the data was carried out using QIAgen Rotor-Gene Q-series software and Origin.

BACTERIAL GROWTH ASSAYS
Paracoccus denitrificans PD1222 was grown aerobically in a minimal-salts medium, at 30°C, as described previously. 5 The nitrogen sources NaNO3 (10 mM) or NH4Cl (10 mM) were added as filter-sterilized supplements to growth media as required. Cultures were also supplemented with the antibiotic rifampicin at 50 μg/mL for selection. Growth curves were performed in 96-well plates where each well contained 100 μL cell culture. Each condition was measured in triplicate, as were bacteria-deficient and nitrogendeficient minimal media control samples. Multi-well plates were open to the atmosphere and subjected to orbital shaking at 200 rpm over the course of the experiment to ensure aeration of cultures. Prior to loading the plate, bacteria were precultured aerobically in minimal medium (5 mL) containing the relevant nitrogen source before being transferred to sterile tubes containing fresh media with required concentrations of quadruplex ligand. Each media condition was inoculated with standardized pre-inoculum at 1% (v/v) mixed thoroughly and then used to load the relevant well of the plate.
A FLUOstar Omega multidetection microplate reader (BMG labtech GmbH, Germany) was used to record bacterial growth over a 24 h period. Attenuance (D) of cultures was monitored at 600 nm, the number of measurements recorded was 48 per growth curve and the sampling interval was 30 mins. The resulting growth curves were normalized relative to the blank media control ( Figure S4, see upper panels) and semilog plots of log10(D600) versus time were produced from which the apparent maximal specific growth rates were calculated ( Figure S5, panel C). Data analysis was performed using Origin® 2016, ver. 9.3 (OriginLab Corp. MA, USA).

CONSTRUCTION OF GENE-REPORTER FUSION STRAINS
To generate reporter plasmids, primer pairs F1nasT/R1nasT and F1nasA/R1nasA were used to amplify upstream regions of nasT and nasA, respectively by PCR using genomic DNA template isolated from P. denitrificans PD1222 (see Table S2 for primer sequences). Escherichia coli JM101 was used to facilitate molecular cloning. DNA fragments for the nasT and nasA regions were ligated as EcoRI-PstI fragments in pMP220 to form separate reporter constructs, which were then conjugated into P. denitrificans using an E. coli helper strain (containing the pRK2013 plasmid) as described previously. 5

GENE EXPRESSION ANALYSIS
Transcriptional activity of the nasT promoter region (containing the nasT' quadruplexforming sequence), and nasA (devoid of putative quadruplex forming sequence, but subject NasT control) were measured by fusing of relevant DNA regions obtained by PCR to a lacZ reporter in the pMP220 vector. 6 Reporter plasmids were then mobilized into P. denitrificans as described previously. 5 β-Galactosidase activity was determined using the method of Miller. 7 Gene expression studies by qPCR were performed essentially as described by Sullivan and co-workers. 9 Here, RNA was extracted from P. denitrificans cultures at mid-exponential phase (D600 ~0.5), by incubating 30 mL of cells with 12 mL of ice-cold 95% ethanol/ 5% phenol (v/v) solution for 30 min. Cells were pelleted at 4,000 x g for according to manufacturer's instructions. RNA integrity was analysed using an Experion Automated Electrophoresis platform (Bio-Rad) and StdSens chips (Bio-Rad). 2 µg of RNA was reverse transcribed to cDNA using SuperScript II reverse transcriptase and random primers (Invitrogen). Resulting cDNA was diluted 1:5 with sterile, nuclease-free water before use in quantitative PCR reactions. Primer pairs F2nasT/R2nasT and F1polB/R1polB were used at a final concentration of 0.4 µM (see Table S2 for sequences). Real-time transcript quantification was achieved using Sensi-FAST SYBR No-ROX kit (Bioline), a C1000 thermal cycler and a CFX96 realtime PCR detection system (Bioline), according to manufacturer's guidelines. Standard curves and primer efficiencies were determined using serially diluted P. denitrificans PD1222 genomic DNA, from a stock concentration of 100 ng/µL. Relative nasT gene expression was determined relative to polB, encoding the β-subunit of DNA polymerase, a constitutively active gene that does not contain G-quadruplex forming sequences and where gene expression was unaffected by either ligand (tested between 0 and 5 µM). Table S1. Thermal melting analysis for nasT' in buffer containing 10 mM sodium cacodylate (pH 7.4) and 100 mM of added cations. Melting temperature, Tm was determined by the baseline method. 2 Tm(VH), enthalpic (ΔHVH), entropic (ΔSVH) and Gibb's free energy (ΔGVH) values were determined using the van't Hoff method. 8 Nd indicates where the melting curve was not complete for further analysis.

Supplementary Data
Cation T m(VH) / T m (°C) ∆H VH (kJ mol -1 ) ∆S VH (kJ mol -1 K -1 ) ∆G VH at 37°C (kJ mol -1 )  ATCTCGGTATCCAGGTCGGT Figure S1. UV melting (solid lines) and annealing (dotted lines) profiles for nasT' in 10 mM sodium cacodylate buffer (pH 7.4) supplemented with either 100 mM KCl, NaCl, NH4Cl or LiCl.      Grad. = µ max (app) Figure S7. Effect of quadruplex-ligands on expression of nasT-lacZ and nasA-lacZ transcriptional fusions during NH4 + -dependent (black) and NO3 --dependent (red) growth of P. denitrificans. Dependence of TmPyP4 on nasT-lacZ and nasA-lacZ expression is shown in panels A and B, respectively. Dependence of 1 on nasT-lacZ and nasA-lacZ expression is shown in panels C and D, respectively.  Figure S8. Effect of quadruplex-ligands on nasT gene expression followed by qPCR analysis of total RNA isolated from NH4 + -dependent (black) and NO3 --dependent (red) grown P. denitrificans cell cultures. Expression data for nasT was normalized relative to that observed for polB, which is a standard housekeeping gene used in previous gene expression studies by qPCR in P. denitrificans.