Visualization of NO₃⁻/NO₂⁻ Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System^,

Abstract

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO₃⁻/NO₂⁻ exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO₃⁻/NO₂⁻ for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology), by employing NO₃⁻/NO₂⁻-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO₃⁻ and NO₂⁻, and its FRET signal is changed in response to NO₃⁻/NO₂⁻ in the micromolar range. Furthermore, both an increase and decrease in cellular NO₃⁻ concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO₃⁻/NO₂⁻ dynamics in various organisms, including plants and animals.

Introduction

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are metabolites in the biological nitrogen cycle. In bacteria and plants, NO₃⁻ is used as a substrate for respiration and/or assimilation. In animals, including humans, NO₃⁻ and NO₂⁻ (NO₃⁻/NO₂⁻) were recognized for a long time as being merely inert oxidants of nitric oxide (NO) (1). NO is a key signaling molecule that regulates a vast range of physiological functions, such as vascular homeostasis, neurotransmission, and host defense (2). Intriguingly, many studies over the last decade revealed that these inert NO₃⁻/NO₂⁻ species are physiologically recycled to form NO and other reactive nitrogen species through the “nitrate–nitrite–nitric oxide (NO₃⁻–NO₂⁻–NO) pathway” (3–5). Currently, NO₃⁻/NO₂⁻ are considered as stable reservoirs for NO-like bioactivity, and several beneficial aspects of NO₃⁻/NO₂⁻ in the treatment and prevention of cardiovascular diseases by restoring NO homeostasis are reported (5, 6).

As the regulation of NO₃⁻/NO₂⁻ in physiological processes is an attractive therapeutic target, it is important to understand NO₃⁻/NO₂⁻ in biological processes, how intracellular levels are regulated, and how they control cellular processes. The most frequently used method for NO₃⁻/NO₂⁻ measurement is based on the Griess reaction (7). The Griess method is also used to assess NO synthesis because of the immediate conversion of NO into NO₃⁻/NO₂⁻ (half-life 2–6 s) (8). Although measurement of NO₃⁻/NO₂⁻ by the Griess assay is simple and convenient, it is difficult to apply this method for in situ measurement in living cells because this method is generally used as the end point assay that involves several chemical reactions. As for mammalian cells, although the NO₃⁻ influx into HeLa-derived cells at low pH conditions was observed by the patch clamp method (9), detections of the dynamics of NO₃⁻/NO₂⁻ in physiological processes are quite difficult by presently available methods.

In some microorganisms, nasST genes are clustered together with other genes involved in NO₃⁻ assimilation (10–13). NasS and NasT are annotated as a NO₃⁻/NO₂⁻-responsive two-component system, where NasS is a NO₃⁻/NO₂⁻ sensor, and NasT is a transcription antiterminator. We have previously demonstrated that the NasS and NasT from the root nodule bacterium Bradyhizobium japonicum form a stable complex (NasST) in the absence of NO₃⁻/NO₂⁻, and the formation of the NasS with NO₃⁻ or NO₂⁻ complex triggers release of the positive RNA-binding regulator NasT (13), which enhances the translation of proteins involved in NO₃⁻ assimilation (Fig. 1A) (11). Herein, we report genetically encoded FRET-based NO₃⁻/NO₂⁻ biosensors that employ NasS and NasT (Fig. 1B). Using this system, we succeeded in monitoring the dynamics of NO₃⁻/NO₂⁻ inside single living cells specifically.

FIGURE 1.

FRET-based NO₃⁻/NO₂⁻ probes, sNOOOpy. A, proposed model of a two-component regulatory system composed of NasS-NasT. NasS plays a negative regulatory role by interacting with NasT. In the presence of NO₃⁻ or NO₂⁻, the putative RNA-binding protein NasT is released from NasS and acts as a transcription anti-terminator that binds the leader sequence in mRNA, preventing hairpin formation and allowing complete transcription of the genes. B, schematic drawing of the sNOOOpy system. CFP and YFP (Venus) are connected with NasT and NasS, respectively. In the NO₃⁻/NO₂⁻-free form (left), the formation of a stable dimer between NasT and NasS draws the two fluorescent proteins close to each other, resulting in high FRET efficiency. In the NO₃⁻- or NO₂⁻-bound form, dissociation of the two proteins separates the two fluorescent proteins, which decreases FRET efficiency. F. I., fluorescence intensity. C, schematic diagram of sNOOOpy proteins, CFP-NasT and NasS-YFP (Venus_cp195). D, FRET/CFP ratio changes in NasS fused with different Venus variants. Fluorescent emissions of NasS fused with Venus variants (1 μm) were measured in the presence of CFP (1 μm) (open square), CFP-NasT (1 μm) (closed square), or CFP-NasT with 2 μm of NO₃⁻ (gray square). The labels 50, 157, 173, 195, and 229 indicate circularly permuted Venus having the 50th, 157th, 173rd, 195th, and 229th amino acid as its N terminus, respectively. E and F, fluorescence emissions of sNOOOpy. Fluorescence was measured by excitation with 410 nm (left and middle) or 475 nm (right) light at various concentrations of NO₃⁻ (E) and NO₂⁻ (F) at 25 °C using protein pairs of 1 μm each of CFP-NasT + NasS-YFP (left), or CFP-NasT + His-tagged NasS (middle), or GST-tagged NasT + NasS-YFP (middle and right) in 100 mm HEPES-NaOH, pH 8.0, and 10 mm KCl. Emissions of sNOOOpy-ΔNasT, which is composed of CFP and NasT-YFP, are shown as broken lines.

Experimental Procedures

Gene Construction

The polymerase chain reaction (PCR) was performed with KOD-Plus Neo polymerase (Toyobo, Japan), and all of the oligonucleotide primers used in this study are listed in Table 1. DNA fragment assembly was performed using the In-Fusion HD cloning kit (Takara Bio, Japan). The NasS and NasT genes were amplified by PCR from a pUC-based clone library of B. japonicum (14). The cDNA of seCFP and YFP (Venus) variants with circular permutation (15) and the pCold I vector (Takara Bio) were amplified by PCR. The amplified genes were assembled to obtain pCold_CFP, pCold_CFP-NasT, and pCold_NasS-YFP for expression in Escherichia coli. CFP⁵ and CFP-NasT were expressed as N-terminal His₆-tagged constructs, whereas NasS-YFP had a His₆-tag added at its C terminus. PCR-based mutagenesis and QuikChange (Stratagene) were used to construct mutants of seCFP (A206K) and NasS (H145A), respectively. The genes CFP-NasT and NasS-Venus(cp195) were cloned into a pFLAG-CMV-1 vector (Sigma) to obtain pCMV_sNOOOpy, which was used for mammalian expression. In pCMV_sNOOOpy, the FLAG sequence was replaced by the nuclear export signal sequence of HIV Rev (LPPLERLTL), and the genes CFP-NasT and NasS-Venus_cp195 were arranged in tandem by self-processing 2A peptides.

TABLE 1.

Oligonucleotide primers used in this study

The letters in boldface represent the overlap sequence in the In-Fusion reaction.

Amplified gene/vector	Forward	Reverse
pCold_CFP, pCold_CFP-NasT
pCold-vector	5′-tag gta atc tct gct taa aag-3′	5′-cat atg cct acc ttc gat atg-3′
seCFP (pCold_CFP)	5′-cga agg tag gca tat ggt gag caa ggg cg-3′	5′-gca gag att acc tat ctg tac agc tcg tcc atg c-3′
seCFP (pCold_CFP-NasT)	5′-cga agg tag gca tat ggt gag caa ggg cg-3′	5′-cag gta cag ctc gtc cat gcc-3′
NasT	5′-cat gga cga gct gta cat gag cgc cga gca g-3′	5′-ctt tta agc aga gat tac cta ttt cag cat ctc cga c-3′
pCold_NasS-Venus
pCold-vector	5′-cac cac cac cac cac cac tag gta atc-3′	5′-ggt gta tta cct c
NasS	5′-gag gta ata cac cat gac cgg acc gct tc-3′	5′-ggc ctt cca gcg acc-3′
Venus	5′-ggt cgc tgg aag gcc atg gtg agc aag ggc-3′	5′-gat tac cta gtg gtg gtg gtg gtg gtg gcc acc gct gcc acc-3′
Venus_cp50	5′-ggt cgc tgg aag gcc acc ggc aag ctg ccc-3′	5′-gat tac cta gtg gtg gtg gtg gtg gtg ggt gca gat cag ctt c-3′
Venus_cp157	5′-ggt cgc tgg aag gcc cag aag aac ggc atc-3′	5′-gat tac cta gtg gtg gtg gtg gtg gtg ctt gtc ggc ggt gat ata g-3′
Venus_cp173	5′-ggt cgc tgg aag gcc atg gac ggc ggc gtg-3′	5′-ctt tta agc aga gat tac cta gtg gtg gtg gtg gtg gtg ctc gat gtt gtg gcg-3′
Venus_cp195	5′-ggt cgc tgg aag gcc ctg ccc gac aac cac-3′	5′-gat tac cta gtg gtg gtg gtg gtg gtg cag cac ggg gcc gtc-3′
Venus_cp229	5′-ggt cgc tgg aag gcc atc act ctc ggc atg-3′	5′-gat tac cta gtg gtg gtg gtg gtg gtg ccc ggc ggc ggt cac-3′
Mutant
NasS(H145A)	5′-ctt tcc gtt ctc gac cgc caa tta cca att gcg g-3′	5′-ccg caa ttg gta att ggc ggt cga gaa cgg aaa g-3′
pCMV_2Apeptide and pCMV_sNOOOpya
pFLAG-CMV1b	5′-cag gct gga gac gtg gag gag aac cct gga cct gca tcc ctg tga ccc ctc ccc agt gcc tct c-3′	5′-ctt cag cag gct gaa gtt agt agc tcc gct tcc ggt aga tca att ctg acg gtt cac taa acg-3′
pCMV_2Apeptide	5′-gtc gga gat gct gaa agg aag cgg agc tac taa c-3′	5′-ggt aga tca att ctg-3′
CFP-NasTc	5′-cag aat tga tct acc atg ctg ccc ccc ctg gaa aga ctg acc ctg agc agc ggc atg gtg agc aag ggc-3′	5′-ttt cag cat ctc cga c-3′
pCMV_CFP-NasT	5′-gca tcc ctg tga ccc-3′	5′-gaa gcg gtc cgg tca tag gtc cag ggt tct c-3′
NasS-Venus_cp195	5′-atg acc gga ccg ctt c-3′	5′-ggg tca cag gga tgc cag cac ggg gcc gtc-3′

^a To construct pCMV_sNOOOpy, a pCMV_2A peptide was primarily constructed. The genes in the order of CFP-NasT and NasS-Venus(cp195) were cloned into the pCMV_2A peptide.

^b The underlined italic letters represent the sequence coding the 2A peptide.

^c The underlined letters represent the sequence coding the nuclear export signal sequence.

Purification of Proteins

The proteins CFP, CFP-NasT, NasS-YFP, GST-tagged NasT, and His-tagged NasS were expressed and purified from E. coli following the same procedures as described previously (13). Appropriate fractions were dialyzed against 10 mm HEPES, pH 8.0. The homogeneity of purified proteins was established by SDS-PAGE analysis. The protein concentrations were determined using A₄₃₅ and a molar extinction coefficient of 32,500 m⁻¹ cm⁻¹ for CFP and CFP-NasT (16) and using A₅₁₅ and a molar extinction coefficient of 84,000 m⁻¹ cm⁻¹ for NasS-YFP (17). The protein concentrations of GST-tagged NasT and His-tagged NasS were determined by the BCA protein assay kit (Pierce) using bovine serum albumin as a standard.

Characterization of the sNOOOpy System

The fluorescence of the sNOOOpy system was investigated in 100 mm HEPES, pH 8.0, and 10 mm KCl using an FP-8200 spectrofluorometer (Jasco) at 25 °C. To obtain the fluorescence spectra, CFP was excited with 410 ± 10 nm light, and emission from 450 to 600 nm was scanned. The NasS-NasT binding assay was performed by using multiwell plates on a TECAN Spark 10M (excitation filter, 405 ± 10 nm; emission filter, 535 ± 10 nm). Emission with various concentrations of NasS-YFP or CFP-NasT was measured as described for the SUMO1 and Ubc9 interaction (18), with some modifications. The FRET emission was fitted using KaleidaGraph (Synergy software) with a single-site binding model.

Titration analyses were performed by FRET/CFP ratios against [NO₃⁻] or [NO₂⁻] (square brackets denote concentration of proteins/ligands). Plots were fitted with Equation 1,

where n is the Hill coefficient, K_D is a [NO₃⁻] or [NO₂⁻] dissociating half of NasST; R = FRET/CFP ratio; R₀ = initial FRET/CFP ratio in the anion-free condition, and R_ΔNasT = FRET/CFP ratio of NasS-YFP with CFP, respectively.

Cell Culture and Microscopy

HeLa cells were obtained from Dr. Takeharu Nagai (Osaka University, Japan) and were cultured in DMEM (Nacalai, Japan) containing 5.5 mm glucose supplemented with 10% fetal bovine serum (Sigma). Cells were transfected with pCMV-sNOOOpy using Lipofectamine 2000 (Life Technologies, Inc.). Then, cells were transferred to a glass-bottom dish (0.17-mm thickness, MatTek) coated with type I-C collagen (Nitta Gelatin, Japan). Cells cultured in phenol red-free DMEM were subject to imaging experiments 40–72 h after transfection.

The cells were maintained on a Ti-E inverted microscope (Nikon Corp., Japan) at 37 °C in a humidified atmosphere containing 5% CO₂ using a stage-top incubator (Tokai Hit, Japan) and were visualized through a Plan Apo 40×, 0.95 numerical aperture, dry objective lens (Nikon). The filters used for dual-emission ratio imaging of sNOOOpy were purchased from Semrock (Rochester, NY) and included an FF01-438/24 excitation filter, an FF458-Di02 dichroic mirror, and two emission filters (an FF01-483/32 for CFP and an FF01-542/27 for YFP). Cells were illuminated using a xenon lamp through 25 and 12.5% neutral density filters. Fluorescence emissions from sNOOOpy were imaged using a scientific CMOS camera (Zyla 4.2, Andor Technologies). CFP and YFP images were obtained by alternating the emission filters with a filter exchanger. The exposure times were 200 ms for CFP and YFP images. The microscope system was controlled by NIS-Elements software (Nikon). Image analysis was performed using MetaMorph software (Molecular Devices). The YFP/CFP emission ratios were calculated by dividing YFP intensity by CFP intensity within a region of interest in a cell.

Results

Construction of the NO₃⁻/NO₂⁻ Biosensor, sNOOOpy

We invented an intermolecular FRET-based NO₃⁻/NO₂⁻ biosensor composed of two proteins, seCFP linked with the N terminus of NasT (19) and YFP (Venus) linked with the C terminus of NasS (Fig. 1C) (20, 21). As a preliminary step in the development, we attempted to find the best combination of the two classes of fluorescent proteins fused with NasT and NasS to improve the dynamic range and signal intensity. Among the combinations summarized in Fig. 1D, the FRET signal of the protein pair composed of CFP-NasT and NasS-Venus_cp195 (a circularly permutated Venus having the 195th amino acid as its N terminus (15)) showed the highest increase in the formation of the NasS-NasT complex (NasST) and the largest change from the addition of NO₃⁻. Therefore, this protein pair was subjected to characterization and further development.

When 1 μm CFP-NasT was mixed with 1 μm NasS-YFP, the FRET/CFP emission ratio increased to 1.8 as assessed by the emission ratio of 527/475 nm, a 3.6-fold increase from the CFP and NasS-YFP protein pairs (Fig. 1, E and F, left panels). Additions of NO₃⁻ and NO₂⁻ abrogated the increase in the emission in a concentration-dependent manner, although these anions showed no effects on emissions of fluorescence proteins (Fig. 1, E and F). The change in FRET signal exhibited a high selectivity for NO₃⁻ and NO₂⁻. Among nine oxoanions summarized in Fig. 2, only NO₃⁻ and NO₂⁻ reduced the FRET ratio. Furthermore, such reductions induced by NO₃⁻ were not interrupted by the presence of the other oxoanions. Thus, the indicator system can specifically detect the change in NO₃⁻ and NO₂⁻ levels as the FRET signal changes, which is caused by association and dissociation of NasS-YFP and CFP-NasT as designed in Fig. 1B. We termed the generated indicator system composed of CFP-NasT and NasS-YFP as sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology). sNOOOpy-cp195, composed of CFP-NasT and NasS-Venus_cp195, was adopted as the wild-type sNOOOpy (sNOOOpy^WT), and the sNOOOpy variants constructed in this study are summarized in Table 2.

FIGURE 2.

Specificities of the sNOOOpy system. FRET/CFP ratio of sNOOOpy in the presence of 0 (gray), 100 μm (green), 10 mm (red), and 100 μm NO₃⁻ with 10 mm of each anion (yellow) is shown. Solid and broken line indicates FRET/CFP ratio of sNOOOpy with 100 μm NO₃⁻ and sNOOOpy-ΔNasT, which is composed of CFP and NasT-YFP, respectively.

TABLE 2.

sNOOOpy variants constructed in this study

Name	Component proteins	Characteristics in vitro
sNOOOpy-ΔNasT	CFP, NasS-Venus_cp195	Negative control
sNOOOpyWT	CFP-NasT, NasS-Venus_cp195	Wild-type sNOOOpy that subjected to characterizations
sNOOOpyH145A	CFP-NasT, NasS(H145A)-Venus_cp195	Form stable dimer that is not dissociated by NO3−/NO2−

In Vitro Characterizations of NasS-NasT Interaction by sNOOOpy

First, we characterized the protein interaction between CFP-NasT and NasS-YFP. Titration of 1 μm CFP-NasT with NasS-YFP showed that apparent dissociation constant (K_ST) between CFP-NasT and NasS-YFP is estimated to be 0.13 μm (Fig. 3, A and B). The FRET ratio of sNOOOpy was decreased by titrate in increasing unlabeled [NasS], indicating that the interaction between NasS and NasT is reversible (Fig. 3C).

FIGURE 3.

Characterizations of NasS-NasT interaction by sNOOOpy system in vitro. A, fluorescence emissions at 535 nm from the NasS-NasT binding assay using multiwell plates on a TECAN Spark 10M (excitation filter, 405 ± 10 nm; emission filter, 535 ± 10 nm). Emission of 1 μm CFP-NasT in the absence of NasS-YFP is shown as a solid line and is labeled (1). Emissions of various [NasS-YFP] in the absence (cyan, labeled (2)) or presence of 1 μΜ CFP-NasT (red, labeled (3)) were plotted. Sums of emissions (labeled (1) and (2)) are shown in blue. B, emissions derived from FRET. FRET emissions were estimated from plots in A by FRET emission = (3) −{(1) + (2)} in A) as in Ref. 18. The plots were fitted with a single binding model (see Equation 2). C, titration analyses of sNOOOpy (1 μm each of CFP-NasT + NasS-YFP) with unlabeled NasS. D, competitive reaction model of sNOOOpy adopted for this study. NasS-YFP is involved in two binding equilibria at steady state as follows: the complex formation with CFP-NasT (Equilibrium 1) or NO₃⁻ or NO₂⁻ (Equilibrium 2). The constants K_ST and K_S-NO3 are dissociation constants from Equations 1 and 2, respectively. E, fluorescence emissions of various [CFP-NasT] in the absence (cyan) or presence of 1 μm NasS-YFP at various [NO₃⁻]. Emissions at 535 nm were measured using multiwell plates as in A. F, emissions derived from FRET were estimated from plots of E as in B. G, relative FRET emissions at various [NO₃⁻] relative to those in the absence of [NO₃⁻] were plotted against [CFP-NasT].

Next, we focused on the NO₃⁻/NO₂⁻-sensing mechanism of NasST at the molecular level. In rhizobial cell function, NO₃⁻/NO₂⁻ induce dissociation of NasST by binding to NasS. Therefore, we inferred that NO₃⁻/NO₂⁻ can be regarded as a competitive inhibitor that competes with NasT for binding to NasS (Fig. 3D). Fig. 3, E and F, shows titration of 1 μm NasS-YFP with CFP-NasT in the presence of various [NO₃⁻]. Although curve-fitting analyses based on the single-site binding model failed because of the progressive decrease of FRET emission at high [CFP-NasT], the inhibitory effects of NO₃⁻ at each [CFP-NasT] were comparable (Fig. 3G). NO₃⁻ decreased FRET emission at low [CFP-NasT], whereas those at high [CFP-NasT] were recovered to the levels of NO₃⁻-free conditions. These results supported that NO₃⁻/NO₂⁻ inhibit NasS-NasT formation competitively.

Sensitivity of the sNOOOpy System in Vitro

Prior to further characterizations, we have prepared sNOOOpy^H145A, which is composed of a NasS(H145A) mutant that was inferred to form a NO₃⁻-binding site based on the NO₃⁻-binding site structure of the NO₃⁻-binding protein NrtA (Fig. 4A) (22). Next, we characterized the sensitivity of the sNOOOpy system. In this study, we used K_D values, the values at which the [NO₃⁻] or [NO₂⁻] dissociates half of the NasST, as the sensitivity determinant of the sNOOOpy system. Because the K_D value corresponds to the half-maximal inhibition concentration (IC₅₀) inhibiting NasST formation in the competitive model as shown in the Fig. 3D, the K_D values were determined by curve-fitting analyses using the Hill equation. Fig. 4B shows the FRET/CFP ratio from titration with NO₃⁻ and NO₂⁻. When 1 μm each of CFP-NasT and NasS-YFP was used, a micromolar [NO₃⁻] decreased the FRET ratio, which is in good agreement with the affinity reported for NasST from Paracoccus denitrificans (K_D ≈ 15 μm) (11). Under our in vitro assay conditions, the K_D values for NO₃⁻ and NO₂⁻ were estimated to be 39.5 and 256 μm, respectively. The FRET signal of sNOOOpy^H145A was not reduced upon the addition of <10 mm NO₃⁻/NO₂⁻, indicating that NasS(H145A)-YFP and CFP-NasT form an extremely stable complex and lost NO₃⁻/NO₂⁻ responsiveness.

FIGURE 4.

Sensitivities of sNOOOpy in vitro. A, NO₃⁻-binding site structure of NrtA. Bound NO₃⁻ is shown as a sphere model. Side chain of the His-196 that forms direct interaction with NO₃⁻ is shown. The residue name and numbers in parentheses indicate the corresponding residue in NasS. This image was prepared using PyMOL (DeLano Scientific, Palo Alto, CA). Amino acid sequence alignment between NasS and NrtA are shown in the bottom panel. The residues involved in binding of a nitrate are indicated by blue dots. The sequences were aligned with Clustal OMEGA (36), and this image was prepared using ESPript (37). B, FRET/CFP ratio of sNOOOpy^WT (WT) and sNOOOpy^H145A (H145A) plotted against increasing concentrations of NO₃⁻ (red) and NO₂⁻ (blue). Values in parentheses indicate the K_D values for NO₃⁻ (red) and NO₂⁻ (blue), the values at which the [NO₃⁻] or [NO₂⁻] dissociates half of the NasST. Concentrations of 1 μm sNOOOpy protein, composed of equal concentrations of CFP-NasT and NasS-YFP, were used. The K_D values are calculated by curve-fitting analysis using the standard Hill equation. C, FRET/CFP ratios at various concentrations of [CFP-NasT] were plotted against increasing [NO₃⁻]. Concentrations of 1 μm NasS-YFP and 0.5–2.0 μm of CFP-NasT were used. D, K_D values were plotted against [CFP-NasT] and fitted by equation: K_D = (1 + [CFP-NasT]/K_ST) × K_S-NO3. From linear fit to the plot, K_S-NO3, K_ST, and K_S-NO3/K_ST, were calculated as 6.0 and 0.15 μm and 39, respectively.

Elevated K_D values corresponding to an increase in the [CFP-NasT] were observed (Fig. 4C). This characteristic in the sensitivities reflects that sNOOOpy is based on an intramolecular FRET system, where CFP-NasT and NO₃⁻ competitively binds to NasS-YFP as shown in Fig. 3D. In a canonical competitive model, IC₅₀ values depend on the substrate concentration, IC₅₀ =(1 + [S]/K_m)K_i, where IC₅₀, S, K_m, and K_i correspond to K_D, CFP-NasT, K_ST, and K_S-NO3 in Fig. 3D, respectively. From the linear fit based on the equation, K_S-NO3 and K_S-NO3/K_ST were calculated to be 6.0 μm and 39, respectively (Fig. 4D). Estimated K_ST value, 0.15 μm, was in good agreement with the value determined by titration of CFP-NasT with NasS-YFP, 0.13 μm, as shown in Fig. 3B and Equation 2,

where E is the fluorescent emission at 535 nm; K_ST is dissociation constant between CFP-NasT and NasS-YFP; [S]_total is the total NasS-YFP; and [T]_total is the total CFP-NasT (= 1 μm) concentrations, respectively. K_ST was calculated by the fit.

The time course of FRET ratio changes at 20–100 μm NO₃⁻ revealed that the rates of NO₃⁻ binding (k_on) and dissociation (k_off) were determined based on the first order fitting to be 2.3 × 10⁻³ μm⁻¹ s⁻¹ and 0.10 s⁻¹, respectively (Fig. 5, A and B). Thus, sNOOOpy system can detect [NO₃⁻] changes on a time scale of seconds.

FIGURE 5.

Characterizations of sNOOOpy. A, time course of the FRET/CFP ratio change in the presence of various concentrations of NO₃⁻. B, apparent rate constants (k_app = k_on[NO₃⁻] + k_off) were plotted against [NO₃⁻]. From linear fit to the plot, k_on and k_off were calculated as 0.0023 μm⁻¹ s⁻¹ and 0.10 s⁻¹, respectively. C, pH and D, temperature dependences of sNOOOpy. The values of R₀ and R_ΔNasT indicate the emission ratio of control (in the anion-free buffer) and that of sNOOOpy-ΔNasT, respectively. The FRET/CFP ratio at 25 °C at 0, 10, 100, and 1000 μm NO₃⁻in the pH range of 6.0–9.0 are shown. The buffer contained 100 mm MES-NaOH (pH 6.0–7.0, open circles) or HEPES-NaOH (pH 7.0–9.0, closed circle). E, kinetic analyses of sNOOOpy^WT in vitro at 25 °C (black) and 37 °C (red). FRET/CFP ratio was plotted against increasing concentrations of NO₃⁻. Concentrations of 1 μm sNOOOpy protein, which was composed of equal concentrations of CFP-NasT and NasS-YFP, were used.

The fluorescence emission ratio was almost invariant from pH 7.0 to 8.5 and over a temperature range of 25 to 40 °C, suggesting that the sensitivity of sNOOOpy will not be affected by small fluctuations in pH and temperature (Fig. 5, C and D). The K_D value for NO₃⁻ at 37 °C (37.9 μm) is almost identical to that at 25 °C (39.5 μm) (Fig. 5E). Thus, these results showed that we can quantify [NO₃⁻] in a range of 1–1000 μm by sNOOOpy under standard in vitro assay conditions.

Imaging of NO₃⁻/NO₂⁻ Levels Inside Single Living Cells

Next, we visualized the NO₃⁻/NO₂⁻ levels inside of a single living HeLa cell expressing sNOOOpy proteins. First, we constructed a mammalian expression plasmid for sNOOOpy, pCMV-sNOOOpy (Fig. 6A), in which cDNAs coding for CFP-NasT, a self-processing 2A peptide, and NasS-YFP were arranged in tandem (23, 24). A single polypeptide of CFP-NasT-2A-NasS-YFP is expected to be cleaved at the 2A site. As a result, CFP-NasT and NasS-YFP were separately expressed (Fig. 6B), which is parallel to the in vitro assay conditions. In addition, a nuclear exporting signal sequence was added to the N terminus of CFP-NasT to prevent importation of the liberated CFP-NasT into the nucleus. sNOOOpy proteins expressed by pCMV-sNOOOpy were properly localized to the cytoplasm and exhibited a high FRET/CFP ratio (1.0–1.6) in the NO₃⁻/NO₂⁻-free medium, indicating the formation of the dimer of CFP-NasT and NasS-YFP (Fig. 6C). The FRET/CFP ratios were reduced in response to an increase in [NO₃⁻] (Fig. 6, C–E, and supplemental Movie 1).

FIGURE 6.

Monitoring the cytoplasmic NO₃⁻ levels of HeLa cells. A, schematic diagram of pCMV-sNOOOpy, the plasmid for mammalian expression, contains a nuclear export signal fused to CFP-NasT and NasS-YFP, and they linked by a self-processing 2A peptide. B, expression of the sNOOOpy system proteins in HeLa cells. CFP-NasT and NasS-YFP were detected by Western blot analysis of the lysates from HeLa cells harboring pCMV-sNOOOpy^WT. pCMV-sNOOOpy was transfected into the HeLa cells with the Lipofectamine 2000 transfection reagent. Western blot analysis was performed 48 h after transfection. CFP-NasT (estimated molecular mass, 69.3 kDa) and NasS-YFP (50.2 kDa) were detected after probing with a GFP(FL) rabbit polyclonal IgG (Santa Cruz Biotechnology, Inc.) followed by an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology). C, sequential images of the FRET/CFP emission ratio (pseudocolored) of HeLa cells expressing sNOOOpy^WT (top and middle) and sNOOOpy^H145A (bottom). Total elapsed time (in minutes) is shown at the top left of the cells. Concentrations of 0.3, 1, and 3 mm NO₃⁻ (top and bottom) and NO₂⁻ (middle) were added to the medium at time = 10, 20, 30 (minutes), respectively. D, time course of the FRET/CFP ratio changes in single cell expressing sNOOOpy^WT over a wide range of NO₃⁻ concentrations (0–10 mm). Concentrations of 0.01, 0.1, 1, and 10 mm NO₃⁻ were added to the medium at time = 15, 30, 45, and 60 (min), respectively. FRET/CFP ratios in each cell and averaged ratio are shown in left and right panels, respectively. Error bars are standard deviations between measurements. E, time course of the FRET/CFP ratio changes in single cell. Concentrations of NO₃⁻ in DMEM were changed as in C. Ratios in each cell with sNOOOpy^WT in (1) NO₃⁻, in (2) NO₂⁻, and (3) sNOOOpy^H145A in NO₃⁻ are shown. Averaged ratios are shown in right panel.

In the HeLa cells expressing sNOOOpy^WT, although up to 0.1 mm NO₃⁻ induced no obvious changes of the FRET/CFP ratio (Fig. 6D), NO₃⁻ at a concentration of 0.3 mm induced a significant and rapid decrease in the FRET/CFP ratio, and the ratio was decreased about 50% by addition of 3 mm NO₃⁻ (Fig. 6E). The HeLa cells containing sNOOOpy^WT were less sensitive to NO₂⁻ compared with NO₃⁻, but apparent changes in the FRET/CFP ratio were detectable at 1 mm NO₂⁻ (Fig. 6C, middle panel). The HeLa cells with sNOOOpy^H145A, which formed stable NasST complexes and lost NO₃⁻/NO₂⁻ responsiveness, were insensitive to a change in ambient [NO₃⁻] (Fig. 6C, bottom panel). It should be noted that the response of sNOOOpy to NO₃⁻ is reversible. Fig. 7 and supplemental Movie 2 show the time course of the FRET/CFP ratio when the medium [NO₃⁻] was alternately changed. The FRET/CFP ratio was reduced from 1.4 to 0.8 (40% decrease relative to that in 0 mm) when the cells were cultured in DMEM containing 1 mm NO₃⁻ for 15 min. Ten minutes after an exchange of the medium to a NO₃⁻-free one, the FRET/CFP ratio had recovered to the initial level. Rates of FRET ratio changes in the cells were determined based on the difference of the ratio at two points of time (Fig. 7B). Rates of changes were estimated to be 0.0011–0.0034 (average 0.0022; 18 cells) by addition of 1 mm [NO₃⁻] to DMEM and 0.0011–0.0022 (average 0.0015; 18 cells) by removal of NO₃⁻.

FIGURE 7.

Detecting the dynamics of cellular NO₃⁻ level. A, sequential images of the FRET/CFP emission ratio (pseudocolored) of HeLa cells expressing sNOOOpy^WT are shown. At time = 0 (min), 1 mm NO₃⁻ was added to the medium. After 15 min, the medium was exchanged for NO₃⁻-free medium. B, time course of the FRET/CFP ratio in a single cell. Concentrations of NO₃⁻ in DMEM were changed as in A. Ratios in each cell and averaged ratio are shown in the left and right panels, respectively. Error bars are standard deviations between measurements. C, graphic representation of sNOOOpy system in living cells. This system enables us to detect both the increase and decrease of NO₃⁻ level in mammalian cell as a FRET signal change.

Discussion

Biosensor Employing a Bacterial Environmental Response System

NasS and NasT in the root nodule bacterium B. japonicum are involved in a two-component response system to environmental [NO₃⁻] and regulate protein levels related to NO₃⁻ assimilation (13). We exploited the change in their association-dissociation behavior depending on [NO₃⁻] to develop a NO₃⁻ biosensor. sNOOOpy is the first FRET-based biosensor derived from a bacterial environmental response system. Typically, bacterial two-component regulatory systems comprise a sensor histidine kinase and its cognate response regulator (25), and their functions are controlled by phosphorylation levels of the response regulator catalyzed by histidine kinase in response to environmental stimuli. However, the two-component system of NasS-NasT is regulated without phosphorylation, meaning the reaction is energetically reversible. The sNOOOpy system does not require any component, such as ATP or metal ions, for detection of the [NO₃⁻] change, and it can furthermore detect both an increase and decrease of [NO₃⁻]. These results provide evidence that a potential biological function of the NasS-NasT two-component system is to respond to not only the increase but also the decrease of cellular [NO₃⁻], allowing the bacteria to presumably suppress the protein transcription level.

An NasS-NasT-like two-component system, AmiC and AmiR, that regulates protein levels involved in the catabolic degradation of aliphatic amides was also found in Pseudomonas aeruginosa (26, 27). When considered with the development of the sNOOOpy system from NasS-NasT, we suggest the possibility of an aliphatic amide sensor derived from AmiC-AmiR.

Sensitivity of the sNOOOpy System in Vitro and in Vivo

NO₃⁻/NO₂⁻ are reported to be in micromolar levels in tissues, blood, and plasma (3–50 μm) (28) and in millimolar levels in urine (8). Therefore, to monitor the change in NO₃⁻/NO₂⁻ levels in physiological processes, indicators must have submicromolar to submillimolar sensitivity. When a protein concentration of 1 μm each of CFP-NasT and NasS-YFP is used, the sNOOOpy system can detect a change in [NO₃⁻] in the micromolar range using our in vitro assay conditions. This detection limit of the sNOOOpy system is similar to that of the Griess method.

To determine whether the sNOOOpy system can work in mammalian cells, we subjected cells harboring sNOOOpy proteins to medium containing physiological concentrations of NO₃⁻ or NO₂⁻ (0.01–10 mm). Although little is known about influx and efflux of NO₃⁻ from mammalian cells, the NO₃⁻ influx and characteristics of sialin, a 2NO₃⁻/H⁺ cotransporter, were investigated only in human submandibular gland cell line cells (9). In the extracellular solution containing physiological concentrations of NO₃⁻ (0.05–0.5 mm), a patch clamp method detected an anion current derived from NO₃⁻ influx into human submandibular gland cell line cells at low pH conditions. Recently, the cell bank found that human submandibular gland cell line cells originated from HeLa cells. Therefore, in this study, we exploited the HeLa cells for live cell NO₃⁻/NO₂⁻ imaging. Although no obvious changes were observed up to 0.1 mm NO₃⁻, the FRET ratio of sNOOOpy was changed in response to 0.3–3 mm NO₃⁻ in the medium even at a neutral pH condition.

Although >90% of CFP-NasT and NasS-YFP were dissociated in the presence of 1 mm NO₃⁻ in vitro assay conditions, the FRET ratios in the HeLa cells showed obvious changes at levels up to 3 mm in the medium. Furthermore, the rate of FRET/CFP ratio changes in vitro is 0.2 s⁻¹ by addition of 40 μm [NO₃⁻] to assay conditions, which was markedly faster than that in HeLa cells, 0.002 s⁻¹ by addition of 1 mm [NO₃⁻] to the medium. Here, we consider some of the reasons for the differences of the sNOOOpy system between in vitro and in vivo. One possibility is that the cellular [NO₃⁻] is not raised to the same level as that in the medium, so concentrations in the medium might not be accurately reflected in the HeLa cells. Another possibility is that the sensitivity of sNOOOpy is affected by some cellular environmental factors. Our results showed that excess of CFP-NasT protein increases K_D values caused by an intermolecular FRET system. Such protein concentration dependence of sNOOOpy may be avoided by developing sNOOOpy to be an intramolecular system.

Among NasS-like proteins, the crystallographic study of NrtA, NO₃⁻-binding protein from cyanobacteria, has been reported (22), and residues involved in NO₃⁻ binding were identified. In this study, we constructed sNOOOpy^H145A, which comprises the NasS^H145A mutant, and we revealed an important role of the residue for NO₃⁻/NO₂⁻ responsiveness by the sNOOOpy system. However, structural bases of the interaction of NasS with NasT are still unclear. Crystallographic studies are desired for further improvement in sensitivity and specificity of the sNOOOpy system.

Furthermore, strong base fluorescence emission might limit the detection sensitivity. Therefore, it might be possible to further improve the FRET signal of sNOOOpy by using a polarized fluorescence excitation and detection technique (29).

Insight for Future Applications of the sNOOOpy System

As NO₃⁻ and NO₂⁻ are accepted as stable reservoirs that can be reduced to bioactive NO through the “NO₃⁻-NO₂⁻-NO pathway,” the potential benefits of NO₃⁻/NO₂⁻ in the health field have received much attention. For example, recent prospective epidemiologic studies have shown that green leafy vegetables protect against coronary heart disease and ischemic strokes (30, 31). Many researchers explained these effects using the biologically plausible hypothesis that NO₃⁻/NO₂⁻ in the diet can provide substrates for NO, which results in vasodilation, decreased blood pressure, and supported cardiovascular function (32–34). Thus, NO/NO₃⁻/NO₂⁻ studies have advanced rapidly in the last decade, and the possibility that these anions can be used as therapy for human diseases (e.g. myocardial infarction, stroke, solid organ transplantation, and sickle cell disease) has been suggested (3, 6, 35). Furthermore, Tang et al. (5) suggested that determining endogenous NO₃⁻/NO₂⁻ levels is expected if these anions can be used as diagnostic biomarkers of disease or treatment regimens. Unfortunately, despite the increased attention to the NO₃⁻-NO₂⁻-NO pathway, no other methodology like that of the Griess reaction has been widely used.

We demonstrate that the sNOOOpy system is a NO₃⁻/NO₂⁻-specific biosensor that enables us to visualize both the increase and decrease in NO₃⁻/NO₂⁻ levels in mammalian cells (Fig. 7C). In particular, because the sNOOOpy system is applicable to living cells, which is unique compared with conventional measurement methods, sNOOOpy makes it possible to observe the intracellular dynamics of NO₃⁻/NO₂⁻ both in situ and in real time. The sNOOOpy system paves the way for the elucidation of NO/NO₃⁻/NO₂⁻ biology. Finally, we refer to the potential usefulness of the sNOOOpy system for applications in bacteria and plants. NO₃⁻/NO₂⁻ serves as an essential nutrient for plant growth and survival, because of its involvement in several crucial biological functions of plants (e.g. tissue development and immune systems). The visualization of NO₃⁻/NO₂⁻ dynamics in the physiological actions of plants by the sNOOOpy system has the potential to contribute to the elucidation of fundamental and applied mechanisms of plant life.

Author Contributions

A. G., M. H., T. S., and H. I. performed the experiments. A. G., M. H., and H. I. analyzed the results. K. M. provided the materials. A. G., M. H., and T. U. were involved in the design of the study. A. G., M. H., H. I., and T. U. wrote the paper.