NirD curtails the stringent response by inhibiting RelA activity in Escherichia coli

Abstract
Introduction
Results
Discussion
Materials and methods
Appendix 1
Data availability
References
Article and author information
Metrics

Abstract

Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.

Introduction

Bacteria have evolved numerous molecular mechanisms to detect and cope with environmental stress, including the use of nucleotide-based signaling pathways to efficiently coordinate cellular processes and provide a fast response. Among these signaling pathways, the stringent response is a general stress response that is mediated by the accumulation of the nucleotides guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-triphosphate 3′-diphosphate (pppGpp), collectively known as (p)ppGpp (Potrykus and Cashel, 2008). These alarmones allow bacteria to rapidly respond and adapt to various conditions of nutritional and environmental stress by affecting gene expression and metabolism. In Gram-negative bacteria, (p)ppGpp binds RNA polymerase, thereby altering its promoter selectivity, which results in genome-wide transcriptional reprograming (Durfee et al., 2008; Ross et al., 2013; Ross et al., 2016; Sanchez-Vazquez et al., 2019; Traxler et al., 2008). Additionally, (p)ppGpp also binds directly and alters the activity of several enzymes including DNA primase, translation factors, lysine decarboxylase, and polyphosphate kinase (Corrigan et al., 2016; Kanjee et al., 2012; Wang et al., 2019a; Zhang et al., 2018). Importantly, this rewiring of cell physiology also appears to play a critical role in the regulation of bacterial virulence, survival during host invasion, and antibiotic resistance and tolerance (Hauryliuk et al., 2015; Hengge, 2020; Irving et al., 2021; Potrykus and Cashel, 2008).

In Escherichia coli, (p)ppGpp synthesis and hydrolysis are regulated by RelA and SpoT, which are members of RelA/SpoT homologue (RSH) (Atkinson et al., 2011) enzymes and share similar domain architecture. The enzymatic N-terminal half (NTD) has the synthetase (SYN) and the hydrolase (HYD) domains, and the C-terminal half (CTD) of the protein contains conserved domains, which play a critical role in sensing and transducing stress signals to the catalytic domains (Gratani et al., 2018; Hauryliuk et al., 2015; Hogg et al., 2004; Mechold et al., 2002; Pausch et al., 2020; Takada et al., 2020; Tamman et al., 2020). SpoT is a bifunctional enzyme with both hydrolytic and synthetic activities while RelA is a monofunctional (p)ppGpp synthetase. Indeed, despite close sequence similarity between RelA and SpoT, RelA maintains a ‘pseudo’-hydrolase domain that is structurally conserved but enzymatically inactive. The regulation of RelA and SpoT activities represents an obvious checkpoint for (p)ppGpp levels which reveals many complexities (reviewed in Irving and Corrigan, 2018; Ronneau and Hallez, 2019).

Activation of the (p)ppGpp synthetase RelA occurs via a ribosomal mechanism in response to amino acid starvation or other stressful conditions, which ultimately result in amino acid starvation, for example, fatty acid starvation, that leads to lysine starvation (Haseltine and Block, 1973; Sinha et al., 2019). Under this condition, deacylated tRNAs accumulate and RelA interacts with uncharged tRNA at the vacant ribosomal A-site causing the activation of its (p)ppGpp synthetic activity (Arenz et al., 2016; Brown et al., 2016; Loveland et al., 2016; Winther et al., 2018). Inversely, RelA is suggested to be inhibited by intra- and/or inter-molecular interactions under replete conditions (Gropp et al., 2001; Turnbull et al., 2019; Yang and Ishiguro, 2001), even though it retains residual activity (Murray and Bremer, 1996). Importantly, the hydrolysis function of SpoT provides an opposing activity that is crucial for balancing the basal activity of RelA. Indeed, disruption of the spoT gene results in a toxic accumulation of (p)ppGpp and is therefore lethal (Xiao et al., 1991).

The regulation of SpoT activities involves sources of nutritional stress other than amino acid starvation. These include the deficiency of fatty acid (Seyfzadeh et al., 1993), phosphate (Spira et al., 1995), carbon (Xiao et al., 1991), and iron (Vinella et al., 2005). Interaction of SpoT with other factors seems to regulate the catalytic balance between the synthetic and hydrolytic activities. During fatty acid starvation, the accumulation of (p)ppGpp required a specific interaction between the acyl carrier protein and SpoT (Battesti and Bouveret, 2006). Similarly, the binding of the protein YtfK with the catalytic domains of SpoT triggers (p)ppGpp accumulation during phosphate or fatty acid starvation (Germain et al., 2019). In addition, stimulation of the SpoT hydrolase activity is driven through direct interaction with the anti-σ⁷⁰ factor Rsd upon carbon downshift (Lee et al., 2018).

Given the importance of (p)ppGpp in stress survival, virulence, and antibiotic tolerance, we have designed a genetic assay for the identification of new protein candidates that can modulate (p)ppGpp homeostasis in E. coli. We thereby discovered that overproduction of NirD, the small subunit of the nitrite reductase, decreases intracellular levels of (p)ppGpp. Importantly, our results show that overexpression of nirD limits RelA-dependent accumulation of (p)ppGpp in vivo and can prevent activation of the stringent response during amino acid starvation. Moreover, our results show that NirD is not involved in (p)ppGpp breakdown. Rather we convincingly show that, mechanistically, NirD inhibits the (p)ppGpp synthetase activity of RelA through a specific and direct interaction with the catalytic domains of RelA in vivo and in vitro.

Results

Identification of genes counteracting the toxicity of (p)ppGpp over-accumulation mediated by RelA

At high concentrations, (p)ppGpp becomes a potent inhibitor of bacterial cell growth. Indeed, rising (p)ppGpp levels by overexpression of the relA gene has been shown to inhibit E. coli growth in amino acid-rich media (Schreiber et al., 1991). To gain further insights into (p)ppGpp homeostasis, we developed a screening assay to identify genes that in multiple copies could reverse this toxicity (Figure 1A). In this study, the toxic accumulation of (p)ppGpp is achieved by induction of the relA gene cloned in the low-copy-number plasmid pBbS2k under the control of the anhydrotetracycline (aTc)-inducible P_tet promoter (Figure 1A). E. coli MG1655 cells harboring the pBbS2k-relA were transformed with a pooled collection of plasmids obtained from the ASKA library containing almost all E. coli K-12 genes (Kitagawa et al., 2005), each cloned into the high-copy-number vector pCA24N downstream of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible P_T5-lac promoter (Figure 1A). This genetic screen led us to identify 88 clones that repetitively grow on non-permissive conditions. Sequence analysis of the pCA24N derivatives from these 88 clones enabled the identification of four different genes. The spoT, gppA, and nirD genes were predominant with each one accounting for almost a third of the genes carried by the sequenced plasmids (33.0, 36.4, and 29.5%, respectively) while uhpA was only identified once (1.1%). Our observations that spoT and gppA revert toxicity associated to (p)ppGpp accumulation is consistent with previous observations (Germain et al., 2019; Murray and Bremer, 1996; Sanyal and Harinarayanan, 2020). Considering the gppA and spoT genes as internal controls of the screen, we further focused our work on nirD.

Figure 1 with 1 supplement see all

Download asset Open asset

NirD can limit the RelA-dependent accumulation of (p)ppGpp.

(A) Schematic representation of the genetic assay using ASKA library to identify genes that in multiple copies would suppress the growth defect associated with toxic over-accumulation of (p)ppGpp. Question marks denote a putative candidate that limits RelA-dependent accumulation of (p)ppGpp. (B) NirD suppresses the growth defect associated with RelA overproduction. Wild-type *E. coli* MG1655 cells were co-transformed with the plasmid derivatives pBbS2k-*relA* and pEG25-*nirD* inducible by anhydrotetracycline (aTc) and isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Serial dilutions of stationary-phase cultures were spotted on NA plates containing the indicated concentrations of inducers. Additional controls and inducer concentrations are shown in Figure 1—figure supplement 1A. The results are representative of three independent experiments with similar results. (**C, D**) In vivo (p)ppGpp dynamic in *E. coli* MG1655 cells carrying pBbS2k-*relA* and pEG25-*nirD* after consecutive expression of the *relA* and *nirD* genes using 100 ng/mL aTc and 1 mM IPTG, respectively. Nucleotides extracted from samples collected at the indicated times were separated by thin layer chromatography. The autoradiogram (C) is representative of three independent experiments, and the curves of the relative levels of (p)ppGpp (D) are represented as the means of the three independent experiments, the error bars depicting the SDs.

Figure 1—source data 1 Raw autoradiogram.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig1-data1-v1.zip
Download elife-64092-fig1-data1-v1.zip
Figure 1—source data 2 Quantification of (p)ppGpp. The amount of (p)ppGpp was normalized to total amount of G nucleotides observed in each sample. Total G is the sum of GTP and (p)ppGpp detected. The source data are provided for the relative levels of (p)ppGpp in Figure 1D, which are represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig1-data2-v1.zip
Download elife-64092-fig1-data2-v1.zip

NirD balances (p)ppGpp level produced by RelA

nirD encodes the small subunit of the cytoplasmic nitrite reductase (Harborne et al., 1992; Peakman et al., 1990a) and has never been linked to the (p)ppGpp metabolism. We first confirmed the result obtained with the high-copy-number vector pCA24N-nirD from the ASKA library by re-cloning the coding region of nirD (untagged) in a more suitable physiological plasmid harboring a tight IPTG-inducible P_T5-lac promoter (pEG25). Indeed, as shown in Figure 1B, NirD can robustly overcome the toxicity associated to (p)ppGpp over-accumulation mediated by RelA. To further assess the role of NirD in the stringent response, we monitored (p)ppGpp levels in vivo after consecutive inductions of RelA and NirD in a double expression system assay. As shown in Figure 1C, D, induction of RelA is associated with a massive accumulation of (p)ppGpp, which is sharply stopped and then decreased upon NirD induction.

The rapid (p)ppGpp decay observed following NirD induction raises the possibility that NirD may trigger SpoT-dependent (p)ppGpp hydrolysis. However, NirD still suppresses toxicity associated to RelA overproduction when the double expression assay experiment is performed in a ΔrelA spoT background (Figure 1—figure supplement 1A). These results show that the hydrolase activity of SpoT is not involved in the reduced (p)ppGpp accumulation observed upon NirD induction. Importantly, overexpression of nirD in a ΔrelA strain failed to phenocopy the well-known amino acid auxotrophic characteristic of a ppGpp⁰ strain (ΔrelA spoT mutant) (Xiao et al., 1991), strongly arguing that NirD does not act as a small alarmone hydrolase (Figure 1—figure supplement 1B). Taken together, our results show that NirD impairs RelA-dependent (p)ppGpp synthesis in E. coli.

NirD can prevent the stringent response in E. coli

Since NirD can efficiently overcome toxicity associated to artificial level of (p)ppGpp when RelA is overproduced (Figure 1B), we next address whether similar effect is observed during physiological condition known to activate RelA. In natural environment, RelA synthesizes the alarmones in response to amino acid deprivation. Indeed during amino acid starvation, deacylated tRNAs accumulate and RelA interacts with uncharged tRNA at the vacant ribosomal A-site causing the activation of its (p)ppGpp synthetic activity (Arenz et al., 2016; Brown et al., 2016; Loveland et al., 2016; Winther et al., 2018). RelA activity is therefore required to survive under amino acid starvation, and as a result a ΔrelA mutant is not able to grow in the presence of 1 mM serine, methionine, and glycine (SMG), which induces isoleucine starvation (Uzan and Danchin, 1978). Interestingly, we observed that overexpression of nirD in wild-type (WT) strain phenocopies the growth defect of a ΔrelA mutant on SMG plate (Figure 2A), suggesting that nirD also prevents (p)ppGpp accumulation from endogenous RelA under these conditions. To further address this possibility, we monitored the dynamics of (p)ppGpp levels under amino acid starvation upon the addition of L-valine (which also induces isoleucine starvation). As shown in Figure 2B, C, and as previously observed (p)ppGpp rapidly increases in WT strain and reaches maximum levels within 5 min (Cashel and Gallant, 1969). Remarkably, when NirD is induced prior to amino acid starvation, no significant accumulation of (p)ppGpp is detected (Figure 2B, C). Taken together, our results show that once induced NirD abolishes the stringent response in E. coli.

Figure 2

Download asset Open asset

NirD inhibits accumulation of (p)ppGpp upon amino acid starvation.

(A) NirD inhibits cell growth on M9-glucose minimal medium supplemented with serine, methionine, and glycine (SMG). Stationary-phase cultures of wild-type *E. coli* MG1655 strain harboring pEG25-*nirD* or pEG25-*spoT* were spotted on NA and SMG plates containing the indicated concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG). The results are representative of three independent experiments. (**B, C**) Exponentially growing cells of wild-type *E. coli* MG1655 harboring pEG25 or pEG25-*nirD* were challenged for amino acid starvation by addition of 500 µg/mL of L-valine. *nirD* is induced by the addition of 1 mM of IPTG. Nucleotides were extracted and separated by thin layer chromatography. The autoradiogram (B) is representative of three independent experiments, and the curves of the relative levels of (p)ppGpp (C) are represented as the means of the three independent experiments, the error bars depicting the SDs.

Figure 2—source data 1 Raw autoradiogram.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig2-data1-v1.zip
Download elife-64092-fig2-data1-v1.zip
Figure 2—source data 2 Quantification of (p)ppGpp. The amount of (p)ppGpp was normalized to total amount of G nucleotides observed in each sample. Total G is the sum of GTP and (p)ppGpp detected. The source data are provided for the relative levels of (p)ppGpp in Figure 2C, which are represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig2-data2-v1.xlsx
Download elife-64092-fig2-data2-v1.xlsx

NirD functionally interacts with RelA in vivo

Regulation of RSH activities through protein–protein interactions has been previously reported (Battesti and Bouveret, 2006; Germain et al., 2019; Hahn et al., 2015; Karstens et al., 2014; Krüger et al., 2020; Lee et al., 2018; Peterson et al., 2020; Raskin et al., 2007; Ronneau et al., 2016; Ronneau et al., 2019; Wout et al., 2004). We therefore tested whether NirD is able to interact with RelA in vivo using a bacterial two-hybrid (BTH) assay (Karimova et al., 1998). For that purpose, the complementary T18 and T25 domains of Bordetella pertussis adenylate cyclase (CyA) were fused to the N-terminus of NirD and RelA proteins using the two compatible vectors pUT18C and pKT25, respectively. When the E. coli BTH101 strain (cya deficient) is co-transformed with plasmids harboring T18 and T25 fusions, NirD exhibited a strong interaction in vivo with RelA (Figure 3A). However, despite the strong sequence homologies between the paralogous RelA and SpoT proteins (Metzger et al., 1989), no physical interaction is observed between SpoT and NirD in vivo by BTH assay (Figure 3A). This result therefore supports a strong specificity of the interaction between NirD and RelA.

Figure 3 with 1 supplement see all

Download asset Open asset

NirD can inhibit RelA-dependent accumulation through physical interaction in vivo.

(A) Bacterial two-hybrid assay using *E. coli* BTH101 cells co-transformed with plasmid derivatives pKT25-*relA* or pKT25-*spoT* and pUT18C-*nirD* or pUT18C-*nirD^E50K*. Stationary-phase cultures were spotted on NA plates containing X-Gal as a blue color reporter for positive interaction. The bars showing β-galactosidase activity are represented as the means of three independent experiments, and the error bars depict the SDs. (B) Growth assay of *E. coli* MG1655 cells co-transformed with the plasmid derivatives pBbS2k-*relA* and pEG25-*nirD* or pEG25-*nirD^E50K* inducible by anhydrotetracycline (aTc) and isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. Serial dilutions of stationary-phase cultures were spotted on NA plates containing the indicated concentrations of inducers. The results are representative of three independent experiments. (**C, D**) In vivo (p)ppGpp level dynamic in *E. coli* MG1655 cells carrying pBbS2k-*relA* and pEG25-*nirD* or pEG25-*nirD^E50K* after successive inductions of expression of the *relA* and *nirD* or *nirD^E50K* genes using 100 ng/mL aTc and 1 mM IPTG, respectively. Nucleotides extracted from samples collected at the indicated times were separated by thin layer chromatography. The autoradiogram (C) is representative of three independent experiments, and the curves of the relative levels of (p)ppGpp (D) are represented as the means of the three independent experiments, the error bars depicting the SDs. (E) Growth curves of wild-type (WT) cells, Δ*nirD,* and *nirD^E50K* mutants under anaerobiosis in M9-glucose minimal medium without amino acids (left panel) or supplemented with 1 mM serine, methionine, and glycine (SMG) (right panel) (see Materials and methods). The growth curves are representative of at least three independent experiments, and the Δ(lag phase) are represented as the means (± SD) of the independent experiments.

Figure 3—source data 1 Determination of β-galactosidase activity. The relative β-galactosidase activity was calculated as follows: $1000 \times \frac{S l o p e (\frac{{Δ O D}_{420 n m}}{Δ t})}{V \times {O D}_{600 n m}}$ . Slope is estimated by linear regression, and V is the volume of culture used (in mL). The source data are provided for the bar chart showing β-galactosidase activity in Figure 3A, which is represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig3-data1-v1.xlsx
Download elife-64092-fig3-data1-v1.xlsx
Figure 3—source data 2 Raw autoradiogram.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig3-data2-v1.zip
Download elife-64092-fig3-data2-v1.zip
Figure 3—source data 3 Quantification of (p)ppGpp. The amount of (p)ppGpp was normalized to total amount of G nucleotides observed in each sample. Total G is the sum of GTP and (p)ppGpp detected. The source data are provided for the relative levels of (p)ppGpp in Figure 3D, which are represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig3-data3-v1.xlsx
Download elife-64092-fig3-data3-v1.xlsx
Figure 3—source data 4 Determination of Δ(lag phase). The Δ(lag phase) corresponds to the time that separates the growth recovery of the wild-type (WT) (lag phase [WT]) from that of the ΔnirD or nirD^E50K (lag phase [ΔnirD, nirD^E50K]). For each strain, the duration of the lag phase was calculated for an OD_600nm corresponding to the entry into the exponential growth phase. The equation for this calculation was estimated by linear regression on the log phase of each growth curve. The source data are provided for the Δ(lag phase) in Figure 3E, which are represented as the means and SDs of at least three experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig3-data4-v1.xlsx
Download elife-64092-fig3-data4-v1.xlsx

To further assess the significance of the NirD-RelA interaction in vivo and its role in (p)ppGpp homeostasis, we randomly mutagenized nirD and searched for defective mutants unable to interact with RelA. One of such mutants that had charge reversal amino acid substitution, glutamate 50 to lysine (E50K), displayed compromised interaction with RelA (Figure 3A). Importantly, we confirm that both T18-NirD and T18-NirD^E50K recombinant proteins were correctly expressed and folded as shown by their ability to strongly interact with the catalytic subunit of the nitrite reductase NirB (Figure 3—figure supplement 1) (Harborne et al., 1992). Importantly and on the contrary to the results obtained with the WT copy of nirD, we observed that overexpression of nirD^E50K is not able to suppress toxicity associated to RelA overproduction on plate (Figure 3B). This result predicts that breaking the interaction with RelA abolishes the inhibitory effect of NirD on (p)ppGpp accumulation. Indeed, as shown in Figure 3C, D, nirD^E50K failed to stop the massive accumulation of (p)ppGpp observed in vivo after RelA overproduction as compared to the fast decay of (p)ppGpp observed when the WT copy of nirD is expressed (Figure 3C and D).

nirD is a second gene of a four-gene operon (nirBDC-cysG) strongly induced under anaerobic growth (Harborne et al., 1992; Peakman et al., 1990b). Interestingly, we observed that ΔnirD mutant presents an extended growth recovery in anaerobic SMG liquid medium consistent with a toxic over-accumulation of (p)ppGpp in this strain (Figure 3E). Indeed, a chromosomal encoded nirD^E50K mutant, which is functional for the nitrite reductase activity (Figure 3—figure supplement 1C) but failed to interact with RelA, displays the same growth defect, suggesting that the NirD-RelA interaction is important to adjust proper (p)ppGpp level under this specific growth condition. Taken together, our results show that a specific functional interaction between RelA and NirD can lower RelA-dependent accumulation of (p)ppGpp in vivo.

NirD targets the catalytic N-terminal region of RelA

RelA consists of several protein domains that have been encompassed in two functional regions, the catalytic NTD and the regulatory CTD. The N-terminal region includes the enzymatically inactive (p)ppGpp hydrolase domain (HYD) as well as the functional (p)ppGpp synthetase domain (SYN), while the CTD comprises the Thr-tRNA synthetase, GTPase and SpoT domain (TGS), the alpha helical domain (AH), the zinc finger domain (ZFD), and finally the RNA recognition motif domain (RRM) (Figure 4A) (Brown et al., 2016; Loveland et al., 2016). To determine which of these domains are required for the interaction with NirD, we assayed the interaction between NirD and several truncated RelA proteins using the BTH assay.

Figure 4 with 1 supplement see all

Download asset Open asset

NirD can interact with the catalytic N-terminal region of RelA.

(A) Schematic representation of RelA and its protein domains. (B) Bacterial two-hybrid assay using *E. coli* BTH101 cells co-transformed with plasmid derivatives pUT18C-*nirD* and pKT25 with the full-length or truncated *relA* gene as indicated. Stationary-phase cultures were spotted on NA plates containing X-Gal as a blue color reporter for positive interaction. The bars showing β-galactosidase activity are represented as the means of three independent experiments, and the error bars depict the SDs.

Figure 4—source data 1 Determination of β-galactosidase activity. The relative β-galactosidase activity was calculated as follows: $1000 \times \frac{S l o p e (\frac{{Δ O D}_{420 n m}}{Δ t})}{V \times {O D}_{600 n m}}$ . Slope is estimated by linear regression, and V is the volume of culture used (in mL). The source data are provided for the bar chart showing β-galactosidase activity in Figure 4B, which is represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig4-data1-v1.xlsx
Download elife-64092-fig4-data1-v1.xlsx

Truncated RelA proteins were fused to the T25 domain of the B. pertussis adenylate cyclase and transformed together with NirD fused to the T18 domain in a cya-deficient E. coli strain (BTH101). BTH analysis revealed that NirD interacts with the RelA^N-terminal fusion comprising the catalytic domains (Figure 4B). Moreover, careful quantification of β-galactosidase activity revealed that none of the four domains encompassing the CTD of RelA is required for this interaction in vivo. However, truncated RelA fusion lacking either the synthetase domain (RelA^1-181) or the ‘pseudo’ hydrolase domain (RelA^181-744) failed to interact in vivo with NirD. Therefore, the catalytic domains of RelA seem necessary and sufficient to interact with NirD in vivo. The specificity of the interaction between NirD and RelA was further reinforced by the absence of interaction observed between NirD and SpoT^N-terminal as well as NirD^E50K and RelA^N-terminal (Figure 4—figure supplement 1A). Interestingly, the C-terminal region of RelA is proposed to play a key role in the autoregulation of the enzymatic activity, and as a result the RelA^N-terminal truncated protein is constitutively active (Schreiber et al., 1991). Given that NirD interacts with RelA^N-terminal, we therefore tested the intriguing possibility that NirD may also be able to inhibit this dysregulated variant. Remarkably, as shown in Figure 4—figure supplement 1B, we observed that NirD but not NirD^E50K inhibits the toxicity associated with (p)ppGpp over-accumulation mediated by the constitutively active RelA^N-terminal. Taken together, our results show that NirD can inhibit RelA activity by interacting with its catalytic N-terminal region in vivo.

NirD directly binds RelA to inhibit the rate of (p)ppGpp synthesis in vitro

To confirm the in vivo interaction of NirD at the catalytic N-terminal region of RelA, we used different complementary in vitro methods. For that purpose, the RelA catalytic domains (RelA^N-terminal), NirD, and NirD^E50K were individually produced and purified by two consecutive chromatographies, affinity and size-exclusion (Figure 5—figure supplement 1A–C). We first performed size-exclusion chromatography (SEC) experiment separating RelA^N-terminal and NirD alone or pre-mixed. As shown in Figure 5—figure supplement 1D, co-elution resulted in an earlier eluting peak, suggesting the formation of a stable complex. Indeed, SDS-PAGE analysis confirmed that the two proteins were co-eluted from the column (Figure 5—figure supplement 1E).

To further characterize the NirD-RelA^N-terminal interaction, we then used biolayer interferometry (BLI), an in vitro approach for measuring biomolecular interactions. Biotinylated NirD was immobilized on streptavidin biosensors as ligand, and RelA^N-terminal was used as the analyte. By adding RelA at concentrations ranging from 0.1 to 30 µM, a dose-response association is recorded and decreased during the dissociation step corresponding to the washing of the sensor (Figure 5A), showing that NirD directly interact with the catalytic N-terminal region of RelA. Moreover, by plotting the maximum binding response against the corresponding concentrations of RelA, we calculated a dissociation constant (K_D) of 0.55 µM (± 0.053 µM) (Figure 5A). In addition, and in agreement with the BTH assays (Figure 4—figure supplement 1A), RelA^N-terminal did not interact with the NirD^E50K variant in vitro (Figure 5A). Finally, to further determine the thermodynamic parameters of the NirD-RelA^N-terminal interaction, we performed isothermal titration calorimetry (ITC) experiment. The assay first confirmed the direct interaction between the two proteins with an estimated K_D of 0.34 µM (Figure 5B), very similar to that estimated by BLI (Figure 5A). This reaction is a favorable spontaneous exothermic reaction with a ΔH kcal/mol of −3.63 ± 0.208 and is enthalpy driven at 25°C. On the contrary and consistent with BLI experiments, no heat exchange was recorded upon titration of RelA^N-terminal by NirD^E50K, further supporting the strong specificity of the interaction between NirD and RelA (Figure 5B). Collectively, these results show that NirD interacts directly and specifically with the catalytic domains of RelA in vitro.

Figure 5 with 1 supplement see all

Download asset Open asset

NirD directly interact with the catalytic N-terminal region of RelA.

(A) Biolayer interferometric assay of RelA^N-terminal on NirD or NirD^E50K. Biotinylated NirD or NirD^E50K were immobilized on streptavidin biosensors and probed with RelA^N-terminal at concentrations ranging from 0.1 to 30 µM. The curves are represented as the means of the subtracted reference binding responses during association and dissociation from three experiments, the error bars depicting the SDs. The inset curve shows the specific biolayer interferometry (BLI) response (nm) 10 s before the end of association as a function of RelA^N-terminal concentration. Data are represented as the means of the three experiments, the error bars depicting the SDs. (**B, C**) Isothermal titration calorimetry profiles corresponding to the binding of RelA^N-terminal30–300 µM NirD (B) or 300 µM NirD^E50K (C) at 25°C. The upper panels show raw data for titration of NirD or NirD^E50K with RelA^N-terminal, and the lower panels show the integrated heats of binding obtained from the raw data. The data were fitted using a ‘One Set of Sites’ model in the PEAQ-ITC Analysis Software.

Figure 5—source data 1 Raw data for biolayer interferometry (BLI) experiments. The source data are provided for the BLI experiments in Figure 5A, which are represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig5-data1-v1.xlsx
Download elife-64092-fig5-data1-v1.xlsx
Figure 5—source data 2 Raw data for isothermal titration calorimetry experiment run with NirD.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig5-data2-v1.xlsx
Download elife-64092-fig5-data2-v1.xlsx
Figure 5—source data 3 Raw data for isothermal titration calorimetry experiment run with NirD^E50K.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig5-data3-v1.xlsx
Download elife-64092-fig5-data3-v1.xlsx

Finally, these results are in line with our in vivo results showing that NirD inhibits accumulation of (p)ppGpp from the constitutively active RelA^N-terminal protein (Figure 4—figure supplement 1B) and therefore support a rather simple model where NirD would directly inhibit RelA synthetic activity through direct binding. To challenge this hypothesis, we performed in vitro pppGpp synthesis assay with purified proteins. We observed that RelA^N-terminal protein could efficiently synthetize [³²P]-pppGpp, but the addition of NirD strongly affected the synthetase activity in vitro. Indeed, 80% of GTP are converted to pppGpp within 60 min by RelA, whereas only 30% are converted when NirD is added (Figure 6A, B). Importantly, addition of the NirD^E50K does not affect the RelA synthetic activity (Figure 6A, B). Finally, careful examination of the initial rates (calculated over the first 150 s) shows a concentration-dependent inhibition of RelA’s (p)ppGpp synthetic activity by NirD with a half-inhibition occurring at ∼1:1 molar ratio and a near complete inhibition for the highest tested concentration of NirD (1:16 molar ratio) (Figure 6C). Taken together, these results show that NirD can block pppGpp synthetase activity of RelA through a direct interaction with the catalytic domains.

Figure 6

Download asset Open asset

NirD inhibits RelA activity in vitro.

(**A, B**) In vitro pppGpp formation by 1 µM RelA^N-terminal in the presence or absence of 4 µM NirD or NirD^E50K. Samples collected at the indicated times were separated by thin layer chromatography (TLC). The autoradiogram (A) is representative of three experiments, and the curves of the relative levels of pppGpp (B) are represented as the means of the three experiments, the error bars depicting the SDs. (C) Relative initial velocity of RelA as a function of NirD:RelA molar ratio. The in vitro formation of pppGpp by 1 µM RelA^N-terminal was assayed in the presence or absence of NirD at concentrations ranging from 0.5 to 16 µM. Samples were collected every 30 s over a period of 150 s and separated by TLC to determine the relative initial velocity (estimated by linear regression using five data points). Results are represented as the means of three experiments, and the error bars depict the SDs.

Figure 6—source data 1 Raw autoradiogram.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig6-data1-v1.zip
Download elife-64092-fig6-data1-v1.zip
Figure 6—source data 2 Quantification of pppGpp. The amount of pppGpp was normalized to total amount of G nucleotides observed in each sample. Total G is the sum of GTP and pppGpp detected. The source data are provided for the relative levels of pppGpp in Figure 6B, which are represented as the means and SDs of three independent experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig6-data2-v1.xlsx
Download elife-64092-fig6-data2-v1.xlsx
Figure 6—source data 3 Determination of the relative initial velocity. The relative initial velocity was estimated on the linear part of the in vitro pppGpp formation by linear regression at the very beginning of the reaction. The source data are provided for the results in Figure 6C, which are represented as the means and SDs of three experiments.: https://cdn.elifesciences.org/articles/64092/elife-64092-fig6-data3-v1.xlsx
Download elife-64092-fig6-data3-v1.xlsx

Discussion

E. coli contains two long RSH enzymes, RelA and SpoT, that synthesize the alarmones (p)ppGpp. While SpoT is a bifunctional enzyme, RelA is monofunctional with a degenerated hydrolase domain, making SpoT the primary source of hydrolysis (Xiao et al., 1991). Therefore, a fine-tune regulation of the intracellular (p)ppGpp synthetic and hydrolytic activities in bacteria is crucial to rapidly adjust (p)ppGpp level in response to environmental stress but also to prevent toxic consequences due to (p)ppGpp over-accumulation (Xiao et al., 1991). Over the past decade, several molecular mechanisms regulating the activity of (p)ppGpp synthetase and/or hydrolase have been characterized (Battesti and Bouveret, 2006; Germain et al., 2019; Hahn et al., 2015; Karstens et al., 2014; Krüger et al., 2020; Lee et al., 2018; Peterson et al., 2020; Raskin et al., 2007; Ronneau et al., 2016; Ronneau et al., 2019; Wout et al., 2004). In this study, we uncovered an unprecedented mode of regulation that can prevent accumulation of (p)ppGpp in E. coli. This regulation implicates physical interaction in vivo and in vitro between the catalytic domains of RelA and the protein NirD, the small subunit of the cytoplasmic NADH-dependent nitrite reductase complex NirBD (Harborne et al., 1992). We show that NirD can functionally and directly inhibit the RelA (p)ppGpp synthetic activity. RelA and SpoT are paralogous proteins; however, despite strong sequence homologies and similar domain architecture we were not able to notice physical interaction between NirD and SpoT (Figure 3A and Figure 4—figure supplement 1A), supporting a specific physiological role of NirD in inhibiting RelA synthetic activity. The absence of a functional link between NirD and SpoT is further reinforced by the observation that NirD does not seem to lower basal (p)ppGpp level produced by the weak (p)ppGpp synthetase activity encoded by SpoT in the absence of RelA (Figure 1—figure supplement 1B).

Stimulation of RelA synthetase activity in E. coli is driven by stalled ribosomes upon amino acid starvation (Haseltine and Block, 1973; Wendrich et al., 2002; Winther et al., 2018). In addition, NtrC-dependent transcription of relA takes an important role in (p)ppGpp accumulation (Brown et al., 2014). Therefore, the identification of a potent intracellular inhibitor of RelA (p)ppGpp synthetase represents an additional level of complexity in the regulation to the current stringent model mediated by RelA. Our results show that the NirD protein level can play an important role for adjusting intracellular (p)ppGpp levels. Indeed, ectopic induction of NirD is sufficient to prevent accumulation of (p)ppGpp mediated by RelA in the absence of nutritional stress (Figure 1C, D). Moreover, we show that once expressed, nirD can also totally abolish activation of the stringent response during amino acid starvation (Figure 2C, D). The estimated concentration of RelA is in the range of 100–400 nM in E. coli (Li et al., 2014; Pedersen and Kjeldgaard, 1977). Importantly, we found in vitro that the NirD-RelA affinity is characterized by a K_D in this concentration range (300–550 nM) (Figure 5A, B). Therefore, at concentrations above the K_D, NirD would interact with RelA to catalytically inhibit its activity. nirD is a second gene of a four-gene operon (nirBDC-cysG) expressed under anaerobic growth (Harborne et al., 1992; Peakman et al., 1990b) and maximally induced in the presence of glucose and nitrate (Tyson et al., 1997; Wang and Gunsalus, 2000). Under these conditions, NirD is predicted to reach µM range (Khlebodarova et al., 2016) and regulation of RelA by NirD would therefore become physiologically relevant. Importantly, while NirB and NirD can form a stable complex needed for the NADH-dependent nitrite reductase activity, we observed that coexpression of nirB and nirD can still counteract RelA-dependent (p)ppGpp accumulation (Figure 3—figure supplement 1B). The cytoplasmic nitrite reductase NirBD has emerged as a key regulator of the nitric oxide (NO) level during nitrate respiration (Bulot et al., 2019). Indeed, by reducing nitrite generated during nitrate reduction, NirBD would limit the level of toxic nitrite and thus its further reduction to NO (Bulot et al., 2019). In addition, our results present evidence that NirD can also promote bacterial fitness by adjusting (p)ppGpp levels in response to amino acid starvation under anaerobic glucose fermentation, a condition where the nitrite reductase is expressed but where the substrate is missing (Figure 3E). This regulation is dependent on the RelA-NirD interaction since NirD^E50K, which is not affected for the nitrite reductase activity (Figure 3—figure supplement 1C) but failed to interact with RelA (Figure 3A), does not enhance bacterial fitness in response to amino acid starvation under anaerobic glucose fermentation (Figure 3E). Therefore, our results suggest that NirD can link (p)ppGpp homeostasis to anaerobic metabolism in E. coli. Additional analyses are currently underway to further investigate the physiological roles of the RelA-NirD interaction in E. coli.

The CTD of RSH enzymes is assumed to play key roles in sensing nutrient starvation and adjusting the enzymatic state of the NTD. In that sense, the RelA structures on stalled ribosomes emphasized the role of CTD in sensing nutrient availability. Once bound to the ribosome, RelA adopts an open extended conformation, which is thought to release the autoinhibitory effect of the CTD on the NTD, therefore promoting (p)ppGpp synthesis (Arenz et al., 2016; Brown et al., 2016; Loveland et al., 2016). Interestingly, we observed that the NTD region of RelA is necessary and sufficient for NirD binding (Figure 4B and Figure 5). Moreover, our in vivo and in vitro results convincingly show that NirD can inhibit the (p)ppGpp synthesis from the constitutively active truncated RelA^N-terminal (Figure 6 and Figure 4—figure supplement 1B), supporting the idea of a ribosome-independent inhibition of the stringent response. Therefore, rather than preventing activation of RelA our results support a simple model of regulation in which NirD directly interacts with the catalytic domains of RelA to inhibit the synthetic activity. Such inhibition might be mediated by directly introducing structural changes in the catalytic domains. Alternatively, NirD may interact with RelA to avoid nucleotide binding by masking the nucleotide-binding site. Structural and functional analyses are currently under investigation to test whether direct binding of NirD inhibits RelA activity.

Finally, (p)ppGpp has emerged as an important regulator of not only the bacterial stress response but also of many aspects of bacterial physiology including virulence, immune evasion, and antibiotic tolerance. Therefore, strategies to inhibit RelA activity and the subsequent production of (p)ppGpp represent an attractive approach for the success of antimicrobial therapy. Currently, there are only a limited number of synthetic inhibitors known to target RelA, mainly based on the design of (p)ppGpp analogues (Wexselblatt et al., 2012; Wexselblatt et al., 2013). Hence, our identification that NirD constitutes a natural potent inhibitor of RelA in vivo and in vitro could represent an important resource for the future design of new drug compounds. Importantly, it is also likely to be a fascinating molecular tool to further tackle the functional and structural determinant of the stringent response.

Reagent type (species) or resource	Designation	Source or reference	Additional information
strain, strain background (Escherichia coli)	BTH101	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752	F^– cya-99 araD139 galE15 galK16 rpsL(Str^R) hsdR2 mcrA1 mcrB1 relA1
strain, strain background (E. coli)	BL21 (DE3)	New England Biolabs	B F^– ompT gal
strain, strain background (E. coli)	MG1655; WT	Lab collection	Wild-type
strain, strain background (E. coli)	nirD^E50K	This paper	MG1655 nirD^E50K
strain, strain background (E. coli)	ΔnirD	This paper	MG1655 nirD::FRT; P1 from KEIO collection (resistance cassette has been flipped out) (Baba et al., 2006)
strain, strain background (E. coli)	ΔrelA	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	MG1655 relA::FRT
strain, strain background (E. coli)	ΔrelA spoT	This paper	MG1655 relA::FRT spoT207::cat; P1 transduction from CF1693 (Baba et al., 2006) in MG1655 relA::FRT
recombinant DNA reagent	pBbS2k-rfp	Lee et al., 2011 doi: 10.1186/1754-1611-5-12	Kan^R, P_tet, rfp
recombinant DNA reagent	pBbS2k-relA	This paper	Kan^R, P_tet, relA; primers 16/17; Maisonneuve lab
recombinant DNA reagent	pBbS2k-relA (sd4)	This paper	Kan^R, P_tet, sd4, relA; primers 163/17; Maisonneuve lab
recombinant DNA reagent	pBbS2k-relA^N-terminal	This paper	Kan^R, P_tet, relA^N-terminal; primers 16/557; Maisonneuve lab
recombinant DNA reagent	pEG25	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Amp^R, P_T5-lac
recombinant DNA reagent	pEG25-nirD	This paper	Amp^R, P_T5-lac, nirD; primers 386/387; Maisonneuve lab
recombinant DNA reagent	pEG25-spoT (sd8U)	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Amp^R, P_T5-lac, sd8U, spoT
recombinant DNA reagent	pEG25-nirD^E50K	This paper	Amp^R, P_T5-lac, nirD^E50K; primers 386/387; Maisonneuve lab
recombinant DNA reagent	pEG25-nirD (WTsd)	This paper	Amp^R, P_T5-lac, WTsd, nirD; primers 555/387; Maisonneuve lab
recombinant DNA reagent	pEG25-nirBD (WTsd)	This paper	Amp^R, P_T5-lac, WTsd, nirBD; primers 553/544; Maisonneuve lab
recombinant DNA reagent	pEG25-relA^N-terminal	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Amp^R, P_T5-lac, relA^N-terminal
recombinant DNA reagent	pKD46	Datsenko and Wanner, 2000 doi: 10.1073/pnas.120163297	Amp^R, P_ara, λ Red
recombinant DNA reagent	pKT25	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752	Kan^R
recombinant DNA reagent	pKT25-zip	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752	Kan^R, zip
recombinant DNA reagent	pKT25-relA	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Kan^R, relA
recombinant DNA reagent	pKT25-spoT	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Kan^R, spoT
recombinant DNA reagent	pKT25-nirB	This paper	Kan^R, nirB; primers 617/618; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^1-663	This paper	Kan^R, relA^1-663; primers 497/502; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^1-580	This paper	Kan^R, relA^1-580; primers 497/501; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^1-470	This paper	Kan^R, relA^1-470; primers 497/500; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^N-terminal	This paper	Kan^R, relA^N-terminal; primers 497/499; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^1-181	This paper	Kan^R, relA^1-181; primers 497/498; Maisonneuve lab
recombinant DNA reagent	pKT25-relA^181-744	This paper	Kan^R, relA^181-744; primers 531/533; Maisonneuve lab
recombinant DNA reagent	pKT25-spoT^N-terminal	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Kan^R, relA^N-terminal
recombinant DNA reagent	pUT18C	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752	Amp^R
recombinant DNA reagent	pUT18C-zip	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752	Amp^R, zip
recombinant DNA reagent	pUT18C-nirD	This paper	Amp^R, nirD; primers 441/443; Maisonneuve lab
recombinant DNA reagent	pUT18C-nirD^E50K	This paper	Amp^R, nirD^E50K; primers 441/443; Maisonneuve lab
recombinant DNA reagent	pET28a(+)	Germain et al., 2019 doi: 10.1038/s41467-019-13764-4	Kan^R, Trx, His
recombinant DNA reagent	pET28a(+)-nirD	This paper	Kan^R, Trx, His, TEV, nirD; primers 558/544; Maisonneuve lab
recombinant DNA reagent	pET28a(+)-nirD^E50K	This paper	Kan^R, Trx, His, TEV, nirD^E50K; primers 558/544; Maisonneuve lab
recombinant DNA reagent	pWRG99	Blank et al., 2011 doi: 10.1371/journal.pone.0015763	pKD46 with Ptet, I-SceI
recombinant DNA reagent	pWRG100	Karimova et al., 1998 doi: 10.1073/pnas.95.10.5752 doi: 10.1371/journal.pone.0015763	pKD3 with I-SceI recognition site

Share this article

Cite this article

NirD can limit the RelA-dependent accumulation of (p)ppGpp.

Figure 1—source data 1

Figure 1—source data 2

NirD inhibits accumulation of (p)ppGpp upon amino acid starvation.

Figure 2—source data 1

Figure 2—source data 2

NirD can inhibit RelA-dependent accumulation through physical interaction in vivo.

Figure 3—source data 1

Figure 3—source data 2

Figure 3—source data 3

Figure 3—source data 4

NirD can interact with the catalytic N-terminal region of RelA.

Figure 4—source data 1

NirD directly interact with the catalytic N-terminal region of RelA.

Figure 5—source data 1

Figure 5—source data 2

Figure 5—source data 3

NirD inhibits RelA activity in vitro.

Figure 6—source data 1

Figure 6—source data 2

Figure 6—source data 3

Author details

Loïc Léger

Contribution

Competing interests

Deborah Byrne

Contribution

Competing interests

Paul Guiraud

Contribution

Competing interests

Elsa Germain

Contribution

Competing interests

Etienne Maisonneuve

Contribution

For correspondence

Competing interests

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Categories and tags

Research organism

Further reading