ARTICLE Received 30 Jan 2014 | Accepted 13 May 2014 | Published 20 Jun 2014 DOI: 10.1038/ncomms5115 OPEN Nitrogen stress response and stringent response are coupled in Escherichia coli Daniel R. Brown1, Geraint Barton2, Zhensheng Pan1, Martin Buck3 & Sivaramesh Wigneshweraraj1 Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria. 1 MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK. 2 Centre for Systems Biology and Bioinformatics, Division of Biosciences, Imperial College London, London SW7 2AZ, UK. 3 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK. Correspondence and requests for materials should be addressed to S.W. (email: [email protected]). NATURE COMMUNICATIONS | 5:4115 | DOI: 10.1038/ncomms5115 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5115 itrogen (N) is an essential element of most macromole- a 1.2 3.5 cules in a bacterial cell, including proteins, nucleic acids N N- and cell wall components. Bacteria can assimilate a variety RO N 1.0 3.0 of N sources and ammonia typically supports the fastest growth, 2.5 serving as the preferred N source for many bacteria including 0.8 E. coli1. During N-limited growth ammonia is converted by NH 2.0 4 glutamine synthetase into glutamine, the primary amine donor Cl (mM) 600nm 0.6 for key amino acid and nucleotide biosynthetic pathways. E. coli OD 1.5 cells respond to N starvation by activating the nitrogen regulation 0.4 (Ntr) stress response, resulting in the expression of B100 genes Growth 1.0 NH Cl N+ to facilitate N scavenging from alternative sources of combined N 4 0.2 0.5 (ref. 2). In enterobacteria, the master transcription regulator of the Ntr stress response is NtrC of the NtrBC two-component 0.0 0.0 system3. N limitation results in the phosphorylation of the 0 50 100 150 200 250 300 response regulator NtrC (product of glnG) by its cognate sensor Time (min) kinase NtrB (product of glnL). N-starved E. coli cells rapidly synthesize ppGpp, the effector b Strain Doubling time (min) 4 WT 49.11 ±0.85 alarmone of the bacterial stringent response . Two enzymes NtrC-3xFLAG 50.86 ±0.91 modulate the levels of ppGpp in E. coli: the ppGpp synthetase ΔglnG 67.74 ±0.92 Δ 5 1.2 glnG::glnGind – IPTG 66.33 ±0.76 RelA and ppGpp synthetase/hydrolase SpoT . RelA and SpoT ΔglnG::glnG + IPTG 49.62 ±2.80 contribute to stress adaptation, antibiotic tolerance, expression of ind virulence traits and the acquisition of persistent phenotype in 1.0 pathogenic bacteria6–9. The regulation of RelA/SpoT enzyme activities has been extensively studied10–13, but the regulatory 0.8 mechanisms that govern transcription of their genes and thereby 600nm 0.6 lead to elevated ppGpp levels during nutritional stresses remain OD elusive. 0.4 Here, we address the links between the Ntr stress response and IPTG added the ppGpp alarmone. To gain insight into NtrC-dependent gene 0.2 networks, we map the genome-wide binding targets of NtrC and RNA polymerase (RNAp) during N starvation in E. coli, using 0.0 chromatin immunoprecipitation followed by high-throughput 0 50 100 150 200 250 300 350 400 450 sequencing (ChIP-seq). Our results reveal that nitrogen stress Time (min) response and stringent response are coupled in E. coli. c 1.2 Results 1.0 Tooling up to study genome-wide binding of NtrC and RNAp. N– To identify genome regions preferentially associated with NtrC, 0.8 we introduced an in-frame fusion encoding three repeats of the 0 FLAG epitope at the 3 end of glnG in E. coli strain NCM3722, a 0.6 prototrophic E. coli K-12 strain. To establish N-starved condi- mRNA levels at tions, we grew batch cultures of the wild-type NCM3722 strain in glnK 0.4 Gutnick minimal medium supplemented with a limiting amount (3 mM) of NH Cl as the sole N source and monitored bacterial 0.2 4 Relative growth as a function of ammonium consumption. In Fig. 1a, the time when wild-type NCM3722 stops growing (t ¼ NRO) coin- 0.0 **** **** cides with the ammonium run out ([ammonium]o0.000625 WT ΔglnG –IPTG +IPTG mM) in the growth medium. To establish that the FLAG epitope ind ind NtrC-3xFLAG lnG had not compromised the activity of NtrC, we measured the glnG doubling times of wild-type NCM3722, NCM3722:glnG-FLAG glnG:: glnG::g and NCM3722:DglnG strains under our growth conditions. The Δ Δ doubling time of the NCM3722:glnG-FLAG strain is close to that Figure 1 | Establishing N-starved growth conditions in E. coli. (a) The of the wild-type NCM3722 strain; however, as expected, the growth arrest of wild-type E. coli NCM3722 cells coincides with ammonium doubling time of the NCM3722:DglnG strain is longer than that run out (at t ¼ NRO) in the minimal Gutnick medium. The time points at of the wild-type NCM3722 strain (Fig. 1b). Further, the expres- which the E. coli cells were sampled for downstream analysis are indicated sion levels of glnK mRNA, an Ntr stress response gene directly (t ¼ N þ and t ¼ N À represents growth under nitrogen replete and starved activated by NtrC, at t ¼ N À (that is, the time-point 20 min after conditions, respectively). (b) The growth curves of wild-type NCM3722, N runs out) are similar in the wild-type NCM3722 and NCM3722:glnG-FLAG (NtrC-3xFLAG), NCM3722:DglnG and NCM3722:glnG-FLAG strains, and as expected is not readily NCM3722:DglnG::glnGind ( À / þ IPTG). The quantitation of the doubling detected in the NCM3722:DglnG strain (Fig. 1c). Complementa- times is also given. (c) Graph showing the relative levels of glnK mRNA tion of the NCM3722:DglnG strain with inducible plasmid-borne expression as fold-change in cells sampled at t ¼ N þ and t ¼ N À . glnG restored both the doubling time and glnK mRNA expression Error bars on all growth curves represent s.d. (where n ¼ 3). Statistical (Fig. 1b,c). In summary, we conclude that (i) FLAG-tag has not significant relationships from One-way ANOVA analysis are denoted adversely affected biological activity of NtrC and (ii) under our (****Po0.0001). experimental conditions t ¼ N À is representative of the time when the E. coli cells are starved for N. 2 NATURE COMMUNICATIONS | 5:4115 | DOI: 10.1038/ncomms5115 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5115 ARTICLE NtrC binds upstream of relA in N-starved E. coli. To identify binding site is located between positions À 838 and À 649 the genomic loci bound by NtrC in E. coli during N starvation, we upstream from the translational start of RelA. Since complex sampled cells for ChIP-seq analysis at t ¼ N À and NtrC-bound formation between NtrC-P and flgN, dicC, fliC, ssrS, soxR and DNA was precipitated with anti-FLAG antibodies. To identify yjcZ probes is not detectable even at a higher concentration of transcriptionally active promoters in E. coli during N starvation, NtrC-P (Supplementary Fig. 2) and the DNA regions are poorly we treated the E. coli cells at t ¼ N À with rifampicin for 15 min enriched by NtrC binding in the ChIP-seq data (Table 1), we (rifampicin inhibits transcription elongation and thus transcrip- consider that flgN, dicC, fliC, ssrS, soxR and yjcZ are unlikely to be tion-initiated RNAp molecules will become trapped at any solely dependent on NtrC. We do not exclude the possibility that functional promoter14,15) and RNAp bound DNA was the upstream regions of flgN, dicC, fliC, ssrS, soxR and yjcZ precipitated with a monoclonal antibody against the b-subunit transcription units contain intrinsically poor NtrC binding sites, of the RNAp. The ChIP-seq data revealed that NtrC bound which function in co-dependence with other transcription factors upstream of at least 21 transcription units at t ¼ N À . RNAp (Supplementary Fig. 2). binding (in the presence and absence of Rif treatment) to promoter regions of all 21 transcription units and differential gene expression analysis (t ¼ N À versus t ¼ N þ ) indicate that NtrC activates relA transcription in N-starved E. coli.To all 21 transcription units are upregulated at t ¼ N À (Table 1, unambiguously establish that relA transcription is activated by Supplementary Figs 1–3). Fourteen of the 21 transcription units NtrC during N starvation, we measured relA mRNA levels in are known members of the NtrC regulon (Table 1)2. However, wild-type NCM3722 and NCM3722:DglnG strains at t ¼ N À none of the seven remaining transcription units (flgMN, dicC, relative to t ¼ N þ . In the wild-type NCM3722 strain, the level of fliC, relA, ssrS, soxR and yjcZ-proP) have been previously relA mRNA expression is B2.2±0.4-fold at t ¼ N À relative to demonstrated to require NtrC for their transcriptional t ¼ N þ , and no detectable increase in the relA mRNA expression regulation.