Anaïs Brosse

Anne Walburger

Axel Magalon

Maude Guillier
email: maude.guillier@ibpc.fr

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. They mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis.

In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent controls. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level.

In contrast, the second control requires only the very 5' end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of posttranscriptional regulation mechanisms in the synthesis of TCSs.

INTRODUCTION

Bacteria have the ability to sense, respond and adapt to a wide diversity of environments and their capacity to regulate gene expression plays a key role in this process. Examples of control have been reported at multiple steps of gene expression. Transcription initiation is, for instance, commonly regulated by proteins that bind to the promoter regions of genes and can activate or repress their transcription [START_REF] Browning | Local and global regulation of transcription initiation in bacteria[END_REF]. Regulators falling in this category are involved in the response to diverse input signals, often via a change in their activity in response to a cognate signal, by phosphorylation in the case of two-component systems (TCS). TCSs are widely used in bacteria and consist of a sensor kinase that can auto-phosphorylate in response to specific stimuli and transfer the phosphate group to a cognate response regulator (RR). In most cases, the phosphorylated form of the RR is the biologically active form and regulates transcription by binding to DNA.

While undoubtedly beneficial for bacterial adaptation, transcriptional regulation is not the only form of control. Indeed, many bacterial genes can also be regulated at the post-transcriptional level and, in these cases, translation is often the regulated step. Although translational control by proteins was described many decades ago, the observation that a myriad of small RNAs, most of which acting as posttranscriptional regulators, exist in virtually all bacteria has confirmed the importance of posttranscriptional control in bacterial adaptation. In the vast majority of cases, sRNAs act by pairing to target-mRNAs via imperfect base-pairing interactions and repress, or more rarely increase, their translation and/or stability [START_REF] Hör | Trans-Acting Small RNAs and Their Effects on Gene Expression in Escherichia coli and Salmonella enterica[END_REF]. Based on the examples studied so far, the most common scenario is that sRNAs of this category pair at or in the vicinity of the ribosome binding-site of their target and repress translation initiation by directly competing with binding of the 30S ribosomal subunit. Several other mechanisms have been described, however, including translation activation or repression by sRNAs pairing outside of the translation initiation region (TIR), or the stabilization or destabilization of target-mRNAs as a direct consequence of sRNAs binding [START_REF] Jørgensen | sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms[END_REF][START_REF] Carrier | Broadening the Definition of Bacterial Small RNAs: Characteristics and Mechanisms of Action[END_REF]. In enteric bacteria such as E. coli and Salmonella, for which many of the details of sRNAs action have been elucidated so far, these imperfectly pairing sRNAs require an RNA chaperone, Hfq or the more recently identified ProQ, for stability and duplex formation with their targets [START_REF] Kavita | New aspects of RNA-based regulation by Hfq and its partner sRNAs[END_REF][START_REF] Holmqvist | RNA-binding activity and regulatory functions of the emerging sRNA-binding protein ProQ[END_REF]. Consistent with this, Hfq is involved in the regulation of a multitude of genes whose expression is under the control of sRNAs. Furthermore, Hfq has also been shown to be involved in the direct control of gene expression, independently of sRNAs [START_REF] Chen | Hfq links translation repression to stress-induced mutagenesis in E. coli[END_REF][START_REF] Ellis | Hfq binds directly to the ribosome-binding site of IS10 transposase mRNA to inhibit translation[END_REF].

Interestingly, transcriptional and post-transcriptional controls do not form completely independent regulatory networks in bacterial cells but rather result in mixed regulatory circuits relying on both proteins and sRNAs acting mostly at the transcriptional and post-transcriptional level, respectively [START_REF] Brosse | Bacterial Small RNAs in Mixed Regulatory Networks[END_REF][START_REF] Nitzan | Integration of Bacterial Small RNAs in Regulatory Networks[END_REF]. Transcription of sRNAs is most often controlled by transcriptional regulators, while sRNAs in turn post-transcriptionally regulate the synthesis of transcriptional regulators. One example is the stress response alternative sigma factor RpoS, which directs transcription of SdsR and SdsN sRNAs in enterobacteria [START_REF] Fröhlich | A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD[END_REF][START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF], while RpoS synthesis is directly up-regulated by at least three sRNAs, namely DsrA, RprA and ArcZ [START_REF] Majdalani | DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription[END_REF][START_REF] Majdalani | Regulation of RpoS by a novel small RNA: the characterization of RprA[END_REF][START_REF] Majdalani | Regulation and mode of action of the second small RNA activator of RpoS translation, RprA[END_REF][START_REF] Mandin | Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA[END_REF]. This control of, and by, sRNAs is also true for regulators of TCSs, including OmpR and PhoP, two of the most studied response regulators in enterobacteria, as well as the LuxO RR involved in quorum-sensing in Vibrio species [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF][START_REF] Coornaert | Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB[END_REF][START_REF] Guillier | Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs[END_REF][START_REF] Guillier | The 5' end of two redundant sRNAs is involved in the regulation of multiple targets, including their own regulator[END_REF][START_REF] Westermann | Dual RNAseq unveils noncoding RNA functions in host-pathogen interactions[END_REF][START_REF] Lenz | The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae[END_REF][START_REF] Tu | Negative feedback loops involving small regulatory RNAs precisely control the Vibrio harveyi quorum-sensing response[END_REF].

NarP is another example of a RR whose synthesis is under sRNA control. Together with its paralog NarX-NarL, the NarQ-NarP TCS regulates the expression of genes involved in the anaerobic respiration on nitrate, the energetically most favourable electron acceptor in the absence of oxygen, and on nitrite, the reduction product of nitrate [START_REF] Stewart | Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli[END_REF]. The translation of narP mRNA is repressed by SdsN137, one isoform of a set of RpoS-dependent sRNAs [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. In addition to this control, we report here that expression of narP is also regulated by Hfq and the RprA sRNA, whose synthesis is primarily controlled by the Rcs phosphorelay, but also responds to the CpxAR TCS and the LrhA regulator of flagellar synthesis [START_REF] Majdalani | Regulation and mode of action of the second small RNA activator of RpoS translation, RprA[END_REF][START_REF] Vogt | The Cpx envelope stress response regulates and is regulated by small noncoding RNAs[END_REF][START_REF] Peterson | LrhA regulates rpoS translation in response to the Rcs phosphorelay system in Escherichia coli[END_REF]. RprA regulates expression of multiple genes involved in diverse stress responses, biofilm formation, formate metabolism, conjugation as well as the gene encoding LrhA, one of its transcriptional regulator [START_REF] Majdalani | Regulation of RpoS by a novel small RNA: the characterization of RprA[END_REF][START_REF] Mika | Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli[END_REF][START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF][START_REF] Lalaouna | Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli[END_REF]. By adding narP to the list of RprA and Hfq targets, our results expand the connections between sRNAs and TCSs, two classes of widespread bacterial regulators, and highlights the high level of integration of the diverse pathways that regulate gene expression. In addition, they demonstrate a dual level of control by a single sRNA, acting both canonically at the translation initiation region (TIR) of narP and, via a distinct sRNA site, and most likely indirectly, at the distant 5'end of the same mRNA.

MATERIALS AND METHODS

Strains, plasmids and general microbiology techniques

All strains and plasmids used in this study are listed in Supplementary Table S1. Cells were grown at 37°C, either in LB medium or in a defined medium (MMGly) composed of potassium phosphate buffer (100 mM) adjusted to pH 7.4, ammonium sulfate (15 mM), sodium chloride (9 mM), magnesium sulfate (2 mM), sodium molybdate (5 µM), Mohr's salt (10 µM), calcium chloride (100 µM), casaminoacids (0.5 %) and thiamine (0.01 %), supplemented with 140 mM of glycerol as sole carbon source. When needed, nitrate was added at a final concentration of 5 mM. Anaerobic growth was performed in gas tight Hungate tubes under Argon atmosphere. When necessary, antibiotics were used at the following concentrations: 100 or 150µg/ml ampicillin (Amp100 or Amp150 in figure legends), 10µg/ml tetracycline, 25µg/ml or 50µg/ml kanamycin and 10µg/ml chloramphenicol. 100 µM of IPTG was also added when required to induce expression of sRNAs from pBRplac derivatives. Amplification of DNA fragments was performed with either Phusion DNA polymerase or LongAmp DNA polymerase (NEB).

Except for cloning, strains are all derivatives of E. coli MG1655, modified by recombineering or P1 transduction when needed. Mutant rprA::tet (from strain NM667, from N. Majdalani, unpublished) and sdsN::kan (strain GS0762 [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]) were obtained from S. Gottesman's and G. Storz's laboratories, respectively, while the DnarP::kan allele was taken from the Keio collection (strain JW2181, [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF]). For the construction of strains carrying hfq point mutants, a Dhfq::cat-sacB purA::kan mutant (strain DJS2604, from D. Schu) was first transduced into the recipient strain, and the different hfq alleles (from strains DJS2927 (wt), DJS2609 (ΔHfq), KK2561 (R16A), KK2562 (Y25D) or KK2560 (Q8A), from D.

Schu carrying alleles from [START_REF] Zhang | Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets[END_REF][START_REF] Schu | Alternative Hfq-sRNA interaction modes dictate alternative mRNA recognition[END_REF]) were then moved into the resulting strain, allowing selection on glucose minimal medium. The various fusions with the lacZ reporter gene were made as follows: a PCR fragment encompassing the sequence to be placed upstream of lacZ, flanked by homology regions, was recombined into the strain MG1508 (or MG2114 for construction of the PnapF-lacZ fusion), carrying the genes for recombineering on a mini-lambda and where a Ptet-cat-sacB cassette has been introduced upstream of lacZ. Recombinant cells were selected on sucrose-containing medium, checked for chloramphenicol sensitivity and the fusion was sequenced. For fusions whose transcription originates from PnarP or PnapF, an rrnBt2 transcription terminator was introduced upstream, either on the PCR fragment (PnarP), or by recombineering into MG2114 strain carrying an rrnBt2-cat-sacB-lacZ construct (for PnapF promoter fusion). Fusions PnarP-207+50-lacZ and Ptet+1+50narP-lacZ have a mutation in the second codon of lacZ (ACC is changed to AAC) which prevents the formation of an inhibitory structure for translation.

For the insertion of a 3x-Flag (sequence DYKDHDGDYKDHDIDYKDDDDK) just upstream of the narP stop codon, a PCR fragment carrying the 3x-Flag sequence preceded by a linker (protein sequence GAGAGAGA) and followed by a kanamycin resistance gene flanked by FRT sites was amplified from plasmid pSUB11 [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF] with homology regions to the end of narP. This fragment was then recombined into strain MG1433 and checked by sequencing after selection and purification on LB-Kan. A control adding only the linker sequence was constructed as well. This narP-3xFlag -FRT-KanR-FRT allele was then transduced into recipient strains as needed.

The plasmids used to overexpress the different Hfq-dependent sRNAs are mostly from (Mandin & Gottesman, 2010), with the addition of pCsrB (from N. de Lay, unpublished), pMcaS, pMicL and pSdsN137 (from G. Storz, [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF][START_REF] Jorgensen | Dual function of the McaS small RNA in controlling biofilm formation[END_REF][START_REF] Guo | MicL, a new sigmaEdependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein[END_REF]) and pCpxQ and pDapZ (this study, based on [START_REF] Chao | A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response[END_REF][START_REF] Chao | An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs[END_REF]; see Fig. S1 for Northern-blot validation of their overexpression). Mutant plasmid pSdsN137-1 [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF] was obtained from G. Storz and pRprAmut2 was constructed by amplification of the pRprA plasmid with mutagenic primers using the Pfu enzyme (Agilent), followed by DpnI digestion and transformation into the cloning strain NEB5-alpha F'I q . Table S2 summarizes the main oligonucleotides used in this study to construct strains or plasmids.

Measure of β-galactosidase activity

Cells were diluted 250-fold into fresh medium from an overnight culture and grown to mid-exponential phase and β-galactosidase activity was measured following Miller's protocol [START_REF] Miller | A Short Course in Bacterial Genetics[END_REF]. Cells were lysed either with toluene (for aerobic extracts) or SDS-chloroform (anaerobic extracts). Results presented here correspond to the average of at least two independent experiments and error bars correspond to the standard deviation.

RNA extraction and northern-blot analysis

The RNAs were extracted with hot phenol as previously described [START_REF] Guillier | Remodelling of the Escherichia coli outer membrane by two small regulatory RNAs[END_REF] from the same cultures as those used for b-galactosidase assays. A constant amount of total RNA (between 3.5 and 14 µg) was loaded on 8% urea acrylamide gel in 1X TBE and transferred to a nitrocellulose membrane (GE Healthcare).

For detection, we used specific 5 ' end biotinylated probes (see Table S2 for sequence), hybridized to the membrane overnight in Ultrahyb buffer (Ambion). After two washes in SSC 2X-SDS 0.1% and two washes in SSC 0.1X-SDS 0.1% at 42°C, the membrane was incubated in nucleic acid detection blocking reagent (ThermoScientific), and then in the same solution in presence of a streptavidin-alkaline phosphatase conjugate (Life Technologies). Membrane was then washed three times in wash buffer (Na2HPO4 29mM, NaH2PO4 8.5mM, NaCl 34mM and SDS 0.05%), equilibrated in assay buffer (NaCl 0.1M, Tris 0.1M pH 9.5) and chemiluminescence was detected using the CDP-star substrate (Applied Biosystems).

Western blot and Phos-tag electrophoresis

The whole procedure for protein extraction, gel electrophoresis and western-blot detection from Phostag containing gels was performed as described previously in [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF], except that precast gels containing Phos-tag and Zn 2+ (Wako) were used. For standard western blots, proteins were separated on precast TGX gels (Biorad). A monoclonal anti-Flag antibody conjugated to alkaline phosphatase (Sigma) was used for the detection of the flagged proteins, with CDP-star reagent to detect chemiluminescence.

RESULTS

Hfq controls the expression of several Escherichia coli response regulators

As mentioned above, several examples of genes encoding regulators of TCSs whose expression is post-transcriptionally regulated by Hfq-dependent sRNAs have been reported. TCSs are widespread in bacteria and more than 30 RR genes exist in the model bacterium E. coli for instance. This raises the possibility that other RRs could be subject to sRNA control. We have begun to address this question by using lacZ reporter fusions to follow the production of 11 E. coli regulators of TCSs (listed in Table 1).

RRs such as OmpR, PhoP or NarP, whose control by sRNAs has been previously described, have been included in this set. It is worth noting that, in agreement with their control by sRNAs, their mRNAs were found deregulated in an hfq null strain and/or enriched following Hfq co-immunoprecipitation [START_REF] Sittka | Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq[END_REF][START_REF] Zhang | Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets[END_REF][START_REF] Holmqvist | Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo[END_REF]. The other RRs included in this study set were BaeR, EvgA, HprR, KdpE, RstA and UvrY, for which various indications of sRNA control have been reported, as well as BasR and PhoB, for which no indication for sRNA control is available to our knowledge (Table 1).

Translational fusions of lacZ to these 11 regulators were constructed at the lacZ locus by recombineering (Fig. 1A). All fusions followed the same general organization. First, their transcription was systematically driven by a constitutively expressed Ptet promoter to exclude regulation at the promoter level. Second, the sequence of each RR gene covering the 5' UTR and all of the coding region with the exception of the stop codon was placed in frame upstream of lacZ starting at the 10 th aminoacid of b-galactosidase; for ompR and phoP however, only the 10 first aa of the coding sequence were included in the fusions as this was previously shown to be sufficient to allow regulation by sRNAs [START_REF] Majdalani | DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription[END_REF][START_REF] Majdalani | Regulation of RpoS by a novel small RNA: the characterization of RprA[END_REF]. Expression of these different fusions was then measured in an hfq + or hfq -background, with the idea that a Hfq effect could indicate potential regulation by sRNAs. Note however that genes whose expression is not affected by Hfq in this experiment could nonetheless be regulated by sRNAs, for instance because these sRNAs are expressed in different experimental conditions than those used here or are Hfq-independent, or because the fusion is not a good reporter in these cases.

Consistent with previous data [START_REF] Coornaert | MicA sRNA links the PhoP regulon to cell envelope stress[END_REF], expression of the phoP fusion was up-regulated almost 2fold in the hfq mutant, even if it is unclear at this stage whether this is due to sRNA control or not. In contrast, expression of ompR, also subject to negative regulation by sRNAs, namely OmrA and OmrB [START_REF] Majdalani | Regulation of RpoS by a novel small RNA: the characterization of RprA[END_REF], was down-regulated 1.5-fold in the absence of Hfq, possibly indicating the existence of sRNAs that can activate ompR expression. Expression of baeR, basR, evgA, kdpE, rstA and uvrY fusions was not significantly changed, while that of phoB and hprR was slightly repressed, 1.6-and 1.4-fold, respectively. The strongest Hfq effect was observed on the narP fusion, since its activity was increased almost 3-fold in the hfq deletion strain. Interestingly, the expression of the narP RR gene was previously shown to be repressed at the post-transcriptional level by SdsN137, one isoform of a set of Hfq-dependent sRNAs involved in nitrogen metabolism [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. However, SdsN sRNAs accumulate mostly in stationary phase as their transcription is dependent on the RpoS sigma factor and the level of SdsN137 is thus not expected to be maximum under the conditions used here. This makes it unlikely that the observed Hfq effect on narP is completely explained by the loss of regulation by SdsN137 in the hfq mutant (see also below), and suggests that other Hfq-dependent sRNAs might regulate narP expression.

narP expression is modulated by several sRNAs

To identify these putative other sRNAs that might regulate narP, we made use of a plasmid library allowing overexpression of most Hfq-dependent sRNAs known to date. This library was initially created by Mandin & Gottesman [START_REF] Mandin | Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA[END_REF], and was completed for the present study with plasmids overexpressing McaS [START_REF] Jorgensen | Dual function of the McaS small RNA in controlling biofilm formation[END_REF], MicL [START_REF] Guo | MicL, a new sigmaEdependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein[END_REF], SdsN137 [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF], CpxQ [START_REF] Chao | A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response[END_REF], DapZ [START_REF] Chao | An atlas of Hfq-bound transcripts reveals 3' UTRs as a genomic reservoir of regulatory small RNAs[END_REF], as well as the non Hfqbinding CsrB [START_REF] Babitzke | Regulation of translation initiation by RNA binding proteins[END_REF] sRNAs. The activity of the Ptet-narP-lacZ fusion was thus measured in presence of all plasmids of the library (Fig. 2). The narP fusion is the same as that used in Table 1; it carries a 150 nt-long 5' UTR that was chosen because this 5' end was the most enriched after treatment with an exonuclease degrading 5'-monophosphate containing RNAs, and thus likely corresponds to the major narP TSS [START_REF] Thomason | Global Transcriptional Start Site Mapping Using Differential RNA Sequencing Reveals Novel Antisense RNAs in Escherichia coli[END_REF]. Four sRNAs had an effect greater than 2-fold: one positively (ChiX) and three negatively (DicF, RprA and SdsN137). Based on previous studies, the effect of ChiX is most probably due to a titration of Hfq [START_REF] Moon | Competition among Hfq-binding small RNAs in Escherichia coli[END_REF][START_REF] Hussein | Disruption of small RNA signaling caused by competition for Hfq[END_REF]. For the repressing sRNAs, the effect of SdsN137 was expected and is in full agreement with the above-mentioned results [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. DicF overproduction led to a strong growth defect and thus the possible repression of narP by DicF was not investigated further. The third repressing sRNA was RprA, whose transcription is under strong control by the Rcs phosphorelay [START_REF] Majdalani | Regulation and mode of action of the second small RNA activator of RpoS translation, RprA[END_REF]. RprA is known to activate the synthesis of the alternative sigma factor σ S and of the RicI protein that inhibits conjugation [START_REF] Majdalani | Regulation of RpoS by a novel small RNA: the characterization of RprA[END_REF][START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF]. Several negative targets of RprA have also been previously described: this sRNA represses expression of the csgD and ydaM (dgcM) genes involved in biofilm formation [START_REF] Mika | Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli[END_REF] and of hdeD, encoding an acid resistance protein [START_REF] Lalaouna | Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli[END_REF]. Interestingly for this study, expression of other genes was found to be modulated in response to RprA pulse-overexpression in Salmonella, including narP, which was repressed [START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF].

Because RprA up-regulates RpoS synthesis, one might hypothesize that the pRprA plasmid used in Fig. 2A could promote SdsN137 synthesis by increasing s S levels, and thereby repress expression of the narP-lacZ fusion. However, the pRprA plasmid still regulated the narP fusion to a similar extent in a strain deleted for the chromosomal copy of sdsN and rprA (3.2-fold vs. 2.9-fold repression, Fig. 2B), indicating that its effect is independent of SdsN. We next investigated in more detail the control of narP by RprA.

RprA directly targets the narP mRNA

Hfq-dependent sRNAs typically regulate gene expression by pairing to their targets, and we therefore looked for possible interactions between RprA and the narP mRNA using IntaRNA (Mann, Wright et al. 2017). The result is shown in Fig. 3A: nts 31-69 of RprA can potentially imperfectly basepair to the TIR of narP messenger, from nts 116 to 154 relative to the narP TSS (i.e. nts -35 to +4 relative to narP start codon). Of note, affinity purification and sequencing of RNAs associated with a tagged version of RprA identified narP mRNA as a potential direct target, although not among the best candidates [START_REF] Lalaouna | Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli[END_REF].

The RprA-narP mRNA predicted interaction (Fig. 3A) was experimentally tested by introducing mutations in RprA and/or in the previously described narP-lacZ fusion. Four possible bps adjacent or inherent to the Shine-Dalgarno (SD) sequence were disrupted by mutating either nts [START_REF] Batchelor | Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system[END_REF][START_REF] Wang | The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite[END_REF][START_REF] Darwin | Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro[END_REF][START_REF] Darwin | Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site[END_REF] of RprA from UGAG to ACUC (RprAmut2) or nts 137 to 140 of narP-lacZ from CUCA to GAGU (narPmut2). RprAmut2 abolished narP control, even though the sRNA accumulates to a level similar to that of the wt (Fig. 3B) while the mut2 change in narP-lacZ reduced control by RprA from 2.8-to 1.6-fold. A likely explanation for this residual control is proposed later in this study. Although the activity of the mutant fusion was strongly decreased, presumably because the mutation reduces the strength of the SD sequence, restoring the pairing by combining these compensatory changes restored control (2.7-fold repression, i.e. similar to the 2.8-fold repression of the wt fusion by wt pRprA), demonstrating that RprA sRNA directly pairs to narP mRNA in vivo. Since the validated interaction includes the Shine-Dalgarno sequence, RprA most likely inhibits narP translation initiation by preventing 30S ribosomal subunit binding to this mRNA.

Overexpression of RprA or SdsN137 decreases NarP protein levels

In most cases, the active form of RRs is the phosphorylated form. However, it has been shown that robustness exists in TCS signaling and that changes in the total levels of a response regulator such as OmpR for instance, either by using an inducible heterogenous promoter or negative control by OmrA/B sRNAs, does not necessarily change the absolute levels of the phosphorylated form [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF][START_REF] Batchelor | Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system[END_REF].

We thus wondered whether controlling narP synthesis by RprA and SdsN137 sRNAs would affect the levels of the phosphorylated form of the NarP RR (NarP-P). For this purpose, a tagged version of the NarP protein was constructed, where a 3xFlag sequence was added at its C-terminus after a short linker. This construction replaces the narP chromosomal copy. The biological activity of this tagged version of NarP was then assessed by measuring its ability to activate transcription from the napF promoter (Fig. 4A).

The E. coli napFDAGHBC operon encodes the periplasmic nitrate reductase (and its accessory proteins) required for nitrate respiration in the presence of low concentration of this substrate [START_REF] Wang | The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite[END_REF]. Expression of the napF operon is induced by FNR and NarP-P, and repressed by NarL, as binding of NarL-P, the phosphorylated form of NarL to the napF promoter prevents binding of NarP-P. Because NarP and NarL are preferentially phosphorylated under low and high nitrate concentration, respectively, napF expression is expected to be higher under low nitrate conditions [START_REF] Darwin | Fnr, NarP, and NarL regulation of Escherichia coli K-12 napF (periplasmic nitrate reductase) operon transcription in vitro[END_REF][START_REF] Darwin | Nitrate and nitrite regulation of the Fnr-dependent aeg-46.5 promoter of Escherichia coli K-12 is mediated by competition between homologous response regulators (NarL and NarP) for a common DNA-binding site[END_REF][START_REF] Stewart | Dual overlapping promoters control napF (periplasmic nitrate reductase) operon expression in Escherichia coli K-12[END_REF]. napF expression was thus followed by using a transcriptional fusion between the napF promoter (from nts -85 to +19 relative to the TSS from the proximal promoter) and lacZ sequence starting 17 nts before the translation initiation codon. In a preliminary experiment, the activity of this fusion was measured in cells grown under anaerobic conditions at different nitrate concentrations and was found to peak at around 5 mM nitrate (data not shown), in agreement with its control by NarL and NarP. The same minimal medium with glycerol as the sole carbon source and supplemented with 5mM nitrate (hereafter MMGly+Ni) was thus used in the next experiments. As expected, expression of the PnapF-lacZ fusion was strongly decreased in the narP deleted strain (Fig. 4B). Furthermore, expression was partially restored in presence of the NarP-3xFLAG protein, showing that this tagged version of NarP retains biological activity, albeit at a reduced level compared to the wt protein (Fig. 4B).

The levels of NarP and NarP-P were then followed by western-blot using antibodies directed against the FLAG sequence upon overexpression of RprA and SdsN137 sRNAs, either wt or variants that are defective in narP control. The western-blots were performed by separating total proteins on a polyacrylamide gel containing Phos-Tag to allow the separation of NarP-P from the non-phosphorylated form of NarP and directly assess the effect of the sRNAs on the two forms of the protein. As shown in Fig. 4C, overexpression of wt RprA and SdsN137 significantly decreased the level of both forms of the NarP protein. Furthermore, this effect was abolished when we used instead the SdsN137-1 mutant that abolishes pairing to narP [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF], indicating that these changes are due to narP post-transcriptional control by this sRNA. Strikingly however, RprAmut2, i.e. the RprA variant that no longer controls expression of the Ptet-narP-lacZ fusion (Fig. 3B), was still very efficient at decreasing NarP and NarP-P levels (Fig. 4C and Fig. S2). This surprising effect of RprAmut2 is further discussed below (see Fig. 5). Changes in total NarP protein levels were also assessed in the same experiment by Western-Blot from a classical polyacrylamide gel where NarP and NarP-P are not separated; the results are in complete agreement with the Phos-Tag data and confirm the reduction in NarP levels in presence of pRprA, pRprAmut2, pSdsN137, but not pSdsN137-1 (Fig. S2).

Overall, narP repression by RprA or SdsN137 results in a clear decrease of both NarP and NarP-P levels.

Downstream effects of RprA and SdsN137 on NarP targets

Previous work has shown that regulating the synthesis of TCSs may have unexpected outcomes on the expression of their targets [START_REF] Batchelor | Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system[END_REF]. For instance, only one of the two sRNAs that repressed PhoP synthesis also repressed expression of PhoP-activated genes, while control of ompR by sRNAs affected only targets that were sensitive to the non-phosphorylated form of OmpR [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF][START_REF] Coornaert | Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB[END_REF]. It was thus of interest to determine how RprA and SdsN137 control affected NarP-targets. For this purpose, we used the previously described PnapF-lacZ transcriptional fusion and measured its activity upon overproduction of wt or mutant RprA and SdsN137 (Fig. 4D). Consistent with the observed reduction in NarP-P levels, RprA and SdsN137 decreased expression of the PnapF fusion by 4.7-and 3-fold, respectively. In contrast, SdsN137-1 had no effect, which was expected since this mutant no longer controls narP. The RprAmut2 variant still decreased expression of the PnapF fusion by 2.2-fold, which is fully consistent with its intermediary effect on the NarP protein levels (Fig. 4C). In other words, controlling NarP synthesis with RprA and SdsN137 sRNAs can impact NarP-targets, as shown here for napF. However, whether this is systematically true for other NarP targets remains to be determined.

A second, independent, regulation of narP by RprA

The previous results clearly established the direct pairing between RprA and the narP translation initiation region (TIR) (Fig. 3). However, mutating RprA in this pairing region (RprAmut2) fully impaired control of a Ptet-narP-lacZ fusion (Fig. 3B) but, surprisingly, did not completely abolish regulation of narP expression when looking at NarP protein levels and napF transcription (Fig. 4C and4D). This could be explained if the mut2 change does not completely prevent the RprA interaction with narP TIR under some conditions, but we also considered the possibility that another regulation of narP by RprA may explain the residual control by RprAmut2. Because the latter effect was observed when narP was expressed from its own chromosomal locus in cells grown anaerobically in the presence of nitrate (Fig. 4), we first envisioned that this second control might target the narP promoter region and/or be observable only in specific experimental conditions.

To get further insight into this question, a narP-lacZ fusion where the Ptet promoter was replaced by the narP promoter was constructed (fusion PnarP-narP-lacZ in Fig. 5A). Interestingly, the residual repression by RprAmut2 was also observed with this fusion, as RprAmut2 decreased its expression by almost 2-fold and wt RprA by more than 6-fold in MMGly+Ni under anaerobic conditions (Fig. 5B).

We then used a fusion containing again the narP promoter but only the first 50 nts of the transcribed region of narP upstream of a lacZ gene carrying its own TIR (fusion PnarP-207+50-lacZ in Fig. 5A). Even though the previously demonstrated interaction site with RprA is completely absent from this fusion, its expression was nonetheless found to be repressed to a similar extent by both wt RprA and RprAmut2

(2.4-and 2.5-fold repression, respectively, Fig. 5B). Thus, RprA also inhibits narP expression by acting either on its promoter or its early 5' UTR, and this second control does not require the narP TIR. To determine whether this was observed only under anaerobic conditions in the presence of nitrate, the same experiment was repeated in aerobically-grown cells in MMGly and in LB medium (Fig. 5C, left panel). In both media, RprA and RprAmut2 repressed expression of the PnarP-207+50-lacZ fusion to a similar extent: about 2.7-fold in MM+Gly and about 2-fold in LB. Hence, this second control of narP by RprA is not specific to anaerobic growth in the presence of nitrate. To explore this phenomenon further, we replaced the narP promoter region with a Ptet promoter (fusion Ptet+1+50narP-lacZ, Fig. 5C, right panel). This did not alleviate control by RprA or RprAmut2, which clearly indicates that only the first 50 nts of the narP 5' UTR are required for this second control by RprA. In addition, the overproduction of several other Hfq-binding sRNAs, including SdsN137, did not repress the expression of this Ptet+1+50narP-lacZ fusion (data not shown), indicating that this effect is specific to RprA.

It is not clear at this stage why this second regulation did not allow control of the Ptet-narP-lacZ fusion by RprAmut2 (Fig. 3B). It is possible that the combination of the experimental settings used in this experiment (aerobic growth in rich medium) with strong transcription from the Ptet promoter and the presence of the two regions of control, i.e. narP TIR and the first 50 nts of narP mRNA, is unfavorable for observing this regulation. Nevertheless, the residual control of the mutant narPmut2-lacZ fusion by the wt pRprA (Fig. 3B) is most likely explained by this second level of control acting on the (+1+50) region of narP mRNA.

Importantly, the effect of RprAmut2 and wt RprA on the shorter PnarP-207+50-and Ptet+1+50narP-lacZ fusions are similar. Thus, this second control most probably relies on a region of RprA that is not affected by the mut2 change, i.e. a different region than the one involved in the pairing to the narP TIR. Like many other sRNAs, RprA exists in vivo both as a full-length primary transcript, and as a shorter, processed form (RprA proc) corresponding to the last 47 nts of the sRNA [START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF][START_REF] Argaman | Novel small RNA-encoding genes in the intergenic regions of Escherichia coli[END_REF]. Because RprAmut2 was as efficient as the wt in repressing expression of the fusions carrying only the first 50 nts of narP mRNA, we wondered whether this second control of narP could also be achieved by RprA proc. This would be consistent with the observation that RprAproc repressed narP in Salmonella even though this short version of the sRNA is devoid of the region interacting with narP TIR [START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF]. The activity of the fusions containing only the first 50 nts of narP mRNA was thus measured in presence of a plasmid overexpressing only the processed version of RprA (Fig. 5C): RprAproc was as efficient as wt RprA, and even more efficient in some cases, to control expression of these two fusions.

Together, these data clearly show that, in addition to the direct pairing of nts 30 to 70 of RprA to narP TIR, this sRNA also represses narP via a second action involving only the first 50 nts of narP mRNA and the 47 last nts of the sRNA.

The Hfq effect on narP-lacZ fusion may be only partially due to sRNA regulation

As described above, we initially focused on the regulation of narP by sRNAs because narP expression was derepressed by about 3-fold in an hfq -background (Fig. 1). Interestingly however, a similar increase in narP expression was observed in the hfq mutant in the absence of either rprA or sdsN genes, or in the double mutant, clearly showing that the Hfq effect is largely independent of these two sRNAs (Fig. 6A). This suggests that other Hfq-dependent sRNAs, not represented in the library used in Fig. 2, could negatively regulate narP expression. Another possibility is that, in addition to being involved in RprA and SdsN action, Hfq could also directly modulate narP expression, this time independently of sRNAs, as shown for the mutS mRNA [START_REF] Chen | Hfq links translation repression to stress-induced mutagenesis in E. coli[END_REF]. To discriminate between these two (nonexclusive) possibilities, we made use of point mutants of Hfq that have been previously described.

Hfq forms a ring-shaped homohexamer that presents different surfaces involved in RNA binding: the proximal, distal, lateral faces and the C-terminal tail. In enterobacteria, several studies pointed to a major role of the proximal face in binding and stabilizing most sRNAs through interaction with the polyU stretch of the terminator, while the distal face displays a preference for (AAN) triplets, found in mRNAs and some sRNAs. The lateral face also participates to the binding of sRNAs or mRNAs via an arginine patch that can interact with UA-rich RNA sequences [START_REF] Zhang | Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets[END_REF][START_REF] Peng | Structural model of an mRNA in complex with the bacterial chaperone Hfq[END_REF][START_REF] Dimastrogiovanni | Recognition of the small regulatory RNA RydC by the bacterial Hfq protein[END_REF][START_REF] Link | Structure of Escherichia coli Hfq bound to polyriboadenylate RNA[END_REF][START_REF] Schu | Alternative Hfq-sRNA interaction modes dictate alternative mRNA recognition[END_REF][START_REF] Updegrove | Hfq: the flexible RNA matchmaker[END_REF]. The expression of the Ptet-narP-lacZ translational fusion was thus measured in point mutants affecting each of three surfaces: Q8A (proximal face), Y25D (distal face) and R16A (lateral face) [START_REF] Zhang | Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets[END_REF]. Q8A and R16A mutants had no effect on the expression of the fusion, while the Y25D change caused a 2.7-fold increase, i.e. similar to the effect of the hfq null allele (Fig. 6B). These data suggest that, as for mutS, narP expression is not only subject to sRNA control, but is also likely directly controlled by Hfq. Since phoP expression was found to be up-regulated in the absence of Hfq, independently of the two known Hfq-dependent sRNAs that repress phoP, i.e. MicA and GcvB [START_REF] Coornaert | Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB[END_REF], the same set of mutants was tested with the Ptet-phoP-lacZ fusion. In this case, the Q8A mimicked the effect of the Δhfq mutation, while R16A and Y25D had no effect, consistent with the hypothesis that phoP expression is regulated by Hfq-dependent sRNAs that remain to be identified.

DISCUSSION

A new connection between sRNAs and nitrate metabolism

Enterobacteria such as Escherichia coli and Salmonella are facultative anaerobes and often encounter low oxygen conditions, e.g. in the gut of mammalian hosts. Their ability to respire on nitrate or nitrite, which are efficient alternative electron acceptors to oxygen, is certainly an advantage under anaerobic conditions and this has been linked to efficient colonization [START_REF] Jones | Anaerobic Respiration of Escherichia coli in the Mouse Intestine. Fang FC[END_REF] and competitive growth in mice large intestine during the host inflammatory response, which generates nitrate [START_REF] Winter | Host-derived nitrate boosts growth of E. coli in the inflamed gut[END_REF]. Several transcriptional regulators ensure control of gene expression in response to the availability of different electron acceptors. In particular, FNR and ArcB-ArcA both respond to anaerobiosis, with the oxidation status of quinones being involved in signaling to ArcB-ArcA. The NarX-NarL and NarQ-NarP TCSs are involved in the response to nitrate and nitrite. While the NarQ sensor phosphorylates both the NarP and NarL RR in response to nitrate and nitrite, NarX preferentially phosphorylates NarL in response to nitrate mostly [START_REF] Rabin | Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate-and nitrite-regulated gene expression in Escherichia coli K-12[END_REF][START_REF] Noriega | Asymmetric cross-regulation between the nitrate-responsive NarX-NarL and NarQ-NarP two-component regulatory systems from Escherichia coli K-12[END_REF]. NarL and NarP regulons partially overlap and include genes for enzymes required for nitrate/nitrite respiration, among them the nitrate reductases NarGHJI (membrane-bound) and NapFDAGHBC (periplasmic), and the nitrite reductases NirBDC and NrfABCDEFG. Other members of the NarP and the NarL regulons, e.g. the hcp-hcr operon, are involved in the response to nitrogen stress and NO detoxification following nitrate respiration [START_REF] Constantinidou | A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth[END_REF][START_REF] Browning | Transcription activation by remodelling of a nucleoprotein assembly: the role of NarL at the FNR-dependent Escherichia coli nir promoter[END_REF][START_REF] Browning | The Escherichia coli K-12 NarL and NarP proteins insulate the nrf promoter from the effects of integration host factor[END_REF][START_REF] Filenko | Transcriptional regulation of a hybrid cluster (prismane) protein[END_REF]. The identification of narP as one of the direct targets of RprA suggests that it may be advantageous for the cell to limit NarP levels under conditions where RprA is expressed, such as cell surface stress that would signal to the Rcs phosphorelay [START_REF] Wall | The Complex Rcs Regulatory Cascade[END_REF]. In line with this, transcriptomic analyses of strains lacking the RcsB regulator identified several genes related to nitrogen metabolism, e.g. napAB, nirBDC, narK or narGH, as repressed by the Rcs system in several bacteria [START_REF] Oshima | Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12[END_REF][START_REF] Meng | Transcriptomic analysis reveals the role of RcsB in suppressing bacterial chemotaxis, flagellar assembly and infection in Yersinia enterocolitica[END_REF].

Reducing NarP levels could facilitate the activation of NarL by NarX or NarQ; this would be true for control by either RprA or SdsN137. Such a precise balance between NarP and NarL functions may be important for a proper response to different nitrate concentrations; for example, NarX-NarL has been found to be more important for intestine colonization than NarQ-NarP, possibly because it corresponds to relatively high nitrate conditions where the NarGHI reductase, whose expression is activated by NarL, plays a key role [START_REF] Jones | Anaerobic Respiration of Escherichia coli in the Mouse Intestine. Fang FC[END_REF]. In this regard, it is interesting to note that SdsN137 levels vary in response to some nitrogen sources [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. Even though RprA and SdsN137 production may not peak under the same conditions, the two sRNAs can be co-expressed, for example in stationary phase where they are both detected [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF][START_REF] Argaman | Novel small RNA-encoding genes in the intergenic regions of Escherichia coli[END_REF][START_REF] Wassarman | Identification of novel small RNAs using comparative genomics and microarrays[END_REF], which could contribute to stronger reduction of narP expression under such conditions.

Interestingly for this study, narL could also be targeted by sRNAs as its mRNA levels were shown to be decreased upon overexpression of DicF, an sRNA that accumulates under micro-aerobic conditions [START_REF] Melson | The sRNA DicF integrates oxygen sensing to enhance enterohemorrhagic Escherichia coli virulence via distinctive RNA control mechanisms[END_REF], and that was identified here as a possible regulator of narP as well (Fig. 2). Furthermore, NarL also promotes synthesis of the NarS sRNA, which is processed from the NarL-activated narK mRNA, encoding a nitrate/nitrite antiporter [START_REF] Wang | The conserved 3′ UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration[END_REF]. In turn, NarS represses the expression of the gene encoding the nitrite transporter NirC. Although this NarS-control does not affect the expression of the other genes of the nirBDC-cysG operon, the synthesis of the NirB subunit of the NADH-dependent nitrite reductase is also subject to sRNA control, in this case by the RyhB sRNA [START_REF] Wright | Comparative genomics boosts target prediction for bacterial small RNAs[END_REF]. The link between sRNAs and nitrogen metabolism is further illustrated by the fact that SdsN137 represses the synthesis of the NfsA nitroreductase and the HmpA nitric oxide dioxygenase [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. Additionally, several global approaches looking at ProQ or Hfq targets, or at RNA-RNA interactions mediated by these chaperones, indicate that yet other genes related to nitrate/nitrite metabolism are likely to be controlled by sRNAs in enterobacteria [START_REF] Sittka | Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq[END_REF][START_REF] Melamed | Global Mapping of Small RNA-Target Interactions in Bacteria[END_REF][START_REF] Melamed | RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles[END_REF].

Lastly, connections between sRNAs and nitrogen are not restricted to enterobacteria and many other examples have been reported in extremely diverse bacteria as well, including the sRNAs related to denitrification in Pseudomonas aeruginosa or Paracoccus denitrificans [START_REF] Tata | The Anaerobically Induced sRNA PaiI Affects Denitrification in Pseudomonas aeruginosa PA14[END_REF][START_REF] Gaimster | A Central Small RNA Regulatory Circuit Controlling Bacterial Denitrification and N2O Emissions[END_REF], the RoxS sRNA that responds to nitric oxide in firmicutes [START_REF] Durand | A nitric oxide regulated small RNA controls expression of genes involved in redox homeostasis in Bacillus subtilis[END_REF] and the sRNAs involved in the control of carbon-nitrogen balance in cyanobacteria [START_REF] Muro-Pastor | Regulatory RNA at the crossroads of carbon and nitrogen metabolism in photosynthetic cyanobacteria[END_REF].

New connections between Hfq and two-component systems

This study adds RprA to the list of sRNAs that include genes for TCSs in their regulons.

Previous work has shown that altering the levels of transcriptional regulators with sRNAs may not always lead to a change in their activity, especially in the case of TCS regulators that must be activated by phosphorylation. In particular, the EnvZ-OmpR TCS was found to be robust, i.e. the level of OmpR-P, the phosphorylated form of OmpR, is insensitive to large changes in total EnvZ or OmpR levels [START_REF] Batchelor | Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system[END_REF].

Consistent with this, repressing ompR expression with OmrA and OmrB sRNAs decreased only the amount of the non-phosphorylated form of OmpR, which allowed these sRNAs to indirectly limit their own synthesis as their transcription responds to both the phosphorylated and the non-phosphorylated forms of OmpR [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF]. Although different, the outcomes of controlling PhoP synthesis with MicA or GcvB sRNA were also surprising: of these two sRNAs that repressed phoP via competition with ribosome binding, only MicA decreased the expression of positive PhoP-targets [START_REF] Coornaert | Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB[END_REF].

In the case of narP, our results show that repression by RprA or SdsN137 sRNAs decreased the level of both the phosphorylated and the non-phosphorylated form of this RR and, consistently, repressed transcription from the NarP-dependent promoter PnapF. This sRNA effect on the phosphorylated form of the RR differs from what has been observed for ompR and indicates that robustness is not true for all TCS. However, for most of the previously reported cases, genes for RRs whose expression is repressed by sRNAs are in an operon with their cognate sensor kinase genes, leading to the prediction that expression of the kinase would also be repressed by the sRNA. This is different for narP since this is one of the few E. coli RR genes that is not part of an operon. Because RprA does not appear to control narQ (our preliminary data), its control of narP should thus change the RR/SK ratio. It would be interesting to determine whether this explains the observed difference in the levels of phosphorylated forms of NarP and OmpR in response to repression by sRNAs, which are decreased or unaffected, respectively (this study and [START_REF] Brosse | Unexpected properties of sRNA promoters allow feedback control via regulation of a two-component system[END_REF]).

Another conclusion of this study is that, in addition to allowing sRNAs function, Hfq could also play a direct role in controlling TCSs expression. Although more direct experiments are required to definitively show that the effect of the Hfq Y25D mutant on narP is due to a defect in mRNA binding, and that sRNAs are not involved, this nonetheless indicates that narP expression could vary in response to signals that affect Hfq synthesis, stability and/or activity. While a similar direct Hfq effect on phoP seems unlikely at this stage, these data also raise the question of whether other TCSs can be controlled by Hfq independently of sRNAs, as previously shown for mutS [START_REF] Chen | Hfq links translation repression to stress-induced mutagenesis in E. coli[END_REF].

In general, it will be important to determine how these post-transcriptional control mechanisms involving Hfq and/or sRNAs affect the various TCS signaling properties and, especially in the case of NarQ-NarP, the crosstalk with other systems.

Two independent actions of RprA to control narP

This study is also interesting from a mechanistic standpoint, with the finding that a sRNA can repress a single target via two pathways, involving different regions of both the sRNA and the mRNA.

The first, canonical, control relies on the pairing of RprA to the narP TIR, which presumably blocks translation initiation. The second control only requires the first 50 nts of narP mRNA, i.e. a region that is distant from the TIR since narP 5'UTR is 150 nt-long. These two pathways are most likely independent as the 5'end region of narP mRNA is sufficient to observe regulation by RprA in the absence of the TIR (Fig. 5) and, similarly, RprA efficiently represses expression of the PBAD-narP-lacZ fusion used in [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF] that carries a 78-nt 5' UTR and is thus devoid of the 5' end of the narP 5' UTR used here (our unpublished results).

The second control can be performed with similar efficiencies by either the full-length or the processed form of RprA carrying only the last 47 nts of the sRNA. Because we could not predict a convincing base-pairing between the 5'end of narP messenger (narP(+1+50) region) and RprA proc, this second control is likely not due to a direct sRNA-mRNA interaction. Instead, one can envision that RprA, or RprA proc, would control the level or the activity of a factor that would act on the narP(+1+50) region. A first possibility is that this factor mediating the observed control of RprA on narP(+1+50) is another sRNA. Such a scenario would be similar to other regulatory circuits involving sponge RNAs (here, RprA) that can titrate and/or destabilize an sRNA and thereby prevent its action [START_REF] Denham | The Sponge RNAs of bacteria -How to find them and their role in regulating the post-transcriptional network[END_REF][START_REF] Bossi | Competing endogenous RNAs: a target-centric view of small RNA regulation in bacteria[END_REF]. We have already tested a possible role of a few candidates sRNAs in the narP control by RprA. These candidates were sRNAs whose levels vary according to aerobic/anaerobic conditions (FnrS, ArcZ, or NarS), sRNAs predicted to interact with RprA based on RIL-seq data (sRNA from the ariR-ymgC intergenic region), or sRNAs whose expression could be related to that of narP (narP 3'UTR). However, our results so far do not support the involvement of any of these sRNAs in this control (our unpublished data). Although possible factor(s) intervening in the effect of RprA on narP(+1+50) remain to be precisely identified, the dual-control mechanism analyzed here is most likely different from the reported examples of RprA pairing to two distinct sites on a single target, such as csgD [START_REF] Mika | Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli[END_REF] or hdeD [START_REF] Lalaouna | Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli[END_REF].

Another question raised by our results is that of the step at which this second control takes place.

Given that the promoter and the TIR of narP are dispensable for this regulation, it is very unlikely to occur at the level of initiation of narP transcription or translation. However, an 8-aa upstream ORF is predicted in the first 50 nts of narP and its translation could be the target of this second control.

Alternatively, the action on narP(+1+50) could primarily affect the stability of narP mRNA, as previously reported for other bacterial sRNAs, possibly by targeting the very 5' end of mRNAs [START_REF] Ramirez-Pena | The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript[END_REF][START_REF] Obana | Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence[END_REF][START_REF] Durand | sRNA-mediated activation of gene expression by inhibition of 5'-3' exonucleolytic mRNA degradation[END_REF]. The effect could also be transcriptional, for instance if RprA indirectly induced a premature termination of narP transcription.

There are several examples of feed-forward regulatory motifs where sRNAs regulate the expression of a target both directly and indirectly; the indirect effect is often mediated by a transcriptional regulator acting on the target promoter. One such example is the activation of the synthesis of the conjugation-inhibiting protein RicI by RprA that relies both on the pairing of RprA to ricI mRNA and on RprA promoting translation of the RpoS sigma factor that activates ricI transcription [START_REF] Papenfort | Small RNA-based feedforward loop with ANDgate logic regulates extrachromosomal DNA transfer in Salmonella[END_REF]. It is likely that the dual control of narP by RprA reported here will also result in a feed-forward regulatory circuit, although with different details. Identifying the missing clues of this circuit, and in particular the nature of the indirect factor(s) involved and the way it acts, will be crucial to assess how this dual control can precisely impact narP expression.

Regardless of the precise mechanism of this second control, it is clear that the processed version of the sRNA is sufficient to promote it, even though this does not necessarily tell us whether the fulllength or the processed form of RprA is preferentially responsible for this regulation when they are both present in the cell. RprA is not the only sRNA that exists under different isoforms, either because of processing, leading for instance to short forms of ArcZ, SdsR, MicL, RbsZ just to name a few [START_REF] Mandin | Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA[END_REF][START_REF] Melamed | RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles[END_REF][START_REF] Guo | MicL, a new sigmaEdependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein[END_REF][START_REF] Papenfort | Specific and pleiotropic patterns of mRNA regulation by ArcZ, a conserved, Hfq-dependent small RNA[END_REF][START_REF] Fröhlich | The target spectrum of SdsR small RNA in Salmonella[END_REF], or of multiple TSS, which explains the different SdsN species [START_REF] Hao | Protection against deleterious nitrogen compounds: role of sigmaS-dependent small RNAs encoded adjacent to sdiA[END_REF]. In the future, it will be interesting to decipher whether full-length and short forms of other sRNAs can also complement each other in the regulation of some targets, as reported here for RprA and narP. this experiment were AB1018 (wt fusion) and AB1037 (mut2) and are deleted for the chromosomal rprA copy. In parallel, RNA was extracted from strains with the wt fusion and Northern-blot analysis was done to assess the overproduction of RprA, wt or mut2, from pBRplac derivatives, using the RprA probe. SsrA was also probed from the same membrane and used as a loading control.

FIGURES LEGENDSFigure 1 :

 1 FIGURES LEGENDS

Figure 2 :

 2 Figure 2: Hfq-dependent sRNAs modulate the expression of narP, most likely at the post-

Figure 3 :

 3 Figure 3: RprA directly pairs to the translation initiation region of narP mRNA. (A). Predicted

Figure 4 :

 4 Figure 4: RprA and SdsN137 decrease the levels of both the phosphorylated and the non-

Figure 5 .

 5 Figure 5. A second control of narP by RprA relies on the 5' end of narP mRNA and the 3' end of

Figure 6 .

 6 Figure 6. The Hfq effect on narP is largely independent of RprA and SdsN137 sRNAs. (A) The bgalactosidase activity of the Ptet-narP-lacZ fusion was measured in LB medium in hfq + and hfq -

Figure S1 :

 S1 Figure S1: Overproduction of DapZ and CpxQ

Figure S2 :

 S2 Figure S2: RprA and SdsN137 decrease the levels of NarP

TABLE 1

 1

		Number of genes	Indication for sRNA regulation 4
	Two-component system (Kinase-Regulator) 1	whose expression is mutant up/down in the TCS	Deregulation in Dhfq	Interaction with Hfq	Portion of the RR gene (relative to start codon) present on the fusion	5
		(based on [33])
	BaeS-BaeR 2			++ [30]	-30 +720
	(response to membrane	14/8		+ for 3' UTR [32]
	stress)
	BasS-BasR (response to iron)	20/12			-18 +666
	EvgS-EvgA			Slight enrichment upon Hfq	-125 +612
	(antibiotic resistance and acid stress)	2/3		IP, and presence in chimeric fragments with sRNAs [34,35]
	(potassium transport) KdpD-KdpE 2	10/10		++ in DproQ [35] ++ [30]	-39 + 675
	(nitrate metabolism) NarQ-NarP 3	NarP 3/9 NarQ 20/25	+++ [30]	+ [32] +++ [30,31]	-150 +645
	PhoR-PhoB (phosphate metabolism)	1/8			-41 +687
	RstB-RstA (acid stress response)	12/22		fragments with sRNAs [35] Presence in chimeric	-13 +726
	BarA-UvrY	BarA 2/24		Slight enrichment upon Hfq +++ [30]	-44 +654
	(global metabolism)	UvrY 86/37		IP, and presence in chimeric
				fragments with sRNAs [35]
	HprS-HprR 3			++ in only one out of two	+1 +669
	(response to oxidative	6/7		experiments [31]
	stress)
	EnvZ-OmpR			+ [31]
	(response to osmotic	71/54			-35+30
	stress and acid stress)
			++ [30,36]	+++ [30,31]
	PhoQ-PhoP (response to magnesium)	4/27		+ [32] Slight enrichment upon Hfq IP, and presence in chimeric	-36+30
				fragments with sRNAs
				[34,35]

Table 1 : TCS investigated in this study.

 1 unless otherwise indicated, TCSs are expressed from a bicistronic operon with the gene for the response regulator being the first cistron.

1

In the BaeS-BaeR and KdpD-KdpE systems the kinase is the first gene in the operon

The two genes of the NarQ-NarP TCS are encoded by different loci of the genome.

Weak, moderate or strong indications for sRNA regulation in different studies are indicated by +, ++ or +++, respectively.

Positions of the transcription start sites (TSS) were chosen based on[START_REF] Thomason | Global Transcriptional Start Site Mapping Using Differential RNA Sequencing Reveals Novel Antisense RNAs in Escherichia coli[END_REF]; even though baeR and kdpE are not the first gene of their operon, they were introduced downstream of the Ptet promoter in the absence of the baeS or kdpD gene, respectively. Note that the start codon is GUG for kdpE and rstA, and UUG for uvrY.

ACKNOWLEDGMENTS

We thank G. Storz, N. Majdalani, S. Gottesman, P. Mandin, E. Solchaga and J. Plumbridge for providing many strains and plasmids in the course of this study. We are grateful to P. Mandin (LCB) for help with RNA extraction from anaerobically-grown cells and to members of the Magalon's lab, especially L. Pieulle, F. Seduk and S. Bulot for assistance. We thank members of the group at the IBPC for support and discussions, and M. Springer, S. Gottesman, C. Condon and C. Chiaruttini for insightful comments on the manuscript.

FUNDING

This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant agreement No. 818750). Research in the UMR8261 is also supported by the CNRS and the "Initiative d'Excellence" program from the French State (Grant "DYNAMO", ANR-11-LABX-0011).