Alexandra Vergnes

Julie P M Viala

Rabah Ouadah-Tsabet

Bérengère Pocachard

Laurent Loiseau

Stéphane Méresse

Frédéric Barras

Laurent Aussel
email: aussel@imm.cnrs.fr

The iron-sulfur cluster sensor IscR is a negative regulator of Spi1 Type III Secretion System in Salmonella enterica

by bacterial pathogens to modulate their infectivity according to iron and oxygen 50

Summary

Iron-sulfur (Fe-S)-containing proteins contribute to various biological processes, including redox reactions or regulation of gene expression. Living organisms have evolved by developing distinct biosynthetic pathways to assemble these clusters, including ISC (Iron Sulfur Cluster) and SUF (Sulfur mobilization). Salmonella enterica serovar Typhimurium is an intracellular pathogen responsible for a wide range of infections, from gastroenteritis to severe systemic diseases. Salmonella possesses all known prokaryotic systems to assemble Fe-S clusters, including ISC and SUF.

Because iron starvation and oxidative stress are detrimental for Fe-S enzyme biogenesis and because such environments are often met by Salmonella during its intracellular life, we investigated the role of the ISC and SUF machineries during the course of the infection. The iscU mutant, which is predicted to have no ISC system functioning, was found to be defective for epithelial cell invasion and for mice infection, whereas the sufBC mutant, which is predicted to have no SUF system functioning, did not present any defect. Moreover, the iscU mutant was highly impaired in the expression of Spi1 TTSS that is essential for the first stage of Salmonella infection.

The Fe-S cluster sensor IscR, a transcriptional regulator matured by the ISC machinery, was shown to bind the promoter of hilD, which encodes the master regulator of Spi1. IscR was also demonstrated to repress hilD and subsequently Spi1 gene expression, consistent with the observation that an iscR mutant is hyperinvasive in epithelial cells. Collectively, our findings indicate that the ISC machinery plays a central role in Salmonella virulence through the ability of IscR to down-regulate Spi1 gene expression. At a broader level, this model illustrates an adaptive mechanism used

Introduction

Iron-sulfur (Fe-S) cluster-containing proteins play major roles in a wide variety of process, including redox reactions, regulation of gene expression or metabolic pathways [START_REF] Kiley | The role of Fe-S proteins in sensing and regulation in bacteria[END_REF]. Assembly and delivery of Fe-S clusters are catalysed by multi-protein systems, ISC (Iron Sulfur Cluster) and SUF (Sulfur mobilzation), conserved in most living organisms. Briefly, sulfur is mobilized from Lcysteine by IscS/SufSE [START_REF] Hidese | Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors[END_REF], and scaffold proteins (IscU/SufBC) bind both sulfur and iron to assemble a cluster before transferring it to the apo-protein via the A-type carriers IscA/SufA [START_REF] Bonomi | Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA[END_REF][START_REF] Vinella | Ironsulfur (Fe/S) protein biogenesis: phylogenomic and genetic studies of A-type carriers[END_REF]Wollers et al., 2010) (Figure S1). In Escherichia coli and most Gram-negative bacteria, the ISC system is encoded by the iscRSUA-hscBA-fdx operon while the SUF system is encoded by the sufABCDSE operon (Fig. S1). Other so-called "non-ISC non-SUF" proteins intervene at the sulfur production step (CyaY), at the transfer steps (ErpA, NfuA, Mrp), or, possibly, at the repair step (YggX).

In the last decade, several studies reported the importance of ISC and/or SUF systems in sustaining virulence of various bacteria. Indeed, iron starvation and oxidative stress are detrimental for Fe-S enzyme biogenesis, and such environments are often met by bacterial pathogens upon infection of their host. Thus, the SUF and the ISC systems are required for full virulence of the plant pathogen Dickeya dadantii [START_REF] Nachin | SoxRdependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase[END_REF][START_REF] Rincon-Enriquez | Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions[END_REF]. In Shigella flexneri, inactivation of the ISC system prevents plaques formation because the strain is non-invasive [START_REF] Runyen-Janecky | Role and regulation of iron-sulfur cluster biosynthesis genes in Shigella flexneri virulence[END_REF]. The SUF system is essential for Mycobacterium tuberculosis survival and therefore required for virulence [START_REF] Huet | Identification of the Mycobacterium tuberculosis SUF machinery as the exclusive mycobacterial system of [Fe-S] cluster assembly: evidence for its implication in the pathogen's survival[END_REF]. In this context, the case of the iscR gene deserves specific attention.

In E. coli, IscR is a [2Fe-2S]-containing protein whose maturation is primarily catalyzed by the ISC system. The holo-form of IscR acts as a repressor of a large number of genes, including the isc operon. The apo-form of IscR acts as an activator of a few genes, including the suf operon. Hence, under iron limitation or in the presence of reactive oxygen species (ROS), IscR exists primarily under its apo-form that activates expression of SUF and no longer represses expression of isc, the amount of Fe-S clusters produced being at its highest level. As IscR maturation is catalyzed by the ISC system, a defect in this latter leads to the accumulation of apo-IscR and activation of the SUF system. This regulatory connection between ISC and SUF must be taken into account when interpreting the phenotype of ISC-defective strains. For instance, a D. dadantii iscR mutant is non-invasive on Saintpaulia ionantha plants because the SUF system can't be activated by IscR, but it presents a wild-type phenotype on Arabidopsis thaliana presumably because the demand in Fe-S clusters to be satisfied for the bacterium to grow is met by ISC in this case [START_REF] Rincon-Enriquez | Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions[END_REF]. In fact, IscR appears to be important for full virulence of several pathogens. Mutations in iscR reduce virulence of Pseudomonas aeruginosa and Xanthomonas campestris [START_REF] Kim | IscR modulates catalase A (KatA) activity, peroxide resistance and full virulence of Pseudomonas aeruginosa PA14[END_REF][START_REF] Fuangthong | IscR plays a role in oxidative stress resistance and pathogenicity of a plant pathogen, Xanthomonas campestris[END_REF]. Moreover, recent studies have shed new light on the role of IscR by expanding its zone of influence beyond the usual Fe-S cluster containing proteins; namely, in enterotoxigenic Escherichia coli, IscR was shown to bind upstream cfaA and to activate CFA/I fimbriae production in response to iron starvation [START_REF] Haines | IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli[END_REF]. In Yersinia pseudotuberculosis, IscR was found to be involved in the regulation of the Type III secretion system (TTSS) master regulator LcrF and was demonstrated to be essential for the virulence of Yersinia in a mouse model of infection [START_REF] Miller | IscR is essential for yersinia pseudotuberculosis type III secretion and virulence[END_REF].

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that is associated with a wide range of infections in mammals, ranging from self-limiting gastroenteritis to severe systemic diseases. During the first stage of infection, the bacterium invades intestinal epithelial cells using a TTSS encoded by Salmonella Pathogenicity Island 1 (Spi1) [START_REF] Galán | Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells[END_REF]. Afterwards, it survives and replicates in macrophages within a membrane-bound compartment called the Salmonella-containing vacuole (SCV) using a TTSS encoded by Spi2 [START_REF] Figueira | Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors[END_REF]. Synthesis of the Spi1 TTSS apparatus and most of its effectors is controlled by a complex regulatory scheme. HilA activates directly the expression of the inv and prg operons, which encode the TTSS apparatus [START_REF] Ellermeier | HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium[END_REF].

Expression of hilA is under the control of many environmental conditions and three transcriptional activators: HilC, HilD and RtsA [START_REF] Ellermeier | HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium[END_REF].

Among the key points leading to the establishment of infection is the ability of the pathogen to adapt to various environments and to resist different stresses, including iron limitation and ROS production. Recently, CyaY and YggX, both involved in ironsulfur biogenesis, were demonstrated to be important for Salmonella virulence in mice [START_REF] Velayudhan | Distinct roles of the Salmonella enterica serovar Typhimurium CyaY and YggX proteins in the biosynthesis and repair of iron-sulfur clusters[END_REF]. Here, we investigated the involvement of ISC and SUF in Salmonella. Whereas no major role was attributed to the SUF system, the ISC machinery was found to be essential for epithelial cell invasion and we explained this by revealing a molecular link between Fe-S cluster and Spi1 TTSS functioning. In particular, we showed that IscR represses the synthesis of the Spi1-carried regulator HilD, decreasing the expression of genes encoding the TTSS apparatus. Thus, the IscR regulator can be exploited by Salmonella as a sensor to modulate its invasiveness depending on the stage of the infection and the compartment infected.

Results

The ISC machinery is essential for HeLa cell invasion and virulence in mice

In order to evaluate the contribution of the ISC and SUF systems to Salmonella intracellular proliferation, we infected activated RAW 264.7 mouse macrophages with Salmonella wild-type strain, iscU and sufBC mutants. Bacterial proliferation was assayed by calculating the proliferation index as a ratio of the intracellular bacteria between 16 and 2 hours post-infection. The proliferation index of the wild-type strain and the iscU mutant were very close (8.8 and 9.4, respectively), whereas it was slightly higher for the sufBC mutant (12.2) (Fig. 1A), reflecting no major role for Fe-S proteins during the intracellular proliferation of macrophages. Next, HeLa cell invasion was carried out. Each strain was incubated for ten minutes with HeLa cells followed by one hour of gentamicin treatment. We found the iscU mutant to invade epithelial cells much less efficiently than the wild-type (Fig. 1B), whereas the sufBC mutant invasion showed no defect compared to the wild-type (Fig. 1B). Next, mixed infections of mice were carried out using wild-type and mutants in order to determine the competitive index (CI) and to compare virulence of the strains [START_REF] Beuzón | Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo[END_REF]. The injection of the inoculum was either intraperitoneal (IP), during which bacterial cells can rapidly reach their target organs (Fig. 1C & Table S1), or oral to force the pathogen to cross the intestinal barrier before accessing the blood system (Fig. 1D & Table S2). By performing intraperitoneal infections, we found the iscU mutant to be affected for its virulence (CI = 0.304) (Fig. 1C & Table S1). In contrast, the CI of the sufBC mutant did not show any significant alteration of virulence compared with the wild-type strain (Fig. 1C & Table S1). Lastly, mice oral infections were carried out. We observed a severe virulence defect of the iscU mutant (CI = 0.011) (Fig. 1D & Table S2), 30-fold less compared to intraperitoneal infection and 90-fold less compared to the wild-type. The sufBC mutation had no effect (CI = 0.758) (Fig. 1D & Table S2). Taken together, these results indicate that the ISC machinery plays a major role in Salmonella virulence during the invasion of epithelial cells and in the mouse model.

The ISC system plays a key role in the regulation of Spi1 Type III Secretion

System gene expression

To test the involvement of Fe-S biosynthesis machineries in Spi1 TTSS regulation, the promoter of hilD -which was shown to be activated to allow Spi1 TTSS biogenesis (Fig. 2A) -was fused to GFP and inserted into the pFPV25 plasmid, yielding the PhilDgfp transcriptional fusion. Such constructs were previously used in Salmonella to report gene expression within host cells [START_REF] Valdivia | Fluorescence-based isolation of bacterial genes expressed within host cells[END_REF][START_REF] Aussel | Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst[END_REF].

Cells were grown in LB and the fusion expression levels were measured after 16 hours of culture. Expression of the PhilD-gfp fusion was found to be 22% lower in an iscU mutant compared with the wild-type, whereas the sufBC mutation had no effect on the expression of this fusion (Fig. 2B). We next tested whether the effect of the iscUmediated reduction had an impact on the expression of the hilD-regulated genes such as rtsA, prgH and invF. Transcriptional fusions were constructed for each gene and their expression assayed in iscU and sufBC backgrounds. Compared to the wild-type strain, the iscU mutant exhibited a decrease in the expression of rtsA (4.1-fold), prgH (5.3-fold) and invF (7-fold) (Fig. 2C, 2D and2E). The sufBC mutation had no effect on the expression level of these genes. Altogether, these results showed that Spi1 TTSS genes are down-regulated in an iscU mutant, conferring a key role to the ISC machinery in the synthesis of this secretion apparatus.

IscR binds on the promoter of hilD

Because the ISC machinery was found to play a role in the regulation of Spi1 TTSS gene expression, we aimed at identifying a protein matured by ISC and involved in the control of the Spi1 TTSS apparatus. Two putative binding sites of IscR -a [2Fe-2S]containing regulator matured by the ISC system -were found in the hilD promoter region. Only three amino acids differ between E. coli and Salmonella IscR proteins (98% identity) and none of these residues are in the DNA binding domain, supporting the idea that identical motifs can be recognized by both proteins [START_REF] Rajagopalan | Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity[END_REF]. The two putative IscR binding sites upstream of hilD resemble Type 2 motifs which can be bound by both holo-and apo-IscR [START_REF] Giel | IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli[END_REF]. Sites 1 and 2 lie, respectively, from -105 to -81 (ATTACCCAAATTTGGGTTCTTTTGG) and from -65 to -41 (ATTGCCAACACACGCTAATAAAGAG) upstream of the hilD gene transcription start site (Fig. 3A). Comparison of these two sites with the IscR sites from E. coli hyaA and Yersinia pseudotuberculosis lcrF shows that they contain five and four of the nine bases in this binding motif, respectively (Fig. 3B) [START_REF] Giel | IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli[END_REF]. Importantly, Site 1 overlaps with a previously identified HilD binding site (Fig. 3A) [START_REF] Olekhnovich | DNA-binding activities of the HilC and HilD virulence regulatory proteins of Salmonella enterica serovar Typhimurium[END_REF]. To test the interaction between IscR and the promoter of hilD (PhilD), electrophoretic mobility shift assays (EMSA) were carried out. When incubated with increasing concentrations of purified His6-IscR, the PhilD probe shifted gradually (Fig. 3C). IscR was able to form four different complexes, consistent with the presence of two Type 2 motifs on PhilD and with the ability of IscR to form dimers (Fig. 3C). Next, three nucleotides were mutated at the positions 1, 5 and 6 of the Type 2 motifs on the hilD promoter (ATTACC was changed to CTTAGG on the site 1 and ATTGCC was changed to CTTGGG on the site 2). When incubated with increasing concentrations of IscR, the resulting DNA fragment, called PhilD*, displayed a more modest shift compared to PhilD, highlighting the importance of these conserved nucleotides in the interaction between IscR and the promoter of hilD (Fig. 3D and3E). Finally, to confirm the direct interaction between IscR and PhilD, DNA-protein interaction experiments were carried out using Bio-Layer Interferometry (BLI), a technique previously used to study protein-protein interactions [START_REF] Arlet | HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia[END_REF]. IscR was also found to interact with PhilD in this assay, whereas no interaction was detected with the promoter of ahpC used as a negative control (Fig. 3F). Interestingly, this experiment allowed us to measure a dissociation constant (Kd) of 390 nM between IscR and PhilD, consistent with previous values of 93 and 112 nM measured between E. coli IscR and the iscR promoter [START_REF] Giel | Regulation of iron-sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe-2S]-IscR in Escherichia coli[END_REF]. Taken together, the results presented in this panel demonstrate a direct interaction between IscR and the promoter of hilD.

IscR represses the expression of hilD gene and HilD targets

Next, to investigate the regulatory role of IscR on hilD, the expression of the hilD fusion was measured and found to be about 1.5-fold up-regulated in an iscR mutant (Fig. 4A), suggesting that IscR acts as a negative regulator. Interestingly, the expression level of hilD was reduced to a similar extent (2.5-fold) in the hilD and in the hilD iscR mutants, consistent with a scenario where IscR could act as an anti-activator on HilD. Because the extent of the regulatory effect was only 1.5-fold, we tested whether the IscRmediated regulation of hilD had any effect on the expression of downstream HilD-regulated genes. The expression of the rtsA, prgH and invF fusions was measured in a wild-type, iscR, hilD and hilD iscR mutant strains. A hilD mutation abrogated the expression of rtsA, prgH and invF (Fig. 4B, 4C and4D). In contrast, the iscR mutant exhibited a 1.5-fold increase in rtsA expression (Fig. 4B) and a 2-fold increase in prgH and invF expression (Fig. 4C and4D). In the hilD iscR mutant, the expression level of the rtsA, prgH and invF fusions was found to be lower than 10 units but higher than the hilD mutant values (Fig. 4B, 4C and4D). The iscR and hilD mutant strains were also complemented by plasmid-borne wild-type copies of iscR and hilD under an arabinoseinducible promoter, respectively (Fig. S2). To investigate further the regulatory effect of IscR, quantitative reverse transcription PCR (qRT-PCR) experiments were carried out with the genes previously analysed. The iscR mutation led to a 5.5-fold increase in hilD mRNA level compared to the wild-type strain, confirming that IscR acts as a repressor of hilD (Fig. 4E). Consistently, rtsA, prgH and invF mRNA levels were found to increase significantly in the iscR mutant (2.5-, 7-and 5.5-fold, respectively) whereas they decreased in the hilD mutant (Fig. 4F, 4G and4H). Noticeably, IscR was not found to shift the PinvF probe in EMSA, supporting the notion that expression of invF is indirectly regulated by IscR (Fig. S3). In the hilD iscR mutant, the rtsA, prgH and invF mRNA levels were 360-, 60-and 460-fold lower compared to the wild-type strain, showing that the expression of rtsA, prgH and invF is totally abrogated in the absence of both iscR and hilD and suggesting a possible Spi1 gene expression reprogramming process in this background (Fig. 4F, 4G and4H). Collectively, these results indicate that IscR acts as a repressor of hilD gene expression, leading to the down-regulation of HilD targets.

Apo-and holo-IscR down-regulate Spi1 TTSS gene expression

To investigate whether IscR acts on Spi1 TTSS gene expression under its apo-or holoform, we built a plasmid encoding an IscR variant (IscR-3CA). This IscR variant has all three cluster-coordinating cysteine residues (Cys92, Cys98 and Cys104) converted into alanine, which prevents binding of the [2Fe-2S] cluster [START_REF] Rajagopalan | Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity[END_REF].

The iscR mutant was transformed with an empty plasmid, with a plasmid encoding IscR or with a plasmid encoding IscR-3CA. In an iscR mutant, expression of the prgH gene was down-regulated 2.4-fold in the presence of piscR while it was down-regulated 1.6-fold in the presence of piscR-3CA (Fig. 5A, left). This indicated that both forms have a negative effect on prgH expression. Similarly, the invF gene was downregulated 1.7-fold in an iscR -/piscR strain and 1.3-fold in an iscR -/piscR-3CA strain, again indicating that apo-and holo-IscR modified invF gene expression (Fig. 5A,left).

The experiment was also carried out in a wild-type strain in which the overexpression of iscR reduced the expression of prgH and invF (2.9-and 2.7-fold, respectively) (Fig. 5A, right). The same observation was made when the iscR-3CA variant was overexpressed (1.9-and 2.1-fold repression compared to the empty plasmid). Taken together, these results show that both apo-and holo-IscR down-regulate the expression of the Spi1 TTSS gene.

IscR mediates iron-dependent control of Spi1 TTSS genes

It was previously shown that iron limitation down-regulates the expression of Spi1 TTSS genes [START_REF] Ellermeier | Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD[END_REF][START_REF] Teixidó | Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro[END_REF]. Therefore, we tested whether IscR could have a role in mediating this regulation. Expression of prgH and invF genes was monitored in bacteria grown in the presence of 2,2'-dipyridyl (dip), an iron chelator (Fig. S4). In the presence of dip, the level of prgH and invF expression dropped 8.8-and 5.7-fold compared with untreated cells (Fig. 5B). In an iscR mutant, the addition of dip was still inhibiting expression of the two genes but to a much reduced extent, i.e. 3.2-fold for prgH and 1.7-fold for invF fusions (Fig. 5B). These data indicate that IscR contributes significantly to the iron-mediated regulation of Spi1 TTSS gene expression.

IscR reduces the invasion of HeLa cells by Salmonella

Because IscR was shown to decrease the expression of Spi1 TTSS genes, we evaluated its contribution to Salmonella virulence. After ten minutes of incubation with HeLa cells, the iscR mutant presented an efficiency of entry 1.44-fold higher than the wild-type strain (Fig. 6A). Finally, the wild-type and the iscR mutant strains were used in mice co-infection experiments. The inoculum was either injected intraperitoneally or ingested orally. Virulence of the iscR mutant was reduced as compared with the wildtype, using both methods: a CI of 0.144 was obtained using intraperitoneal infections (Fig. 6B left & Table S3), whereas a value of 0.255 was found after oral ingestion (Fig. 6B right & Table S3). These results show that during the first step of the infection, IscR can reduce HeLa cell invasion by Salmonella, consistent with its negative role in Spi1 TTSS expression.

Discussion

In this study, we showed that the Iron Sulfur Cluster (ISC) machinery is required for the virulence of Salmonella enterica. The reduced invasion of an iscU mutant in HeLa cells combined to its drastic virulence attenuation during oral mouse infections confer a central role to the ISC system in the establishment of the Salmonella infectious process. One reason underlying the reduced virulence of an iscU mutant could be a decrease in Spi1 TTSS gene expression. Indeed, we showed that apo-IscR, which prevails in an iscU mutant, downregulates the expression of the master regulator hilD and thereby of several downstream HilD-regulated genes. Our findings support the idea that the ISC machinery is important for Salmonella virulence through the ability of the Fe-S cluster sensor IscR to regulate Spi1 gene expression (Fig. 7). They also reflect an adaptive mechanism used by Salmonella to favour its infectivity in the gut lumen and tissues, where oxygen is rare and iron available to the bacterium [START_REF] Kortman | Nutritional iron turned inside out: intestinal stress from a gut microbial perspective[END_REF][START_REF] Jennewein | Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence[END_REF].

In a strain lacking the ISC machinery, the expression of iscR increases significantly (Fig. S5) and the synthesized IscR is mostly under its apo-form. Apo-IscR binds on Type 2 DNA-binding motifs and such motifs were found in the promoter region of hilD (Fig. S6). Therefore, the increase in iscR expression observed in an iscU mutant should lead to a decrease in Spi1 gene expression. This prediction was demonstrated experimentally as Spi1 TTSS genes were found to be down-regulated in an iscU mutant and up-regulated in an iscR mutant. Moreover, our results using an Fe-S cluster-free variant indicated that the apo-form of IscR negatively regulates the expression of the Spi1 TTS genes. Recently, Yersinia IscR was shown to bind upstream of the operon encoding the TTSS master regulator LcrF, which endowed IscR with a positive role in transcription of Yersinia TTSS genes [START_REF] Miller | IscR is essential for yersinia pseudotuberculosis type III secretion and virulence[END_REF].

Thus, both pathogens appear to have recruited IscR with a primary role in their virulence program. Based on the phenotypes exhibited by iscR mutants, it appears that IscR targets TTSS regulation in opposite ways, as an activator in Yersinia and a repressor in Salmonella. Further studies are required to characterize the physiological significance of the TTSS regulation by IscR in both species.

In recent years, studies from different groups reported that the presence of iron in the environment induced the expression of hilA [START_REF] Ellermeier | Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD[END_REF] and hilD [START_REF] Teixidó | Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro[END_REF]. The question arose whether the iron sensing regulator Fur was involved in this iron-mediated activation [START_REF] Ellermeier | Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD[END_REF][START_REF] Teixidó | Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro[END_REF]. Slauch and colleagues proposed two models to explain Fur activation of Spi1: either by indirect control of the HilD protein or by affecting the threshold value of HilD concentration required to activate the hilD promoter [START_REF] Ellermeier | Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD[END_REF].

More recently, Campoy and colleagues proposed a different model in which Fur would bind upstream of the hilD promoter and directly stimulate the expression of hilD [START_REF] Teixidó | Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro[END_REF]. In the present study, we also found iron to be required for Spi1 gene expression. Expression of prgH and invF decreased 9-and 6-fold under iron limited conditions, this regulatory mechanism being largely mediated by Fur. In an iscR mutant, the expression of these two genes was also reduced under iron limited conditions compared to LB, but to a much reduced extent (3-and 2-fold, respectively).

Thereby, we wondered if IscR could be involved in the iron regulation of Spi1 genes.

Our results show that under iron replete conditions, IscR represses Spi1 expression 1.5-fold only (WT vs. ΔiscR in LB), whereas under iron limited conditions, IscR exerts a 5-fold repression effect (WT vs. ΔiscR in LB + dip). So going from iron limited to iron replete conditions will shift the equilibrium from apo-to holo-IscR, thereby repressing iscR and inducing Spi1 gene expression by alleviating IscR repression. Altogether, these results suggest that IscR could act as an additional iron-sensor in the regulation of Spi1 TTSS.

Expression of Spi1 TTSS is one of the most important factors leading to the success of Salmonella invasion of intestinal epithelial cells. The composition of such an environment is a complex issue because in vivo studies in the gut are extremely challenging and because bacterial infections trigger an inflammatory response which modifies the nature of this environment. Nevertheless, recent studies shed light on oxygen and iron availability. Except for a relatively aerobic zone adjacent to the mucosal surface, the gut lumen contains low levels of oxygen [START_REF] Marteyn | Modulation of Shigella virulence in response to available oxygen in vivo[END_REF][START_REF] Jennewein | Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence[END_REF]. In contrast, iron is present in sufficient amounts to sustain bacterial replication in the gut, although the production of siderophores by enteric pathogens suggests a limited availability [START_REF] Kortman | Nutritional iron turned inside out: intestinal stress from a gut microbial perspective[END_REF]. Both limited oxygen tensions and available iron define optimal conditions for Fe-S cluster stability. Hence, IscR is predicted to be mostly under its holo-form and at low cellular concentrations, favoring Spi1 TTSS gene expression (Fig. S5) [START_REF] Schwartz | IscR, an Fe-S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe-S cluster assembly proteins[END_REF][START_REF] Vinella | In vivo [Fe-S] cluster acquisition by IscR and NsrR, two stress regulators in Escherichia coli[END_REF]. In previous studies, we have shown that ROS were produced by macrophages and that intracellular Salmonella were capable of sensing these ROS [START_REF] Aussel | Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst[END_REF]. Moreover, an increase in iron efflux was observed in Salmonella-infected phagocytes, reducing both the cytoplasmic labile iron and the ferritin storage within macrophages, thus restricting the acquisition of iron by intracellular Salmonella [START_REF] Nairz | The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium[END_REF]. Under such environmental conditions -iron starvation and oxidative stress -, the apo-IscR form is predicted to dominate, decreasing Spi1 TTSS gene expression and limiting energetic expenses for the pathogen during macrophage infection. This model is supported by transcriptomic and deep-sequencing analysis showing reduced expression for most of the invasion-associated Spi1 genes in macrophages [START_REF] Eriksson | Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica[END_REF][START_REF] Srikumar | RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium[END_REF]. Indeed, in macrophages, hilD gene expression decreased around 3-fold whereas iscR gene expression increased around 5-fold compared to LB medium [START_REF] Eriksson | Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica[END_REF]. Thus, this scenario describes an adaptive mechanism used by S. enterica to favor its virulence in the gut and to succeed in epithelial cell invasion. From this viewpoint, IscR is predicted to play a key role in sensing oxygen and iron levels since its expression and maturation can be modulated as a function of the host environmental conditions.

Iron-sulfur clusters are used by a large number of protein species -over 130 in E. coli and Salmonella -which participate in diverse biological processes [START_REF] Py | Building Fe-S proteins: bacterial strategies[END_REF]. In this study, the defect of the iscU mutant in HeLa cell invasion and oral mice infection can be partially attributed to the down-regulation of Spi1 TTSS gene expression by IscR. Other cellular processes dependent on enzymes carrying Fe-S clusters, like motility, adhesion or respiration, could also be affected as shown in other pathogens [START_REF] Lim | IscR is a global regulator essential for pathogenesis of Vibrio vulnificus and induced by host cells[END_REF]. Conversely, the iscR mutant was shown to be more invasive in HeLa cells and less virulent in mice compared to the wild-type, consistent with a possible regulation of other genes involved in Salmonella pathogenesis by IscR.

Analysis of iscR and iscU contributions to Salmonella virulence at the genomic level might identify additional actors and will constitute the scope of future studies.

Experimental Procedures

Ethics Statement

Animal experimentation was conducted in strict accordance with good animal practice

Bacterial strains and growth conditions

The bacterial strains used in this study are described in Table 1. Strains were routinely grown at 37°C in Luria-Bertani (LB) medium. Ampicillin (50 µg/ml), kanamycin (25 µg/ml) and chloramphenicol (25 µg/ml) were added when necessary. Deletions of various genes and concomitant insertion of an antibiotic resistance cassette was carried out using Lambda-Red-mediated recombination [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF]. Mutations were moved to 12023 wild-type strain by P22 transductions.

Plasmid constructions

The cloning vector used to monitor gene expression was pFPV25, carrying promotorless gfpmut3a gene [START_REF] Valdivia | Fluorescence-based isolation of bacterial genes expressed within host cells[END_REF]. The inserts carrying 300 bp upstream prgH, invF, rtsA or hilD start codon were PCR-amplified from S. enterica 12023 by using the primers listed in Table S4. PCR products were digested using XbaI and KpnI, and cloned into pFPV25 vector, yielding PprgH-gfp, PinvF-gfp, PrtsA-gfp and PhilD-gfp plasmids (Table 1). For iscR and hilD complementation, the coding sequence of each gene was digested using XbaI and NcoI and cloned into pKI*, yielding piscR and philD (Table 1). For IscR purification, the coding sequence of iscR was digested using NdeI and XhoI and cloned into pet22b(+), yielding pHis6-iscR (Table 1). The plasmids expressing IscR variant (piscR-3CA and pHis6-iscR-3CA) were PCR-amplified in two steps using two central oligonucleotides carrying the mutations (C92A/C98A/C104A), the iscR-3CA fw and iscR-3CA-rv oligonucleotides (Table S4).

All the inserts were verified by DNA sequencing.

Bacterial infection of macrophages

RAW 264.7 macrophages were seeded at a density of 410 5 cells per well in 6-well tissue culture plates containing DMEM with 10% fetal bovine serum (FBS) (HyClone).

Macrophages were supplemented with IFN- (10 U/ml, ImmunoTools) 24 hours before use when indicated. Bacteria were cultured over-night at 37°C with shaking, and opsonized in DMEM containing FBS and normal mouse serum (10%, Perbio) for 30 minutes. The macrophages were activated with 0.2 µM phorbol 12-myristate 13acetate (PMA) (Sigma Aldrich) before infection when indicated. Bacteria were added to the monolayers at a multiplicity of infection 10:1, centrifuged at 400 g for 5 min at 4°C, and incubated for 30 min at 37°C in 5% CO2. The macrophages were washed three times, and incubated with DMEM containing FBS and 100 µg/ml gentamicin for 60 minutes, after which the gentamicin concentration was decreased to 10 µg/ml for the remainder of the experiment. For enumeration of intracellular bacteria, macrophages were washed two times with PBS, lysed with 0.1% Triton X-100, and a dilution series was plated on LB agar.

Invasion assay of HeLa cells

HeLa cells were seeded at a density of 5.10 4 cells/ml in 6-well plates and incubated overnight at 37°C with 5% CO2 in DMEM with 10% FBS. Bacterial strains were cultured aerobically overnight at 37°C in LB. Bacteria were diluted 100-fold and sub-cultured for 3 h at 37°C. HeLa cells were infected for 10 minutes with bacterial strains at MOI 100:1. Cells were washed three times with PBS and incubated for 1 h in DMEM supplemented with 100 μg/ml gentamicin. After incubation, HeLa cells were washed two times with PBS and lysed with 0.2% Triton X-100. Colony-forming units (cfu) of intracellular bacteria were counted by plating the appropriate dilution on LB agar plates.

Competition assays

Eight-to 10-week-old C57/B6 mice were inoculated intraperitoneally or orally with equal amounts of two bacterial strains for a total of 10 5 bacteria per mouse. The spleens were harvested 48 hours after inoculation (i.p. infection) or 5 days after ingestion (oral infection), then homogenized. Bacteria were recovered and enumerated after plating a dilution series onto LB agar and LB agar with the appropriate antibiotics.

Competitive indexes (CI) were determined for each mouse [START_REF] Beuzón | Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo[END_REF]. The CI is defined as the ratio between the mutant and wild-type strains within the output (bacteria recovered from the mouse after infection) divided by their ratios within the input (initial inoculum). A one-sample t test was used to determine whether the CIs were significantly different. All statistical analyses were performed by using Prism (GraphPad, San Diego, CA). The two-tailed P value was calculated.

Purification of His6-iscR and His6-iscR-3CA E. coli strain BL21(DE3) / piscR-His6 was grown at 37°C in LB medium. At an OD600nm = 0.6, 0.5 mM IPTG was added and growth was carried on for 2 h. Cells were harvested by centrifugation, and the pellet was resuspended in 8 ml of buffer A (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 10 mM imidazole). Resuspended cells were broken by two passages through an ice-chilled French pressure cell at 0.5 tons. The resulting crude extract was centrifuged twice at 10000 x g for 30 min at 4°C. The supernatant was applied to a 1-ml His-trap column prepacked with nickel (GE Healthcare life Sciences) and equilibrated with buffer A. Proteins were eluted with a 40-ml gradient running from 10 to 500 mM imidazole. Fractions were analysed by a sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-PAGE). After elution, the His6-IscRcontaining fractions were pooled and the solution was concentrated by ultrafiltration on Amicon ultra 10 K (Millipore) (2500 x g, 4°C). Protein concentration was determined spectrophotometrically at 280 nm (NanoDrop 1000; Thermo Fisher Scientific). Identical protocol was used to purify the His6-IscR-3CA protein.

Mutagenesis and preparation of DNA substrates

Substrates for EMSA were produced using 5′-Cy5 labelled primers (Eurogentec) at one extremity. The resulting DNA fragments include 200 nucleotides upstream and 40 nucleotides downstream the start codon of hilD and invF genes, yielding PhilD and PinvF. PhilD* was obtained in two steps using two central oligonucleotides carrying the mutations (See Table S4 for details). Sequence accuracy of the PCR products was checked by sequencing.

Electrophoretic Mobility Shift Assays (EMSA)

Reaction mixes (10 μl) contained Cy5 labelled DNA (100 nM) in Tris-HCl, pH 7.5, 0.5 M KCl buffer containing 0.3 mg/ml sonicated calf thymus DNA. Appropriate protein concentrations indicated in figure legends were added prior to incubation at 25 °C for 30 min. Samples were loaded on a 6% native PAGE in TBE 1× at 90 V, and ran for 5 hours at 135 V. Migration profiles were analysed by scanning the gel using a 635 nm laser and a LPR filter (FLA5100, Fujifilm).

BioLayer Interferometry (BLI)

Protein-DNA interaction experiments were conducted at 25°C with the BLItz instrument from ForteBio (Menlo Park, CA, USA). The BLI consists in a real time optical biosensing technique exploits the interference pattern of white light reflected from two surfaces to measure biomolecular interactions [START_REF] Concepcion | Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization[END_REF]. Purified His6-IscR protein ligand was immobilized onto two different Ni-NTA biosensors (ForteBio) in duplicate at 6 μM. PCR amplified DNA fragment of hilD and ahpC promoters were used as the analytes throughout the study at the 240 nM. The assay was conducted in 25 mM TRIS-HCl, pH 7.5, 0.5M NaCl. The binding reactions were performed with an initial baseline for 30 seconds, an association step at 120 seconds and a dissociation step of 120 seconds with lateral shaking at 2200 rpm. A double reference subtraction (sensor reference and His6-IscR) was applied to account for non-specific binding, background, and signal drift to minimize sensor variability.

RNA Preparation and Reverse Transcription

RNAs were prepared from 1 ml culture of S. enterica grown in LB until OD600nm = 1.5.

The cells were harvested and frost at -80°C. Total RNAs were isolated from the pellet using the SV Total RNA Isolation System (Promega). The RNA quality was assessed by an Experion chip (Bio-Rad) and the absence of DNA contamination was confirmed by PCR. For cDNA synthesis, 650 ng total RNA and 0.5 μg random primers were used with the GoScript™ Reverse transcriptase (Promega).

Quantitative Real-Time-PCR for Transcriptional Analyses

Quantitative real-time PCR (qPCR) analyses were performed on a CFX96 Real-Time System (Bio-Rad). The reaction volume was 15 μl and the final concentration of each primer was 0.5 μM. The cycling parameters of the qRT-PCR were 98°C for 2 min, followed by 45 cycles of 98°C for 5 s, 56°C for 10 s, 72°C for 1s. A final melting curve from 65°C to 95°C is added to determine the specificity of the amplification. The yejA gene was used as a reference for normalization. For each point, a technical duplicate was performed. The amplification efficiencies for each primer pairs were comprised between 75 and 100%. All of the primer pairs used for qRT-PCR are reported in the table S4.

  as defined by the French animal welfare bodies (Law 87-848 dated 19 October 1987 modified by Decree 2001-464 and Decree 2001-131 relative to European Convention, EEC Directive 86/609). All animal work was approved by the Direction Départementale des Services Vétérinaires des Bouches-du-Rhône (authorization number 13.118 to SM, Application number AR 1A09382857717).

Figure 1 .

 1 Figure 1. The ISC machinery is essential for HeLa cell invasion and mice

Figure 2 .

 2 Figure 2. The ISC system is required for the expression of Spi1 Type III Secretion

Figure 3 .

 3 Figure 3. IscR binds to the hilD promoter in vitro.

Figure 4 .

 4 Figure 4. IscR represses hilD gene expression.

Figure 5 .

 5 Figure 5. Apo-and holo-IscR negatively regulates the expression of Spi1 TTSS

Figure 6 .

 6 Figure 6. Salmonella iscR mutant is hyperinvasive in HeLa cells.

Figure 7 .

 7 Figure 7. Simplified model for the regulation of Spi1 TTSS.

Table 1 . Bacterial strains and plasmids.

 1

	Strain	Relevant genotype	Source or reference
	12023	Wild-type	Laboratory stock
	ST360	iscU::Kn	This study
	ST332	sufB-sufC::Cm	This study
	ST491	iscR::Kn	This study
	ST532	iscR (Kn S )	This study
	ST520	hilD::Cm	This study
	ST533	hilD::Cm iscR::Kn	This study
	Plasmids
	pFPV25	GFP reporter fusion vector (Ap r )	(Valdivia and Falkow, 1997)
	PprgH-gfp	pFPV25 derivative carrying the prgH-gfp promoter (Ap r )	This study
	PinvF-gfp	pFPV25 derivative carrying the invF-gfp promoter (Ap r )	This study
	PrtsA-gfp	pFPV25 derivative carrying the rtsA-gfp promoter (Ap r )	This study
	PhilD-gfp	pFPV25 derivative carrying the hilD-gfp promoter (Ap r )	This study
	pET-22b(+)	Expression vector carrying a C-terminal His tag	Novagen
	pHis6-iscR	pET-22b(+) derivative carrying the His6-iscR (Ap r )	This study
	pHis6-iscR-3CA	pET-22b(+) derivative carrying the His6-iscR-3CA (Ap r )	This study
	pKI*	pBAD24 derivative carrying a kanamycin resistance cassette (Kn r )	This study
	piscR	pKI* derivative carrying iscR (Kn r )	This study
	philD	pKI* derivative carrying hilD (Kn r )	This study

Acknowledgments

Thanks are due to the members of the FB group for fruitful discussions, in particular to Béatrice Py and Béatrice Roche for extremely helpful advice. We thank Sandrine Ollagnier-de Choudens (LCBM Grenoble) for biochemical expertise. We are grateful to Paul Hoffman (University of Virginia) and Josep Casadesús (University of Seville) for providing plasmid and strain. We thank Yann Denis (Transcriptomics Facility, IMM), Deborah Byrne (Protein Expression Facility, IMM), Rebecca Stevens (GAFL, Avignon) and Kévin Forin for technical assistance. This work was funded by the Agence Nationale de la Recherche (ANR, Programme M.I.E. "Salmo-sensor", Grant ANR-08-MIEN-025-01 and ANR Blanc SPV05511), the Centre National de la Recherche Scientifique (CNRS, PICS07279), and Aix-Marseille Université. BP was funded by the Fondation pour la Recherche Médicale (FRM).


Fabrice Sthal
email: fsthal@ens2m.fr

Xavier Vacheret

Patrice Salzenstein

Serge Galliou

Gilles Cibiel
email: gilles.cibiel@cnes.fr

E Rubiola

Advanced bridge instrument for the measurement of the phase noise and of the short-term frequency stability of ultra-stable quartz resonators

Advanced bridge instrument for the measurement of the phase noise and of the short-term frequency stability of ultra-stable quartz resonators

INTRODUCTION

High-stability quartz oscillators are needed in a number of space applications. A short-term stability of parts in 10 -14 [Allan deviation σ y (τ) ] is sometimes required, for integration time τ of approximately 1-10 s. The Centre National d'Etudes Spatiales (CNES) and the FEMTO-ST Institute have been collaborating for many years in this domain, aiming at measuring and at understanding the oscillator noise [START_REF] Galliou | A program to analyze origins of noise in ultra stable resonators[END_REF]. The highest stability has been observed on 5 MHz and 10 MHz bulk acoustic-wave resonators. Yet this stability is still not sufficient, or the manufacturing method is not reproducible. Recently, the analysis of a few premium-stability oscillators has demonstrated that the oscillator frequency instability is due to the fluctuation of the resonator natural frequency, rather than to the noise of the sustaining amplifier via the Leeson effect [START_REF] Rubiola | On the 1/f frequency noise in ultra-stable quartz oscillators[END_REF]. It is therefore natural to give attention to the measurement of the resonator fluctuations. This paper presents improvements of an instrument intended to test 5 MHz and 10 MHz high-stability resonators packaged in HC40 enclosure that used in ultra stable oscillators. The instrument is based on some known ideas, namely: (1) the frequency stability is measured through the phase noise spectral density S φ (f), converted into S y (f), and then into σ y (τ); and (2) S φ (f) is measured with the bridge (interferometric) scheme. This scheme provides the lowest background noise, and enables the measurement at low drive level. Improvements concern the design of new double ovens, the reduction of the residual carrier, a lower drift, and a modified calibration process.

II. BASIC PRINCIPLE ON MEASUREMENT BENCH

Fig. 1 shows the principle of resonator phase noise measurement bench using carrier suppression technique. The carrier signal of the driving source is split into two equal parts to drive both devices under test (DUT). The DUTs can be resistors (to measure the noise floor of the system) or crystal resonator pairs. The resonant frequency of each arm of the bridge is tuned to the driving source frequency with a series tuning capacitor. The carrier signal is canceled when both signals are combined 180° out of phase. Since phase noise is defined relative to the carrier power, reducing the carrier has the effect of amplifying the phase noise of the DUT. The output signal is amplified and then detected by the phase noise detector.

Calibration of the measurement system is obtained by injecting a known side band on one of the arms of the bridge. The noise of the DUT, as seen on the fast Fourier transform analyzer, is corrected using the calibration factor determined with the side band. Fig. 2 shows the measurement system. The new bench developed here has been obtained from two previous systems used in our laboratory experiences [START_REF] Sthal | Phase noise measurements of 10 MHz BVA quartz crystal resonator[END_REF][START_REF] Rubiola | Flicker noise measurement of HF quartz resonators[END_REF]. Any measurement requires a data acquisition.. A specific acquisition interface has been developed. It is based on HP3561A FFT analyzer. The main interface window is presented in Fig. 3. Frequency range and numbers of average can be well adapted according to the measurement time.

III. IMPROVEMENTS

A. Resonator ovens

The main improvement of ovens is shown in Fig. 4. Fig. 4a shows the crystal box of the new ovens. It consists of a double enclosure and two thermally controlled ovens, used in order to control the quartz crystal temperature. The temperature of the crystal oven is controlled by means of a tuning resistor chip. A resistor step of about 30 Ω allows a temperature adjustment of 0.05 °C in the 60 to 90 °C temperature range. In our new crystal ovens, the tuning capacitors of the quartz crystals are inserted in the crystal oven. Fig. 4b shows the crystal oven and the inserted location of the tuning capacitor. Fig. 4c shows the cable ovens.

By this way, a remote temperature control can be easily achieved without opening the overall device. The cable oven temperature is set at a fixed value between the room temperature and the crystal temperature. Performances of these crystal ovens have been measured according to the procedure describe in a previous paper [START_REF] Sthal | Thermal characterization of crystal ovens used in phase noise measurement system[END_REF]. The thermal stability is about 2 µ°C at 1 s and about 20 µ°C at 10 s. The relative frequency fluctuation of Δf/f of the bench due to the thermal effects on the resonator is about 2⋅10 -15 at 1 s.

B. Temperature effect on tuning capacitors

The crystal tuning capacitor, located in the enclosure of quartz, is subjected to the fluctuations of temperature brought by the thermal regulation of the thermostat. The capacitor varies according to a thermal coefficient. In our case, standard variable capacities 5600 Johanson having a temperature coefficient of ±30 ppm/°C are used. Quartz crystal and its tuning capacitor are represented in Fig. 5.

) ( 2 0 0 s m C C C f f + ≈ Δ (1)
With C s : Serial tuning capacitor associated to the quartz crystal and f 0 the resonant frequency of the resonator.

Thus, the resonant frequency of the resonator and its tuning capacitor is given by:

⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + + × = ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ Δ + = Δ + = ) ( 2 1 1 0 0 0 0 0 s m C C C f f f f f f f (2)
The fluctuations ΔC s of C s due to the thermal fluctuations give a relative frequency variation expressed by: In the worst case, for C s = 2.9 pF, the relative frequency fluctuations are about 2.4⋅10 -10 for a temperature variation of 1 °C. According to the thermal stability of the ovens at 10 s, the relative frequency fluctuation of Δf/f of the bench due to the thermal effects on the tuning capacitor will not be above 5⋅10 -15 . This oven configuration is well adapted to measure resonators with an Allan standard deviation stability around 1⋅10 -14 .

( ) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ Δ × × ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + - = Δ s s s s m C C C C C C f f 2 0 0 2 (3)

C. Calibration

Calibration of the measurement system is obtained by injecting a known side band on one arm of the bench. This sideband simulates an equivalent phase noise. When quartz crystal resonators are measured, the calibration sideband must be injected inside the bandwidth of the resonator. Fig. 7 shows the FFT level obtained from the known sideband versus the difference between the source frequency and the sideband frequency. Values are given according to the powers dissipated by the quartz crystal. The frequency of the calibration sideband is moved from 0.2 Hz to 1.2 Hz from the carrier frequency. The maximum of the curves is located towards a frequency offset of 0.4 Hz. The level difference between 0.4 Hz and 1 Hz can vary between 0.3 dB and 1 dB. The calibration side band is very important. Indeed, the measured noise is directly modified by the correction factor as shown in Fig. 8. The calibration sideband must be to the maximum amplitude of quartz crystal transfer function to not generate an error of measurement and increase the uncertainties of measurements. The value of the calibration sideband was thus fixed at 0.4 Hz from the source frequency instead of below 2 Hz.

D. Output offset

The FFT analyzer input is sensitive to the DC voltage. The calibration range of the analyzer can become inappropriate if this voltage fluctuate during the measures. Before the measurement, the delay line of the measurement bench is tuned to give a signal at the phase detector output with no DC voltage. Unfortunately, the experiment duration to get low frequency (about 1 hour for 0.01Hz) shows us that the output offset can drift.

Better understanding of this parameter has been obtained by measuring its variations. For that, we used the FFT HP3621A analyzer which is able to measure the DC residual voltage. An interface allowing a acquisition of the offset was developed (Fig. 9). The output offset is measured during an integration time of 2 seconds. Its evolution is observed with a space time 5 seconds interval between each measurement. The observation time can be higher than 7 days. Initially, we examined the offset stability of the DC-100 kHz amplifier used after the phase detector. Its offset variations are below 2 mV which is remarkably stable.

The second analysis consists in measuring the offset during the phase of adjustment in a simple arm of the bench. Resistor is used to measure the noise floor of the system. The delay line is tuned to have the simple arm and the pump signal 90° out of phase. Fig. 10 shows the offset measurements with a dissipated power in resistor equal to 75 µW. Curves are given for a time up to 2 hours which is above classically phase noise measurement.

A first problem is revealed by these measurements. The nature of the connection between the element to be measured and its support has been examined. It is easier to use a mechanical contact to facilitate the assembly and disassembly of the DUT that must be measured. Commercial benches often use this kind of support. Unfortunately, saved time is with the detriment of the stability of the system and the quality of measurement carried out. It is preferable in this case to weld the connections between the DUT (resonator) and the support. Another series of test have been achieved in order to limit the offset drift during the measurement time. An answer is come from the attenuator-phase converter units of each arm. These units are used to adjust the carrier suppression. Fig. 11 shows the offset drift for two kind of attenuator. The 0-20 dB attenuators were used in our previous version of our bench. Usual adjustment in term of amplitude level is always around 3 dB. We thus replaced the 0-20 dB attenuator by a 0-8 dB attenuator. This enables us to still have a comfortable margin of adjustment and to decrease the effect of the thermal relaxation by 60% what is a considerable profit is appreciable taking into consideration expected performance. Offset variations are a good indicator of reliability of measurements. A null offset indicates a state of quasi-perfect balance between both arms of the bench. It is necessary to seek a limit point to consider that the measurement is not affected by the offset. Fig. 12 shows 5 MHz quartz crystal resonator phase noise measurements with a voluntary "degraded" value of offset (about 200 mV) compared with a measurement having an offset adjustment. Insignificant differences are observed between both curves. Thus, an offset below 200 mV warrants a good measurement.

E. Carrier suppression

The carrier suppression is a critical phase in the bench adjustment. It depends on many factors (dissipated power, phase tuning, stabilization time, measurement environment) that can interact on stability, adjustment facilities or attenuation level. If the ideal adjustment exists in theory, in practice, the total carrier suppression without fluctuations during a long time is really difficult to obtain, even impossible to realize. We carried out comparative measurements on power levels and carrier suppression. We have sought a range of adjustment in which a reliable and reproducible measurement is guarantee. A limit was established concerning the minimal value of the carrier suppression level. Table II gives carrier suppression level that can be acceptable to get a good phase noise measurement.

Minimum value -65 dB Accepted Minimum value

-75 dB Mean value -82 dB Maximum value -98 dB Fig. 13 shows the measurement of a quartz crystal pair with both different carrier suppression levels. A same behavior is observed according to the power applied on the resonator. It should be noted that in practice the average carrier suppression level is in the neighbourhoods of -82 dB. According to many measurements that we have done, a safety margin of about -10 dB can be considered.

F. Measurement environment

Influence of the environment to the phase noise measurement is illustrated in Fig. 14. Undesirable peak appeared around 7 Hz. This sporadic and random phenomenon has been observed several times. It is a student festival of music which revealed us the sensitivity of our device to the low-frequency vibrations. The loudness and the test periods of the loudspeakers allow us to establish the link between the effect and the cause. The measurement frequencies to get the phase noise of the resonator are classically in the bandwidth 0.01 Hz to 10 Hz. This frequency range is particularly sensitive to mechanical vibrations. Vibration isolation tables must be chosen very carefully regarding their cut-off frequencies which are in this frequency band. With active control tables, it is also possible to amplify parasitic signals inside the bandwidth. This problem must be taking into account very carefully. After several tests, localization of the measurement bench was chosen according to this phenomenon.

G. Trend curves

Resonator noise is measured through the phase noise spectral density and then converted into Allan standard deviation. The Allan variance is computed from the Fourier frequency and from the power spectral density of the frequency fluctuations [START_REF] Rutman | Characterization of phase and frequency instabilities in precision frequency source: Fifteen years of progress[END_REF]:

∫ ∞ = 0 2 4 2 ) ( sin 2 ) ( ) ( df f f f S y y τ π τ π τ σ (4)
with f = Fourier frequency. The standard Allan variance can not be calculated from the phase noise of the resonator unless one knows the loaded Q L of the resonator. The equations below show how σ y (τ) is calculated for the flicker phase noise in a resonator. The resonator is considered as a low pass filter. Thus, we have:

) ( 2 1 ) ( 2 f S Q f S L y φ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ = (5)
Here S φ (f) is the measured phase noise of the resonator, Q L is the loaded quality factor of the resonator and S y (f) is the power spectral density of the frequency fluctuations of the resonator. The flicker phase in S φ (f) gives flicker frequency in S y (f).

As expected σ y (τ) is independent of τ for flicker frequency noise, σ y (τ) is:

) 1 ( ) 2 ln( 2 ) ( 2 Hz f S y y = = τ σ ( 6 )
Thus the Allan deviation is:

) 1 ( 2 1 ) 2 ln( 2 ) ( 2 2 Hz f S Q L y = ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ = φ τ σ (7)
The Allan deviation calculated this way represents the Allan deviation of an oscillator containing the test resonator in which the only source of flicker frequency noise is the test resonator.

The loaded quality factor Q L is given by means of the Lesson cutoff frequency which is equal to:

L L Q f 2 0 ν = (8)
The cut-off frequency is traditionally obtained graphically. This treatment is often subject to interpretation which can vary from one operator to another. To avoid dispersion, we propose a calculation approach by trend curves with f -1 and f -3 slopes (Fig. 15). These slopes correspond to the low-pass filter effect of the frequency flicker resonator noise. The ranges of the frequencies are chosen according to the slope variations. This method can be applied directly in the phase noise measurements (Fig. 16). A better choice can be obtained by injecting a known white noise at resonator input and observe its low-pass filter effect (Fig. 17). The curve corresponds to the resonator transfer function. In this last case, due to the level of the known noise source, the frequency ranges to get the trend curves are more important and give more precision in cutoff frequency determination.

Classically, for 5 MHz quartz crystal resonator, the frequency range of the f 0 slope is 0.2 Hz-0.8 Hz and the frequency range of the f -2 slope is 2 Hz-6 Hz.

Values of σ y are compared in Table 3 and4 Finally, σ y is given by two extreme values. This method is reproducible and allows the same treatment in a resonator batch. Comparison between resonators is really simplified.

IV. CONCLUSION New double ovens have been designed to improve the ultimate noise floor of our carrier suppression bench. These new crystal ovens present an Allan standard deviation of about 2⋅10 -15 at 1 s in terms of relative frequency fluctuations.

Several improvements have been presented in order to increase the reliability of the phase noise measurement bench of 5 MHz and 10 MHz crystal resonators. The developed bench is now suitable to measure manufacturing production of resonators using this passive method.

ACKNOWLEDGMENT

Authors thank CNES for the funding support.

Figure 1 .

 1 Figure 1. Principle of the measurement bench.

  concept gives a lot of facilities to build the system.

Figure 2 .

 2 Figure 2. Resonator phase noise measurement bench.

Figure 3 .

 3 Figure 3. Data acquisition interface.

Figure 4 .

 4 Figure 4. Double quartz crystal ovens. (a) oven top view, (b) crystal oven, (c) cables oven.

Figure 5 .

 5 Figure 5. Quartz crystal model and its serial tuning capacitor. R m , L m and C m are the motional parameters of the quartz crystal and C 0 is the static capacitor. Typical equivalent parameters of 5 MHz quartz crystal resonator are: Quartz crystal static capacitor: C 0 ≈ 3 pF Quartz crystal motional capacitor: C m ≈ 0.2 fF The relative variation of the frequency is classically given by the following equation:

Fig. 6

 6 Fig. 6 shows the relative frequency fluctuations according to the tuning capacitor C s for a temperature variation of 1 °C.

Figure 6 .

 6 Figure 6. Δf/f due to the tuning capacitor C s for 1°C.

Figure 7 .

 7 Figure 7. Fluctuation of the calibration sideband level obtnained at the FFT analyzer output in dBV² according to the frequency offset between the carrier frequency and the sideband frequency.

Figure 8 .

 8 Figure 8. Calibration side band effect on phase noise measurment of 5 MHz quartz crystal resoantor (crystal dissipated power: 20 µW).

σ

  Y VARIATION ACCORDING TO SIDEBAND CALIBRATION ERROR (QUARTZ CRSYTAL POWER: 20 µW). σ y (τ) -calibration side band 0.4 Hz 1.3⋅10 -13 σ y (τ) -calibration side band 1 Hz 1.8⋅10 -13

Figure 9 .

 9 Figure 9. Offset HP3561A acquisition window.

Figure 10 .

 10 Figure 10. Offset stability according to the connection nature between DUTs and support.

Figure 11 .

 11 Figure 11. Offset drift according different attenuators.

Figure 12 .

 12 Figure 12. Quartz crystal phase noise measurement according to ouptut offset Quartz crystal dissipated power (100 µW).

Figure 13 .

 13 Figure 13. Effect of the carrier supression level on phase noise measurement of a quartz crystal pair (PQ = 20 µW).

Figure 14 .

 14 Figure 14. Effect of the measurement environment.

Figure 15 .

 15 Figure 15. Frequency bandwidth for f -1 et f -3 interpolation.

  . Both measured resonators are considered identical that means S φ (f) is equal to the single sideband power spectral density of phase fluctuations L(f). A significant difference can be observed in term of cut-off frequency. A precision of about 0.1 Hz is obtained on the Leeson frequency. According to the analyzer, L(f) can be measured with ± 2 dB of precision.

Figure 16 .

 16 Figure 16. L(f) of both 5 MHz Quartz crystal resonators (P Q = 20 µW).

Figure 17 .

 17 Figure 17. Measurement of the resonator transfer function.

  1,12E-13 8.87E-14 7,05E-14

Table 1

 1

gives the corresponding fluctuations in term of Allan standard deviation σ y .

TABLE I .

 I

TABLE II .

 II ACCEPTABLE CARRIER SUPPRESSION LEVEL.

TABLE III .

 III ALLAN STANDARD DEVIATION OBTAINED WITH fL FROM FIG. 16.

	σy	-130	-132	-134

L(1 Hz) 1,6 1,19E-13 9,46E-14 7,52E-14 1,7 1,27E-13 1,01E-13 7,99E-14 f L 1,8 1,34E-13 1,06E-13 8,46E-14

TABLE IV .

 IV ALLAN STANDARD DEVIATION OBTAINED WITH f L FROM FIG. 17.