Véronique Monteil

Annie Kolb

Jacques D'alayer

Pierre Beguin

Françoise Norel
email: francoise.norel@pasteur.fr

Identification of conserved amino acid residues of the Salmonella σ σ σ σ S chaperone Crl involved in Crl-σ σ σ σ S interactions

Keywords: Salmonella, crl, rpoS, sigma factor, RNA polymerase

Proteins that bind σ factors typically attenuate σ factor function by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary phase sigma factor σ S (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of σ S -dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than is rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a ∆crl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56 and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crlσ S interaction and that residues 1 to 71 in σ S are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur, and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.

INTRODUCTION

In bacteria, transcription depends on a multisubunit RNA polymerase (RNAP) consisting of a catalytically active core enzyme (E) with a subunit structure α 2 ββ'ω, that associates with any one of several σ factors to form different holoenzyme (Eσ) species. The σ subunit is required for specific promoter binding, and different σ factors direct RNAP to different classes of promoters, thereby modulating the gene expression patterns [START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF]. The RNA polymerase holoenzyme containing the σ 70 subunit is responsible for the transcription of most genes during exponential growth [START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF]. When cells enter stationary phase or are under specific stress conditions (high osmolarity, low pH or high and low temperatures) during exponential growth, σ S , which is encoded by the rpoS gene, becomes more abundant, associates with the core enzyme, and directs the transcription of genes essential for the general stress response and for stationary phase survival [START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF][START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF][START_REF] Klauck | The σ S subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli[END_REF].

Sigma factors compete for binding to a limited amount of the core polymerase [START_REF] Grigorova | Insights into transcriptional regulation and σ competition from an equilibrum model of RNA polymerase binding to DNA[END_REF][START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF][START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF][START_REF] Nyström | Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition?[END_REF]. σ 70 is abundant throughout the growth cycle and has the highest affinity of all sigma factors for E in vitro [START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF]. In contrast, levels of σ S only reach about one-third of the σ 70 levels upon entry into stationary phase, and σ S exhibits the lowest affinity for E of all sigma factors in vitro [START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF]. The cell uses at least two strategies to ensure the switch between σ 70 and σ Sassociated RNA polymerases and to allow gene expression to be reprogrammed upon entry into stationary phase. Several factors (Rsd, 6S RNA, ppGpp and DksA) indirectly increase σ S competitiveness by decreasing the ability of σ 70 to bind to E [START_REF] Klauck | The σ S subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli[END_REF]. In addition, the unconventional regulatory protein Crl increases the performance of σ S .

The crl gene product is a regulator of σ S activity in Escherichia coli [START_REF] Bougdour | Crl, a low temperature-induced protein in Escherichia coli that binds directly to the stationary phase sigma subunit of RNA polymerase[END_REF][START_REF] Gaal | Crl facilitates RNA polymerase holoenzyme formation[END_REF][START_REF] Pratt | Crl stimulates RpoS activity during stationary phase[END_REF][START_REF] Typas | Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σ S activity and levels[END_REF] and Salmonella [START_REF] Robbe-Saule | Effect of growth temperature on Crl-dependent regulation of σ S activity in Salmonella enterica serovar Typhimurium[END_REF][START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]41). In both species, the Crl protein binds σ S (2, 12) and facilitates RNA polymerase holoenzyme Eσ S formation [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF][START_REF] Typas | Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σ S activity and levels[END_REF], thereby enhancing σ S effectiveness [START_REF] Dong | RpoS regulation of gene expression during exponential growth of Escherichia coli K12[END_REF][START_REF] Gaal | Crl facilitates RNA polymerase holoenzyme formation[END_REF][START_REF] Lelong | The Crl-RpoS regulon of Escherichia coli[END_REF][START_REF] Robbe-Saule | Effect of growth temperature on Crl-dependent regulation of σ S activity in Salmonella enterica serovar Typhimurium[END_REF][START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]41,[START_REF] Typas | Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σ S activity and levels[END_REF]. The Crl protein of Salmonella binds σ S with a stoechiometry 1:1 and increases the affinity of σ S for the core enzyme 7-fold [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF]. In contrast, Crl does not bind σ 70 and does not modify the affinity of σ 70 for the core enzyme [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF]. Lelong et al. [START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF] reported that, in E. coli W3110, Crl can interact with the ferric uptake regulator Fur, a key protein for the control of intracellular iron concentration (8 and references therein) and that Fur represses crl transcription [START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF].

In an attempt to gain further insight into Crl function, we searched for homologues of Crl in protein databases. Residues conserved in all members of the Crl family were substituted by site-directed mutagenesis experiments to identify residues important for Crl activity in Salmonella. We used the bacterial adenylate cyclase two-hybrid system (BACTH, [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF] to determine whether these residues and specific regions of Crl are required for efficient interaction with σ S . We also used the BACTH system to assess the effect of the Crl mutations on the interaction of Crl with Fur.

MATERIALS AND METHODS

Bacterial strains, plasmids and growth conditions. Strains and plasmids are listed in Table 1. Bacteriophage P22HT105/1int was used to transfer mutations between Salmonella strains by transduction [START_REF] Schmieger | Phage P22-mutants with increased or decreased transduction abilities[END_REF]. Green plates, for screening for P22-infected cells or lysogens, were prepared as described previously [START_REF] Sternberg | Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium[END_REF]. Strains were routinely cultured in Luria Bertani medium (LB; 43). Minimal medium was M9 [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] containing 20 mM glucose. For routine monitoring of multicellular behaviour (rdar morphotype), Salmonella strains were grown at 28°C on LB agar plates without NaCl (LB0) supplemented with Congo Red (40 µg ml -1

) as described (CR plates,[START_REF] Römling | Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae[END_REF]. Antibiotics were used at the following concentrations: ampicillin (Ap), 100 µg ml -1

; carbenicillin (Cb), 100 µg ml Oxidative shock survival assay. Cells were grown to stationary phase in LB (optical density at 600nM (OD 600 ) of 3.5 to 4), washed, resuspended in PBS and mixed with H 2 O 2 at a final concentration of 15 mM. Aliquots of bacteria were removed at time intervals, and numbers of colony forming cells were determined on LB plates.

DNA manipulations and sequence analysis. Standard molecular biology techniques were used [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. Oligonucleotides were obtained from Sigma-Aldrich (France). DNA sequencing was performed by Cogenics (France). DNA and amino acid sequence analyses were conducted using the BLAST programs at the NCBI (National Center for Biotechnology Information).

Cloning of the rpoS T2 allele. We demonstrated that the nucleotide sequence of a PCRamplified rpoS gene from Salmonella enterica serovar Typhi strain T2 (rpoS T2 ) is identical to that in Salmonella ATCC14028 (41) except for a G/A mutation at position 318, which does not change the amino acid sequence, and a deletion of 8 nucleotides starting at position 109, which results in a frameshift and appearance of a premature stop codon in rpoS T2 . Total DNA from S. Typhi T2 was cleaved with BglII and the resulting fragments were cloned into the BamHI site of pACYC184. Recombinant plasmids were then transformed into the E. coli rpoS mutant strain MC1061K carrying an rpoS-dependent spvRAB-lacZ fusion on pSTF4 [START_REF] Kowarz | The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes[END_REF]. Selection of Cb R Cm R Lac + clones and subsequent examination of their plasmid DNA by restriction analysis and DNA sequencing of rpoS produced pACrpoS T2 containing a 6 kb BglII fragment carrying rpoS T2 . A 3.3 kb SphI-ScaI fragment carrying rpoS T2 was subsequently cloned into the SphI and SmaI sites of pUC19 to yield pUCrpoS T2 .

Construction of S. Typhimurium 2922KrpoS T2

. The rpoS allele in S. Typhimurium ATCC14028 was replaced by the rpoS T2 allele using the following strategy (41). pUCrpoS T2 contains the rpoS T2 allele of S. Typhi strain T2 and the downstream genes, including a gene encoding a putative decarboxylase (named STY3047 in S. Typhi and STM2922 in S. Typhimurium) that is not regulated by rpoS [START_REF] Bang | Alternative sigma factor interactions in Salmonella: σ E and σ H promote antioxidant defences by enhancing σ S levels[END_REF]. In pUCrpoS T2 K, a kanamycin resistance (Km) cartridge is located in the SmaI site of STY3047. pUCrpoS T2 K was introduced by electroporation into ATCCrpoS, where it was unstable. Recombination of the Km cartridge into the host genome with simultaneous loss of pUCrpoS T2 K resulted in recombinants that were resistant to kanamycin and sensitive to carbenicillin. A Km R Cb S Cm S recombinant was selected, checked by PCR for the presence of the STM2922::Km mutation and simultaneous replacement of the ∆rpoS::Cm mutation by the rpoS T2 allele. The rpoS T2 and STM2922::Km alleles were then co-transduced to ATCCrpoS. Transductants that were Km R but Cm S were selected, and transduction of the STM2922 ::Km mutation and simultaneous replacement of the ∆rpoS ::Cm mutation by the rpoS T2 allele were confirmed by PCR. One Km R Cm S strain, designated 2922KrpoS T2 , was also checked by DNA sequencing for the presence of the rpoS T2 allele. with buffer A containing 20 mM imidazole. σ S T2 -His 6 was eluted with buffer A containing 300 mM imidazole. After electrophoresis in a 10% polyacrylamide-SDS gel, the σ S T2 -His 6

Determination of the

protein was transferred on a PVDF membrane, stained with Amido Black and excised from the membrane for N-terminal sequence analysis. Amino-terminal Edman degradation was carried out on an Applied Biosystems sequencer.

Construction of a chromosomal crl-lac transcriptional fusion. We previously created a chromosomal mutation in the crl gene of Salmonella ATCC14028 using PCR-generated linear DNA fragments and the λRed recombination method (ATCCcrl, 40). A single-copy crl-lacZY transcriptional gene fusion was constructed in ATCCcrl using conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site as described [START_REF] Ellermeier | Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria[END_REF]. PCR assays were used to ensure integration of the plasmids in the correct location and to exclude the presence of multiple plasmid integrants (using standard test primers, such as those described in 11). We also used flanking locus-specific primers to amplify junction fragments that were subsequently analysed by DNA sequencing. Isogenic strains were constructed by P22 HT int mediated transduction of the mutations into the appropriate strains.

Enzymatic assays. β-galactosidase activity was measured as described by Miller [START_REF] Miller | Experiments in Molecular Genetics[END_REF] and is expressed in Miller units.

BACTH assays. The bacterial adenylate cyclase-based two hybrid (BACTH) assay is dependent upon the functional reconstitution of the Bordetella pertussis adenyl cyclase T18 and T25 subdomains by two interacting partners [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF]. MacConkey maltose plates supplemented with carbenicillin, kanamycin and 0.5 mM IPTG to assess the Mal + phenotype and on LB plates supplemented with 5-bromo-4-chloro-indolyl-β-D-galactoside (X-Gal, 40 µg ml -1 ), carbenicillin, kanamycin and 0.5 mM IPTG to assess the Lac + phenotype and β-galactosidase activity. Plates were incubated at 30°C for 3 days and colonies were then collected and their β-galactosidase activities were measured as described by Miller [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. For immunodetection of the Crl-T18 and T25-σ S chimeras, BTH101

derivatives were grown to stationary phase (OD600= 3.5) in LB in the presence of 2 mM cAMP and 0.5 mM IPTG to induce fully the lac promoter on pKT25, pKNT25 and pUT18.

Construction of plasmids with mutated crl genes

Site-directed mutagenesis was performed using the QuikChange II Site-directed mutagenesis kit (Stratagene) as recommended by the manufacturer. Site-directed mutagenesis of plasmid pACcrl-1 yielded plasmids pACcrl Y22A , pACcrl F35A , pACcrl C28A, pACcrl C37A, pACcrl C41A, pACcrl F53A, pACcrl W56A, pACcrl G74A, pACcrl G80A, pACcrl W82A, pACcrl F103A, pACcrl C28-37 , pACcrl C28-41 , pACcrl C37-C41 and pACcrl C28-37-41 (Table 1). Plasmids pUTCrl Y22A, pUTCrl F53A, pUTCrl W56A, pUTCrl W82A and pUTCrl G80A (Table 1) were obtained by cloning PCR amplified DNA fragments from plasmids pACcrl Y22A , pACcrl F53A, pACcrl W56A, pACcrl W82A , and pACcrl G80A, respectively, between the PstI and EcoRI sites of pUT18 using primers Crl-Pst and Crl-Eco (as described above). Plasmids pUTCrl R51A, pUTCrl E52A, pUTCrl G55A, pUTCrl W57A were obtained by site-directed mutagenesis of plasmid pUTCrl (Table 1). All plasmids were confirmed to be correct by DNA sequencing.

Electrophoresis and immunoblot analysis of proteins. Whole-cell extracts were prepared and SDS-polyacrylamide gel electrophoresis was carried out as described by Silhavy et al. [START_REF] Silhavy | Experiments with Gene Fusions[END_REF]. The amount of protein in whole-cell lysates was determined using the DC Protein Assay kit (Bio-Rad). Equal amounts of protein were loaded in each slot. The molecular sizes of the proteins were estimated using molecular size standards (Fermentas, France). Rabbit antibodies against the Crl protein of Salmonella were from Robbe-Saule et al. [START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]. Rabbit antibodies against the σ S protein of S. enterica serovar Typhimurium were from Coynault et al. [START_REF] Coynault | Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon[END_REF]. Proteins were transferred to Amersham Hybond P membranes (GE Healthcare) and incubated with the polyclonal rabbit antibody serum as previously described [START_REF] Coynault | Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon[END_REF]. Bound antibodies were detected using a secondary anti-rabbit antibody linked to peroxidase and the Amersham ECL plus western blotting detection system kit (GE Healthcare). Gel retardation assays. The DNA encoding rpoS was amplified using primers HK1 5'-AGGCTCGGATCCATGAGTCAGAATACGCTGAAAGTTCAT-3' and HK2 5'-

Overproduction and purification of

TTCCGAAAGCTTTTACTCGCGGAACAGCGCTTCGATATT-3' and cloned into the

BamHI and HindIII sites of pQE30 to yield pQE30rpoS. The nucleotide sequence of the rpoS insert in pQE30rpoS was checked by DNA sequencing. S. Typhimurium His 6 -σ S produced from pQE30rpoS was purified using Ni-affinity chromatography as described for His 6 -σ S T2 .

E. coli core (Epicentre) was used to reconstitute the σ S -holoenzyme, which was incubated with variant Crl proteins (wild-type or mutants) or buffer. 9 µl of the protein complexes were then added to 3 µl of [ 32 P]-labelled katN fragment in buffer A (40 mM Hepes pH 8.0, 10 mM MgCl 2 , 100 mM K-glutamate, 2 mM DTT) containing 500 µg ml -1 BSA, and incubated at 28°C for 20 min. The final concentrations of core, His 6 -σ S , Crl and katN fragments were respectively 8 nM, 32 nM, 3 µM and 1 nM. After addition of 3 µl loading buffer (buffer A containing 50% sucrose, 0.025 % xylene cyanol blue and 150 µg ml -1 of heparin) the mixture was loaded onto a 6 % native polyacrylamide gel run in TG buffer (25 mM Tris, 192 mM Glycine pH 8.5) at 8V/cm. The gel was dried before being autoradiographed and quantified using a PhosphorImager (Molecular Dynamics).

Abortive initiation assays.

A mixture of the 207-bp lacUV5 fragment (10 nM), ApA (2 mM), and 80 µM [α-32 P] UTP was added to an equal volume of the holoenzyme (60 nM, His 6 -σ S : core ratio = 2.5 : 1) preincubated with buffer or Crl (wild-type or variants) at 6 µM concentration at 28° C. Aliquots were removed at various times. The aliquots were spotted onto Whatman 3MM paper prespotted with 100 mM EDTA, and chromatograms were developed as previously described [START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF].

RESULTS

Distribution and sequence conservation of Crl in microbial sequenced genomes

A Blast search in 1264 sequenced bacterial genomes (www.ncbi.nlm.nih.gov) revealed 143 genomes containing a crl gene. They are all γ-Proteobacteria and contain an rpoS gene (Table 2). Crl was absent from many rpoS-containing bacteria, for example Pseudomonas, in which RpoS plays an important role [START_REF] Schuster | The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing[END_REF]. Thus, crl is not as widely distributed as rpoS and it is also less conserved at the sequence level (Table 2). Sequence comparison of the 143 Crl proteins identified 60 Crl sequences that differed by at least one amino acid. Alignment of these 60 Crl sequences by clustalW revealed 17 residues that are conserved in all members of the Crl family (Figure 1). Analysis of the 16 completed genomic sequences of Salmonella (www.ncbi.nlm.nih.gov) indicated that the Crl sequence is highly conserved (Figure 1 and Table 2). Interestingly, the crl gene in S. enterica serovar Paratyphi B SPB7 lacks a G at position 73 in the open-reading frame, causing the appearance of a premature stop codon and the likely production of a truncated, non-functional Crl protein.

The first 71 amino acids of σ σ σ σ S are non-essential for Crl binding

Crl is less conserved than the full length RpoS protein (Table 2). However, the first 55 residues of RpoS are poorly conserved, whereas the remaining 275 residues of RpoS are highly conserved (data not shown). For instance, the level of sequence identity between RpoS STM and the RpoS proteins from the Enterobacteria Proteus mirabilis and Providencia stuartii is 43% and 40% for aminoacids 1-55 compared to 87% and 86% for amino acids 56-330, respectively. These first 55 residues define a specific domain of σ S that is not well conserved in other sigma factors besides σ 70 (corresponding roughly to region σ 1.1 , see also Discussion section and 32, 51), and might thus play a role in the specificity of recognition of σ S by Crl. In addition, σ 1.1 and Crl might play complementary roles in the formation of Eσ .

Crl increases the affinity of σ S for the core enzyme [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF]. σ 1.1 stabilizes the interaction between σ 70 and core RNAP [START_REF] Hinton | The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA polymerase more rapidly in the absence of σ 70 region 1.1: Evidence that region 1.1 stabilizes the interaction between σ 70 and core[END_REF] and might play a similar role in σ S -core interaction. These possibilities prompted us to determine whether this domain of σ S is required for σ S activity and for Crl activation.

We first examined the sensitivity to Crl activation of a σ S mutant lacking the first 39 residues of σ S (σ S T2 , Figures 2 and3) and thus lacking most of region σ 1.1 [START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF][START_REF] Vassylyev | Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6A resolution[END_REF]. rpoS T2 is a natural rpoS allele from S. Typhi strain T2 that contains a deletion of eight nucleotides in the 5' end of the coding sequence resulting in the appearance of a premature stop codon (Figure 2A). Surprisingly, rpoS T2 was able to direct the synthesis of a 35 kDa σ S protein (σ S T2 , Figure 3A). N-terminal sequence analysis indicated that σ S T2 has a N-terminal Met residue followed by a sequence that is identical to that downstream of residue 39 in native σ S (Figure 2B). We inferred that σ S T2 is encoded from the mutant gene in which translation initiates at a GTG start codon preceding codon 40 of σ S in rpoS T2 , and upstream of which is a putative ribosome binding site (SD, Figure 2B). Translational reinitiation within the E. coli rpoS gene has also been reported previously [START_REF] Gowrishankar | In vitro properties of RpoS (σ S ) mutants of Escherichia coli with postulated N-terminal subregion 1.1 or C-terminal region 4 deleted[END_REF]. To facilitate characterization of the rpoS T2 allele in isogenic backgrounds, the rpoS gene from S. Typhimurium ATCC14028 was replaced by rpoS T2 yielding strain 2922KrpoS T2 . During exponential growth, σ S T2 was produced in higher amounts than the wild-type σ S protein (OD600 0.1 to 1.4, Figure 3B), likely because the Nterminal 11 residues of σ S involved in ClpX recognition and σ S proteolysis by ClpP [START_REF] Flynn | Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpXrecognition signals[END_REF][START_REF] Stüdemann | Sequential recognition of two distinct sites in σ S by the proteolytic targeting factor RssB and ClpX[END_REF] were not present in σ S T2 . σ S is required for bacterial resistance to various stresses during stationary phase (the so-called general stress resistance) [START_REF] Ishihama | Functional modulation of Escherichia coli RNA polymerase[END_REF][START_REF] Klauck | The σ S subunit of RNA polymerase as a signal integrator and network master regulator in the general stress response in Escherichia coli[END_REF]. The σ S -dependent gene katE encodes a catalase, an enzyme that detoxifies hydrogen peroxide (H 2 O 2 ) and contributes to the resistance of Salmonella to oxidative stress in stationary phase [START_REF] Robbe-Saule | Effect of growth temperature on Crl-dependent regulation of σ S activity in Salmonella enterica serovar Typhimurium[END_REF]. Despite the high amount of σ S T2 produced, strain 2922KrpoS T2 was slightly less resistant to H 2 O 2 than the wild-type strain 2922K and exhibited lower expression of the katE-lacZ fusion (Figure 2CD). This suggested that σ S T2 is less active than wild type σ S . In vitro transcription experiments with σ S T2 confirmed this hypothesis (data not shown). Interestingly, the levels of H 2 O 2 resistance and the expression of the catalase gene katE were more dependent on Crl activation in 2922KrpoS T2 than in the wild-type strain (Figure 2CD). This indicated that the σ S T2 protein is able to interact with Crl and that its activity is highly dependent on Crl.

We previously used the bacterial two-hybrid system (BACTH system, 22) to detect interaction between Crl and σ S (12). C-terminal fusions of S. Typhimurium σ S to T25 (T25σ S ) and N-terminal fusions of S. Typhimurium Crl to T18 (Crl-T18) were produced in the E.

coli cya lac + strain BTH101 from pKTRpoS and pUTCrl, respectively, and the resulting βgalactosidase activities were measured (Table 3). As expected [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF], T25-σ S interacted with

Crl-T18 (Table 3). In this system, the first 38 residues of σ S were dispensable for efficient binding of Crl (compare T25-σ S and T25-σ S ∆1-38 in the presence of Crl-T18, Table 3).

Moreover, deletion of residues 1 to 38 from σ S resulted in increased levels of the chimera and β-galactosidase activity in the BACTH assay (Figure 3C lanes 2 and 5, Table 3). This is probably again due to the absence of the ClpX recognition site in T25-σ S ∆1-38 . Interestingly, deletion of residues 1 to 71 in T25-σ S ∆1-71 , and deletion of residues 12 to 71, which does not remove the ClpX recognition site [START_REF] Flynn | Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpXrecognition signals[END_REF][START_REF] Stüdemann | Sequential recognition of two distinct sites in σ S by the proteolytic targeting factor RssB and ClpX[END_REF] in T25-σ S ∆12-71 , had a more pronounced effect on σ S levels and β-galactosidase activity than deletion of residues 1 to 38 (in T25-σ S ∆1-38 ) (Figure 3C lanes 4-6, Table 3). This indicated that the first 71 residues in σ S are non-essential for Crl binding, and further suggested that the deletion of this region induces a conformational change in σ S that favors its stability. Altogether, these results suggested that Crl interacts with the conserved amino acid region (72-330) of σ S .

Identification of conserved residues important for Crl activity

Alanine substitution mutagenesis was performed to investigate the functional relevance of the conserved residues in Crl. The conserved residues are located in four regions of Crl (one third of them are aromatic amino-acids, Figure 1). As a first step, one or two residues in each conserved region were mutated. We also mutated the three cysteine residues in Crl. pACcrl-1 expresses the crl gene from S. Typhimurium under the control of the vector cat promoter, and is able to complement a ∆crl mutant of Salmonella [START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]. Site-directed mutagenesis of pACcrl-1 yielded eleven derivatives expressing altered Crl proteins (Table 1 and Figure 4A). The ability of the mutated crl genes to complement the Salmonella ∆crl mutant was assessed using three different tests (Figures 4 and5). The development of the rdar morphotype depends on Crl [START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF] and was used as a qualitative test (Figure 5). This colony morphology is caused by production of curli and cellulose and is correlated with biofilm formation and expression of the regulatory gene csgD [START_REF] Römling | Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae[END_REF]. In ATCC14028, the expression level of a csgD-lacZ fusion and the H 2 O 2 resistance level at 28°C both depend on Crl [START_REF] Robbe-Saule | Effect of growth temperature on Crl-dependent regulation of σ S activity in Salmonella enterica serovar Typhimurium[END_REF][START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF], and were used as quantitative assays (Figure 4BC).

Derivatives of pACcrl-1 expressing the Crl proteins with F35A, C28A, C37A, G74A, G80A and F103A substitutions complemented ∆crl as well as did pACcrl-1 (Figures 45and data not shown). Two substitutions, W56A and F53A, located in a conserved motif of Crl (residues 48 to 57, Figure 1), substantially diminished complementation in all three tests (Figure 4 and Figure 5 spots 7-8). Two additional substitutions, Y22A and W82A, also decreased the complementation levels but to a lesser extent (Figure 4 and Figure 5 spots 3 and 11). These four substitutions did not significantly decrease the cellular level of Crl (Figure 6A) and, thus, likely affected its activity.

The Salmonella Crl protein contains three cysteines, (C28, C37, C41), one of which (C41) is conserved in all Crl members. Alanine substitution of cysteine residues (C28A, C37A and C41A) did not significantly affect the levels of Crl protein or its activity (Figures 4, 5 and 6A). Only a slight effect of C41A on H 2 O 2 resistance was observed (Figure 4B).

Interestingly, substitution of all three cysteine residues substantially decreased Crl levels mutant protein were lower than that of Crl C37-41 (Figure 6A, lanes 19-20). This suggests that the C28A substitution further decreased the stability of Crl C37-41 .

Screening of the crl mutations in the BACTH system and in vitro assays

We next determined whether the mutations selected above affected Crl-σ S interaction.

DNA encoding proteins with Y22A, F53A, W56A or W82A substitutions or the neutral G80A substitution were introduced into pUTCrl and the ability of the Crl-T18 derivatives to interact with σ S was assessed in the presence of T25-σ S (Table 3). Interestingly, the Y22A, F53A, W56A, W82A substitutions, but not the G80A substitution, all abolished the interaction between Crl-T18 and T25-σ S (Table 3). These substitutions did not affect levels of Crl-T18 (Figure 6B). The altered Crl-T18 hybrid proteins were also unable to interact with the T25-σ S derivative lacking amino acids 12 to 71 (data not shown). In conclusion, the Y22A, F53A, W56A and W82A substitutions all abolished the interaction between Crl and σ S either directly or indirectly through conformational changes in Crl.

The activities of the variant proteins were also probed in vitro by gel retardation and abortive initiation assays, based on the ability of Crl to increase the amount of promotercomplexes formed by the σ S -RNA polymerase (Eσ S ) and activate transcription initiation [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF][START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]. As shown on the autoradiogram of the native polyacrylamide gel (Figure 7A between Crl variants and σ S . Therefore, abortive initiation assays, which do not require heparin addition and can be exploited for more quantitative studies, were conducted using the well-characterised lacUV5 promoter, which can be transcribed in vitro by Eσ 70 and Eσ S and, unlike katN, has the advantage of producing high amounts of abortive products [START_REF] Malan | Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter[END_REF]. As expected, Eσ S produced less ApApUpU tetranucleotide from lacUV5 in the presence of the Crl variants than with the wild-type protein (Figure 7B). The results were more obvious than in gel retardation assays since wild-type Crl stimulated Eσ S -lacUV5 abortive transcription by 4-fold under our conditions. After 30 min incubation at 28°C, the observed decrease in ApApUpU synthesis was 35 % for Y22A, 40% for W82A and 90% for F53A. The addition of W56A Crl resulted in an even lower activity than in the absence of Crl, suggesting a slight inhibitory effect of this variant on σ S -dependent transcription. Altogether, these results emphasise the role of the REF-GWW motif (Figure 1) in Crl activation of σ S -dependent promoters. Consistent with this hypothesis, the R51A, W57A substitutions and to a lesser extent the E52A and G55A substitutions affected the interaction between Crl and σ S in the BACTH assay (Table 3) but did not affect levels of Crl-T18 (Figure 6B).

Fur-Crl interactions in Salmonella

To determine whether the four substitutions studied above affected Crl-σ S interaction specifically, we assessed their effect on the interaction of Crl with another partner. In a previous study using E. coli W3110, an interaction between Crl and the ferric uptake regulator Fur was detected by co-purification [START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF]. To investigate the interaction between the Salmonella Crl and Fur proteins in the BACTH assay, we constructed plasmids pUTFur encoding a Fur-T18 protein and pKTFur encoding a T25-Fur protein. Significant interactions between the Crl-and the Fur-hybrid proteins were not detected (Table 3). The Fur hybrid proteins were stable, as indicated by the fact that the expected dimerization of Fur-T18 and T25-Fur (8 and references therein) gave a positive signal (Table 3).

crl transcription in E. coli W3110 is repressed by Fur (by 100-fold) and by Crl (by more than 10-fold) [START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF]. To assess the possible relationship between Crl and Fur in Salmonella ATCC14028, we examined the effects of Fur and Crl on crl expression. We first compared Crl levels in the wild-type and fur strains by immunodetection of the Crl protein (Figure 6C). Cells were grown to stationary phase in LB at 30°C and 37°C, and also on LB agar at 30°C, a growth condition used to study the regulation of crl expression by Fur in E.

coli W3110 [START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF]. The amount of Crl detected in the fur strain was the same as in the wild-type under all three conditions (Figure 6C, compare lanes 1 and 2, 4 and 5, 7 and 8). In addition, the levels of crl-lacZ-encoded β-galactosidase were not significantly affected either by the fur

DISCUSSION

Analysis of the protein sequence databases revealed the narrow distribution of Crl homologues in bacteria. In contrast, RpoS homologues are found in many gram-negative bacteria of the γ-, δand βsubdivisions (www.ncbi.nlm.nih.gov). Several hypotheses might explain the absence of Crl in bacteria containing a RpoS homologue. Crl compensates for a low affinity of σ S for the E, and might be dispensable in bacteria expressing a σ S protein with a high affinity for E. Moreover, the physiological impact of Crl is greatest at low concentrations of σ S [START_REF] Robbe-Saule | Crl activates transcription initiation of RpoSregulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium[END_REF]41), and the environment can modulate the impact of Crl by affecting σ S level [START_REF] Robbe-Saule | Effect of growth temperature on Crl-dependent regulation of σ S activity in Salmonella enterica serovar Typhimurium[END_REF]. Thus, depending on the bacterial lifestyles and environment encountered, cellular physiology and the mechanisms of regulation of rpoS expression, Crl might be dispensable. Alternatively, functional homologues of Crl might exist in some rpoScontaining bacteria or some species might use alternative strategies to favor σ S interaction with E. Interestingly, we did not find Crl homologues in bacteria that do not contain rpoS.

This is consistent with previous work suggesting that the main function of Crl is to favor σ S interaction with RNA polymerase (41,[START_REF] Typas | Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of σ S activity and levels[END_REF].

Of the 16 Salmonella strains whose genome sequence is completed, one, S. Paratyphi SB7, contains a frameshift mutation in crl that results in the appearance of a premature stop codon. This observation is reminescent of previous findings that rpoS null mutants can be found in natural isolates of S.Typhi [START_REF] Coynault | Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon[END_REF][START_REF] Robbe-Saule | Characterization of the RpoS status of clinical isolates of Salmonella enterica[END_REF][START_REF] Robbe-Saule | The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses[END_REF], and is consistent with our previous observation that a ∆crl mutation increases the competitive fitness of Salmonella by attenuating σ S activity (41). The selection of rpoS mutants in bacterial populations likely results from a growth advantage of rpoS mutants in the absence of environmental stress [START_REF] King | A regulatory trade-off as a source of strain variation in the species Escherichia coli[END_REF][START_REF] Notley-Mcrobb | rpoS mutations and loss of general stress resistance in Escherichia coli populations as a consequence of conflict between competing stress responses[END_REF][START_REF] Nyström | Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition?[END_REF]. However, in environments where bacteria encounter mild stress, ∆rpoS mutants are outcompeted by the wild-type strain, whereas rpoS attenuated mutants exhibit increased fitness [START_REF] Notley-Mcrobb | rpoS mutations and loss of general stress resistance in Escherichia coli populations as a consequence of conflict between competing stress responses[END_REF]41,[START_REF] Zambrano | ATCCfur sdhA-lacZ-k ATCC14028 ∆fur-4l::cat sdhA-lacZY-Km ATCCcrl-lacZ ATCC14028 crl-lacZY-Km ATCCrpoS crl-lacZ ATCC14028 ∆rpoS::Cm crl-lacZY-Km ATCCfur crl-lacZ ATCC14028 ∆fur-41::cat crl-lacZY-Km ATCCrpoSfur crl-lacZ ATCC14028 ∆rpoS ∆fur-41::cat crl-lacZY-Km 2922K ATCC14028 ∆STM[END_REF]. We can predict that the beneficial effect of a crl mutation is significant when the survival strategy requires "enough but not too much" σ S activity.

We showed that four conserved residues (Y22, F53, W56, W82) are important for Crl activity and for Crl-σ S interaction but not for Crl stability. In contrast, a Crl protein in each of its three cysteine residues was substituted was unstable. The Y22A and W82A substitutions had a more dramatic effect in the BACTH system than in the complementation tests and the in vitro assays (Table 3 andFigures 4, 5 and7). The IPTG-inducible lac promoter on pKT25 and pUT18 is induced by cAMP and, thus, a mutation that decreases the efficiency of the interaction between T25-σ S and Crl-T18 also lowers the expression level of the hybrid proteins, thereby amplifying the effect of the mutation (24 and Daniel Ladant personal communication).

The F53A and W56A substitutions are located in a conserved motif in the middle part of the protein (REF-GWW Figure 1). Analysis of four additional substitutions (R51A, E52A, G55A W57A) in the BACTH assay confirmed the role of the REF-GWW motif in Crl-σ S interactions (Table 3, Figure 6B). The two other substitutions, Y22A and W82A, are outside of this motif. At least some of these mutations likely affect Crl binding to σ S indirectly through conformational changes. The three-dimensional structure of Crl would provide new insight into this issue. Unfortunately, our attempts to crystallize Crl have been unsuccessful.

In addition, deletion experiments in the BACTH system did not allow us to delineate a subregion of Crl involved in σ S binding. Crl might spread along σ S or, more likely, a specific conformation of Crl might be required for efficient interaction with σ S .

The primary determinants of σ S involved in the binding of Crl are unknown, but the lack of interaction between Crl and σ 70 [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF] suggests that the interaction involves sequence determinants or structural features that are specific to σ S . The vast majority of σ factors, including σ S , belong to the so-called σ 70 family, reflecting their relationship to the principal σ factor of E. coli, σ 70 . Sequence alignments of σ 70 family members of groups 1 and 2, to which σ S belongs, reveal that they have four conserved regions (regions 1-4) [START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF][START_REF] Paget | The σ 70 family of sigma factors[END_REF]. Among these, region 2 (subregions 2.1 to 2. σ 1.1 shows little conservation between σ 70 and σ S and is not found in the alternative sigma factors of the σ 70 family [START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF][START_REF] Paget | The σ 70 family of sigma factors[END_REF]. Therefore, the specificity of recognition between Crl and σ S might involve σ 1.1 . However, our findings that (1) σ S T2 , a protein lacking residues 1 to 39, including most of region σ 1.1 in native σ S (32, 51) is dependent on Crl activation (Figure 2), and (2) deletion of residues 1 to 71 of σ S , which removes region σ 1.1 and part of subregion 1.2 [START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF][START_REF] Vassylyev | Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6A resolution[END_REF] does not abolish Crl-σ S interaction (Table 3), ruled out this possibility.

Experiments are in progress to determine which region of σ S is involved in Crl binding. In addition to its autoinhibitory role, σ 1.1 stabilizes the interaction between σ 70 and core RNAP [START_REF] Hinton | The bacteriophage T4 inhibitor and coactivator AsiA inhibits Escherichia coli RNA polymerase more rapidly in the absence of σ 70 region 1.1: Evidence that region 1.1 stabilizes the interaction between σ 70 and core[END_REF]. Our finding that σ S T2 is -less active but -more dependent on Crl activation than wild-type σ S (Figure 2CD and data not shown) is consistent with a similar role for σ 1.1 in σ S -core interaction. Crl increases the affinity of σ S for core RNAP [START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF] and might partially compensate for the absence of a full region σ 1.1 in σ S T2 . cultures (optical density at 600 nm = 0.5) of Salmonella were diluted into LB0 pre-warmed at 28°C to prolong the exponential phase. Aliquots were removed at various time intervals and β-galactosidase activity measured (lines) according to the method of Miller [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. The growth phase was determined by measuring the culture turbidity at an optical density of 600 nm (dashed line, the growth curve was similar for all the strains). The measurements were repeated twice, and a representative experiment is shown.

FIGURE 5. Rdar morphotypes of Salmonella ATCCcrl harbouring different plasmids.

The ∆crl mutant harbouring the vector pACYC184, the wild-type crl gene on pACcrl-1 and mutated pACcrl-1 derivatives (

- 1 ; 1 ; 1 .

 111 chloramphenicol (Cm), 15 µg ml -1 for the chromosomal resistance gene and 30 µg ml -1 for the plasmid resistance gene; kanamycin, (Km) 50 µg ml and tetracycline (Tet) 20 µg ml -

  N-terminal sequence of σ σ σ σ S T2 . pQErpoS T2 , which produces the rpoS T2 gene with a C-terminal His 6 extension under the control of the pQE60 IPTG-inducible promoter, was constructed as follows. Primers specific to the ends of rpoS (5were used to amplify a 1 kb fragment of S. Typhi T2 total DNA by PCR. EcoRI and BglII restriction sites were incorporated at its 5' and 3' ends, respectively. After digestion with EcoRI and BglII, the PCR-amplified fragment was ligated into the EcoRI and BglII sites of pQE60. The nucleotide sequence of the rpoS insert in pQErpoS T2 was verified. In this construct, rpoS T2 is expressed from its own translation sequences. E. coli JM109 carrying pQErpoS T2 was grown in LB containing ampicillin at 37°C to an optical density of 0.9, when 0.7 mM isopropyl-β-Dthiogalactoside (IPTG) was added. The cells were harvested after 5 h, resuspended in buffer A (50 mM Na 2 HPO 4 pH 8, 300 mM NaCl) and then disrupted in by French Press. The crude cell extract was supplemented with Benzonase (1250 U) and imidazole (10 mM) and centrifuged at 27,000 g for 30 min. The supernatant was added to 1.5 ml Ni-NTA column and washed

  The resulting cAMP binds to and activates the transcription activator CRP, a positive regulator of t lac and mal operons involved in lactose and maltose catabolism. Derivatives of pUT18, pKT25 and pKNT25, used in the BACTH assays, were constructed by cloning PCR amplified DNA fragments encoding the protein of interest from S. Typhimurium between the PstI and EcoRI sites of pUT18 and pKNT25 and the XbaI and KpnI sites of pKT25. The DNA encoding Crl was amplified using primers Crl-Pst 5'-CA TGC CTG CAG AAG GAG ATC GCA ATG ACG TTA CCG AGT-3' and Crl-Eco 5'-T CGA TGA ATT CGA TGC CGA CAG TTT TAC CGG CTC GTC GT-3', restricted by PstI and EcoRI and cloned into the PstI and EcoRI sites of both pUT18 and pKNT25 to yield pUTCrl and pKNTCrl, respectively. The DNA encoding RpoS was amplified using primers RpoS-Xba 5'-TCG ACT CTA GAT ATG AGT CAG AAT ACG CTG AAA GTT CAT-3' and RpoS-Kpn 5'-AC TTA GGT ACC TTA CTC GCG GAA CAG CGC TTC GAT-3' restricted by XbaI and KpnI and cloned into the XbaI and KpnI sites of pKT25 to yield pKTRpoS. The DNA encoding Fur was amplified using primers Fur-Pst 5'-CAT GCC TGC AGC ATG ACT GAC AAC AAT ACC GCA-3' and Fur-Eco 5'-T CGA TGA ATT CGA TTT AGT CGC GTC ATC GTG CGC GT-3' for cloning into the PstI and EcoRI sites of pUT18 (yielding pUTFur) and using Fur-Xba 5'-TCG ACT CTA GAC ATG ACT GAC AAC AAT ACC GCA TTA AAG A-3' and Fur-Kpn 5'-AC TTA GGT ACC TTA TTT AGT CGC GTC ATC GTG CGC GT-3' for cloning into the XbaI and KpnI sites of pKT25 (yielding pKTFur). pKTRpoS ∆12-71 is a derivative of pKTRpoS with an in frame deletion of an internal HpaI-DraI fragment. pKTRpoS ∆1-38 contains a DNA fragment amplified with primers RpoST2-Xba 5'-TCG ACT CTA GAC CTG GCT GAA GAA GAG CTG TTA TCG CAA-3' and RpoS-Kpn (see above) and cloned into the XbaI and KpnI sites of pKT25. pKTRpoS ∆1-71 contains a DNA fragment amplified with primers RpoS-Xba5 5'-TCG ACT CTA GAA ACA GCC GAA GAA GAA GTC TAT TTT GCG CGT-3' and RpoS-Kpn (see above) and cloned into the XbaI and KpnI sites of pKT25. All plasmids were confirmed to be correct by DNA sequencing. The E. coli cya strain BTH101 was transformed with derivatives of plasmids pKT25, pKNT25, and pUT18 encoding proteins fused to the T25 and T18 fragments of Bordetella pertussis adenyl cyclase. Co-transformants were plated onto

His 6 -

 6 Crl variants. A 250 ml of JM109 carrying pQEcrl wild-type or mutant was grown in LB medium containing carbenicillin at 30°C to an optical density of 0.6, and then 1 mM IPTG was added. After 30 min, the temperature was lowered to 23° C. The cells were harvested after 4 h, washed, resuspended in 10 ml of buffer A (25 mM Tris pH 8.0, 1 M NaCl, 0.01% NP-40) supplemented with "Complete EDTA free anti-protease" as described by the manufacturer (Roche) and then disrupted in a French press (Aminco). The crude cell extract was centrifuged at 15,000 g for 30 min. The supernatant was adjusted to 3 mM imidazole, added to 1.5 ml of Ni-nitrilotriacetic acid agarose (QIAGEN) and gently mixed for 1 h. The slurry was packed onto an Econo-Pac column (Bio-Rad) and washed with 15 ml of buffer A containing 20 mM imidazole. His 6 -Crl was eluted with buffer A containing 250 mM imidazole, dialyzed against buffer B (20 mM Tris-HCl pH 8, 50 mM NaCl, 0.01% NP-40), and added to DNA-cellulose equilibrated in the same buffer (0.3 ml, Sigma D-8515). The DNA-cellulose was removed by centrifugation and the concentrations of the proteins determined by a Bradford assay.

(

  Figure 6A lanes 17 and 20). Examination of the Crl family members indicated that their all contain at least 2 cysteine residues. Altogether, these results suggested that the presence of at least one, and of probably two, cysteine residue(s) is required for Crl stability. Double mutations in the cysteine residues in Crl (Crl C28-37 , Crl C28-41 , Crl C37-41 ) were constructed to determine what combination of cysteine substitution affected Crl stability. The combinations of C28A and C37A substitutions and C28A and C41A substitutions did not significantly affected the levels of Crl protein (Figure 6A lanes 14 and 15). In contrast, alanine substitution of both C37 and C41 substantially decreased Crl levels (Figure 6A lane 16) suggesting that these two cysteine residues are important for Crl stability. Levels of the triple Crl C28-37-41

  lane 3), Eσ S binding to the [ 32 P]-labelled katN promoter was stimulated 2-fold by wild-type Crl. The Y22A and W82A variants were still able to increase the formation of Eσ S -katN complexes by about 1.8 and 1.6 fold (Figure7A lanes 4 and 6). In contrast, the F53A and W56A variants of Crl (Figure7Alanes 5 and 7) had hardly any effect, in agreement with their highly reduced ability to stimulate csgD-lacZ expression or hydrogen peroxide resistance. The gel retardation methodology is only semi-quantitative, however, and the addition of heparin to disrupt nonspecific core-DNA binding before electrophoresis might have disturbed weak interactions

4 )

 4 and region 4 (subregions 4.1 and 4.2) contain DNA-binding domains that mediate recognition of the conserved -10 and -35 elements of σ 70 -dependent promoters respectively. The linear division of σ 70 factors into functionally distinct regions is largely confirmed by structural data, which revealed that primary sigma factors have four flexibly linked domains, σ 1.1 , σ 2 , σ 3 and σ 4 , containing regions 1.1, 1.2-2.4, 3.0-3.1, and 4.1-4.2, respectively[START_REF] Campbell | Regulation of bacterial RNA polymerase σ factor activity: a structural perspective[END_REF][START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF]. Free σ factors generally do not specifically bind promoter DNA, and the N-terminal σ 1.1 region is autoinhibitory(4,[START_REF] Dombroski | Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA[END_REF]. σ 1.1 might act indirectly to inhibit promoter binding by stabilizing a compact conformation of σ that is incompatible with promoter recognition[START_REF] Sorenson | Disulfide cross-linking indicates that FlgMbound and free σ 28 adopt similar conformations[END_REF]. Binding to core RNAP induces large movements of the σ domains (3), converting σ into an active conformation in which the DNA binding determinants in σ 2 and σ 4 are exposed[START_REF] Murakami | Bacterial RNA polymerase: the wholo story[END_REF].

3 FIGURE 2 .

 32 FIGURE 2. Characterization of the rpoS mutant allele rpoS T2 .

FIGURE 3 .FIGURE 4

 34 FIGURE 3. Expression of σσ σ σ S wild-type and mutant proteins.

FIGURE 7 .

 7 FIGURE 7. Effects of the Crl variants on Eσ σ σ σ S promoter binding and abortive

FIGURE 8 . 40 .

 840 FIGURE 8. Expression of crl-lacZ and sdhA-lacZ gene fusions in Salmonella.

Table 1 : Bacterial strains and plasmids used in this study ___________________________________________________________________________

 1

	Strain or plasmid	Characteristics	Source or reference a
	Escherichia coli
	MC1061K	araD139 ∆(ara-leu)-767 ∆(lacIPOZY)X74 rpsL	26
		galU galK rpoS::kan
	JM109	recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi	43
		∆(lac-proAB) F'(traD36 proAB + lacI q lacZ∆M15)
	BTH101	F-cya-99 araD139 galE15 galK16 rpsL1 (Str R )	D. Ladant
		hsdR2 mcrA1 mcrB1
	Salmonella serovar Typhi
	T2	Wild-type		CIS	b
	Salmonella serovar Typhimurium
	C52	Wild-type		26
	C52K	C52 ∆rpoS::kan		26
	ATCC14028	Wild-type		ATCC	c
	ATCCrpoS	ATCC14028 ∆rpoS		41
	ATCCcrl	ATCC14028 ∆crl::Cm		40
	JS402	∆fur-41::cat		19
	ATCCfur	ATCC14028 ∆fur-41::cat
	ATCCrpoSfur	ATCC14028 ∆rpoS ∆fur-41::cat
	ATCCcrl csgD-lacZ	ATCC14028 ∆crl::cm csgD-lacZY		40
	ATCCsdhA-lacZ-k	ATCC14028 sdhA-lacZY-Km		41

Table 2 : Distribution and sequence conservation of Crl in bacterial sequenced genomes.

 2 Range of percentage of identity between the aminoacid sequence of the Crl proteins in strains belonging to the indicated genus and that of the S. Typhimurium Crl protein (Crl STM , 133 residues, NP-459316 Figure1 line 16). Range of percentage of identity between the aminoacid sequence of the RpoS proteins in strains belonging to the indicated genus and that of the S. Typhimurium RpoS protein (RpoS STM , 330 residues, X77752, 26).

	________________________________________________________________________
	Genus	a	identity with Crl STM (%) b	identity with RpoS STM (%) c
	Salmonella	96-100	99-100
	Citrobacter	90	100
	Escherichia	83-84	98-99
	Shigella	83-84	99
	Enterobacter	74-77	97
	Klebsiella	75-76	98	Enterobacteriaceae
	Serratia	69	94
	Erwinia	66	95
	Yersinia	63-66	84-97
	Pectobacterium	64	91
	Proteus	49	79
	Providencia	40-45	78
	Aeromonas	43-44	85-84
	Vibrio	38-50	72-82
	Unclassified Vibrionales	46	81
	Photobacterium	42-49	79-80
	Moritella	38	76
	Psychromonas	35-44	77
	a bacterial sequenced genomes containing a crl gene

c

Table 3 : BACTH analysis of Crl interactions with

 3

	σ σ σ σ S and Fur.

Table 1 )

 1 were grown five days on CR plates at 28°C.

	Plasmids are 1 : pACYC184, 2 : pACcrl-1, 3 : pACcrl Y22A , 4 : pACcrl F35A , 5 : pACcrl C37A , 6 :
	pACcrl C41A , 7 : pACcrl F53A , 8 : pACcrl W56A , 9 : pACcrl G74A , 10 : pACcrl G80A , 11 :
	pACcrl W82A , 12 : pACcrl F103A , 13 : pACcrl C28A , 14 : pACcrl C28-37-41 . Complementation of the
	crl mutation was observed with pACcrl-1 and its derivatives containing mutations F35A,
	C28A, C37A, C41A, G74A, G80A, F103A.
	FIGURE 6.

Expression of Crl wild-type and mutant proteins.

  , 14: pACcrl C28-37, 15: pACcrl C28-41, 16: pACcrl C37-41, 17: pACcrl C28-37-41 . 5 µg of total protein was loaded in slots 18 to 20. 19: pACcrl C37-41, 20: pACcrl C28-37-41, 19: a stationary phase culture of ATCC14028 in LB at 37°C was used as control. B) Expression of wild-type and altered Crl-T18 hybrid proteins. The E. coli cya strain BTH101 harboring pUT18 and its derivatives were grown to stationary phase (OD600= 3) in LB in the presence of 2 mM cAMP and 0.5 mM IPTG to fully induce the lac promoter on pUT18. 5 µg of total protein was loaded in each slot and analysed by western blotting with anti-Crl antibodies. 1: no plasmid, 2: pUT18, 3: pUTCrl, 4: pUTCrl Y22A , 5: pUTCrl F53A , 6: pUTCrl W56A , 7: pUTCrl G80A , 8: pUTCrl W82A, 9: pUTCrl R51A, 10: pUTCrl E52A, 11: pUTCrl G55A, 12: pUTCrl W57A .C) Detection of Crl in Salmonella ATCC14028[START_REF] Bang | Alternative sigma factor interactions in Salmonella: σ E and σ H promote antioxidant defences by enhancing σ S levels[END_REF] 4,[START_REF] Coynault | Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon[END_REF][START_REF] Dong | RpoS regulation of gene expression during exponential growth of Escherichia coli K12[END_REF][START_REF] Gowrishankar | In vitro properties of RpoS (σ S ) mutants of Escherichia coli with postulated N-terminal subregion 1.1 or C-terminal region 4 deleted[END_REF] and its mutant derivatives ATCCfur (2, 5, 8, 13), ATCCcrl[START_REF] Callaci | Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit[END_REF][START_REF] Chang | Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid[END_REF][START_REF] Dombroski | Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA[END_REF][START_REF] Ellermeier | Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria[END_REF], ATCCrpoS[START_REF] England | Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery[END_REF], and ATCCrpoSfur[START_REF] Gaal | Crl facilitates RNA polymerase holoenzyme formation[END_REF].Cultures were grown in different conditions, as indicated, and analysed by western blotting with anti-Crl antibodies. 1 µg of total protein was loaded in each slot. LB 37°C: overnight LB cultures at 37°C. LB 30°C: overnight LB cultures at 30°C. LB agar 30°C: colonies grown on LB agar at 30°C as described[START_REF] Lelong | Mutual regulation of Crl and Fur in Escherichia coli W3110[END_REF].

	A) Detection of Crl produced from pACcrl-1 and its mutant derivatives in Salmonella
	ATCCcrl. ATCCcrl strains, harboring the vector pACYC184 and the pACcrl-1 derivatives as
	indicated below, were grown to stationary phase (OD600=4) in LB at 37°C and analysed by
	western blotting with anti-Crl antibodies. 1 µg of total protein was loaded in slots 1 to17. 1:
	pACcrl-1, 2: pACYC184, 3: pACcrl Y22A , 4: pACcrl F35A , 5: pACcrl C37A , 6: pACcrl C41A , 7:
	pACcrl F53A , 8: pACcrl W56A , 9: pACcrl G74A , 10: pACcrl G80A , 11: pACcrl W82A , 12: pACcrl F103A ,
	13: pACcrl

C28A

Acknowledgments:

We are grateful to Anthony Pugsley for critical reading of the manuscript. We thank James Slauch for providing the Salmonella fur strain. We are grateful to Gouzel Karimova and Daniel Ladant for providing the cya strain and BACTH vectors and for helpful discussions.

mutation or by the production of Crl in trans from plasmid pACcrl-1 (Figure 8AB). These results suggested that crl transcription is not repressed by Fur and Crl in ATCC14028.

In the presence of iron, Fur negatively controls transcription of a small RNA, RyhB, which facilitates degradation of the sdhCDBA mRNA encoding succinate dehydrogenase [START_REF] Massé | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF]. Consistent with this finding, sdhA-lacZ -encoded β-galactosidase in ATCC14028 was dependent on both iron and Fur (Figure 8C). This demonstrated that regulation by Fur is indeed functional in ATCC14028. Fur did not exert any effect on the crl-lacZ fusion (Figure 8C). Also, no effect of Fur on crl-lacZ expression was detected in LB supplemented with iron (data not shown).

E. coli W3110 has many genes that are differentially expressed in E. coli MG1655 [START_REF] Vijayendran | The plasticity of global proteome and genome expression analysed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12[END_REF], and many laboratory stocks of W3110 contain mutations in rpoS (21, 52 and www.ncbi.nlm.nih.gov). These findings prompted us to evaluate the effect of the fur mutation on crl expression in an rpoS background. Crl production and crl-lacZ-encoded β galactosidase levels were not affected by the fur mutation in the Salmonella rpoS mutant (Figure 6C