Tatiana Rochat

Philippe Bouloc

Qi Yang

Lionello Bossi

Nara Figueroa-Bossi

Lack of interchangeability of Hfq-like proteins

ABSTRACT

GcvB is a conserved 200 nucleotide RNA that downregulates several genes involved in amino acid uptake or biosynthesis in bacteria. The physiological role of GcvB action is not entirely clear, but it is likely aimed at balancing of nutritional resources under fast growth conditions. GcvB inhibits translation of target messenger RNAs by pairing with sequences inside or upstream of ribosome binding sites. In the present study, characterization of a novel GcvB-regulated locus revealed some unique features in the mode of functioning of this regulatory RNA. We found that GcvB represses yifK -a highly conserved locus encoding a putative amino acid transporter -b y targeting a translational enhancer element. Two ACA motifs within the target sequence are the main determinants of the enhancer activity. Replacing either of these motifs with random triplets caused up to a 10-fold decrease in yifK expression regardless of the GcvB allele (deleted or suitably modified to recognize the mutated target). It thus appears that GcvB effectiveness as a regulator results from countering the enhancer activity. When the enhancer is removed, GcvB action no longer constitutes a rate-limiting factor for yifK expression. Overall, this study is relevant not only to a better understanding of GcvB function but it also provides insight into an elusive aspect of the translation initiation process.

Besides the GcvB control, the yifK locus is regulated at the transcriptional level by the leucine responsive regulator Lrp, and by HdfR (YifA) a poorly known transcriptional regulator, that appears to require the product of the adjacent, divergently oriented gene, yifE, for expression or activity. Transcription initiating at the yifK promoter extends into the adjacent argX-hisR-leuT-proM tRNA operon yielding an unusual primary transcript which both a messenger RNA and a tRNA precursor. This chimeric RNA si rapidly processed by RNAse E.

RÉSUMÉ

GcvB est un ARN bactérien conservé de 200 nucléotides, qui régule négativement l'expression de plusieurs gènes impliqués dans l'import et la biosynthèse des acides aminés. Bien que le rôle physiologique de GcvB ne soit pas complètement élucidé, il contribuerait vraisemblablement à équilibrer les ressources nutritionnelles en conditions de croissance rapide. GcvB inhibe la traduction des ARNm cibles en s'appariant avec des séquences à l'intérieur ou en amont du site de liaison du ribosome. Dans cette étude, la caractérisation d'un nouveau locus régulé par GcvB a permis de dévoiler des aspects singuliers du mode de fonctionnement de cet ARN régulateur. Nous avons découvert que GcvB réprime yifK -un gène très conservé, codant pour un transporteur d'acides aminés putatif -en ciblant un élément activateur de la traduction sur l'ARNm. Deux motifs ACA dans la séquence cible sont les déterminants principaux de la fonction activatrice. Le remplacement de l'un ou l'autre avec des triplets aléatoires, provoque une diminution de 10 fois du niveau d'expression de yifK, quelque soit l'allèle de GcvB (délétion ou changement de séquence permettant la reconnaissance de la cible mutante).

Il apparait ainsi que l'efficacité de GcvB à réguler négativement sa cible serait liée a sa capacité d'antagoniser l'élément activateur. Lorsque l'activateur est éliminé, l'action de GcvB n'est plus un facteur limitant pour l'expression de yifK. Dans son ensemble, cette étude apporte une meilleure compréhension de la fonction de GcvB et révèle un nouvel aspect du processus d'initiation de la traduction.

En plus du contrôle par GcvB, le locus yifK est régulé au niveau transcriptionnel par Lrp (leucine-responsive regulatory protein) et par HdfR (YifA) un régulateur transcriptionnel peu connu qui requerrait le produit du gène adjacent orienté de façon divergente, yifE, pour son expression ou activité. Enfin, la transcription initiée au niveau du promoteur yifK s'étend dans l'opéron d'ARNt argX-hisR-leuT-proM adjacent, donnant lieu à un transcrit primaire qui est à la fois un lARNm et un précurseur des ARNt. Cet ARN chimère est rapidement maturé par l'ARNase E.

I. INTRODUCTION I.1.Salmonella: general features

Salmonella is a Gram-negative, rod shaped Gamma proteobacterium of the Enterobacteriacea family. Almost a century ago, the classification system of various Salmonella serotypes based on surface antigens was established by Kauffman and White (Le [START_REF] Minor | Supplement No. XXVII (1983) to the Kauffmann-White scheme][END_REF]. The World Health Organization Collaborating Centre for Reference and Research on Salmonella (WHOCC-Salm) has reported in 2007 (supplement data in 2010), that there are 2610 serotypes in the genus Salmonella [START_REF] Patrick | ANTIGENIC FORMULAE OF THE SALMONELLA SEROVARS[END_REF], Guibourdenche e t a l . , 2010). All these serotypes can be grouped into two species: Salmonella enterica a n d Salmonella bongori.

Chromosomal DNA hybridization experiments [START_REF] Crosa | Molecular relationships among the Salmonelleae[END_REF][START_REF] Minor | The taxonomy of Salmonella][END_REF][START_REF] Minor | Characterization of a 7th subspecies of Salmonella: S. choleraesuis subsp. indica subsp. nov][END_REF], and multi-locus enzyme electrophoresis (MLEE) [START_REF] Reeves | Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb[END_REF][START_REF] Beltran | Toward a population genetic analysis of Salmonella: genetic diversity and relationships among strains of serotypes S. choleraesuis, S. derby, S. dublin, S. enteritidis, S. heidelberg, S. infantis, S. newport, and S. typhimurium[END_REF] have led to a further subdivision of Salmonella enterica into six subspecies: I (enterica), II (salamae), IIIa (arizonae), IIIb (diarizonae), IV (houtenae) and V (indica).

In order to harmonize the designation and writing of Salmonella species and serovars, it has been agreed that species are written in italics following the international convention and the serovar is specified after the species, non-italicized and starting with a capital letter [START_REF] Brenner | Salmonella nomenclature[END_REF], Tindall e t a l . , 2005[START_REF] Maloy | Strain collections and genetic nomenclature[END_REF] For example, "Salmonella enterica serovar Typhimurium".

Salmonella is a common and important pathogen found all worldwide [START_REF] Galanis | Web-based surveillance and global Salmonella distribution, 2000-2002[END_REF] that can infect a wide range of hosts, including humans, animals and plants [START_REF] Gu | Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants[END_REF]. Particularly important is the incidence of Salmonella-caused foodborne diseases in industrialized countries (Le [START_REF] Hello | Foodborne outbreak and nonmotile Salmonella enterica variant, France[END_REF][START_REF] Scallan | Foodborne illness acquired in the United States--major pathogens[END_REF][START_REF] Majowicz | The global burden of nontyphoidal Salmonella gastroenteritis. Clinical infectious diseases : an official publication of the[END_REF]. The pathogenicity of Salmonella largely relies on its ability to corrupt the functioning of host cells by secreting virulence factors and effector proteins through specialized type III secretion systems (TTSS). Two distinct type III secretion systems, encoded by Salmonella pathogenicity islands (SPI) 1 and 2 are the most important virulence-related TTSS. The function of secretory system encoded by SPI-1 is to provide ability to invade epithelial cells causing the inflammatory response response (Hapfelmeier e t a l . , 2004). The function of secretory system encoded by SPI-2 allows bacteria to survive and reproduce within epithelial cells and phagocytes [START_REF] Hensel | Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12[END_REF][START_REF] Shea | Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion system on bacterial growth in the mouse[END_REF][START_REF] Hensel | Salmonella pathogenicity island 2[END_REF].

Phylogenetically, Salmonella is related to Shigella, Citrobacter a n d Escherichia [START_REF] Groisman | How Salmonella became a pathogen[END_REF]. For example, the two laboratory model strains Escherichia coli K 1 2 a n d Salmonella enterica serovar Typhimurium (hereafter referred to as S. Typhimurium) strain LT2 can be considered highly related at the genomic level [START_REF] Ochman | Evolution in bacteria: evidence for a universal substitution rate in cellular genomes[END_REF][START_REF] Doolittle | Determining divergence times of the major kingdoms of living organisms with a protein clock[END_REF]. The sequence of the genome of S. Typhimurium LT2, isolated in the 1940s and used in the first studies on phage-mediated transduction, was completed in 2001 [START_REF] Mcclelland | Complete genome sequence of Salmonella enterica serovar Typhimurium LT2[END_REF]. It comprises a circular chromosome of 4,657,432 base-pairs (bp) and the so-called virulence plasmid pSLT (93,939 bp). DNA sequence identity between S. Typhimurium LT2 and E. coli K12 is around 80%, amino acid similiarity of these two organisms reaches to 90% [START_REF] Mcclelland | Complete genome sequence of Salmonella enterica serovar Typhimurium LT2[END_REF].

Besides being an important model pathogen, Salmonella h a s constituted a model system for genetic and biochemical analysis since the 1950s [START_REF] Sanderson | Genetic map of Salmonella typhimurium, edition VIII[END_REF]. Many tools have been developed and some of the key bacterial cellular mechanisms have been discovered by work in this organism.

I.2. Gene Expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes, the products also can be ribosomal RNA (rRNA), transfer RNA (tRNA) and small non-coding RNA (sRNA). The passage from gene to protein, comprises two main steps: transcription and translation.

I.2.1. Transcription

Transcription is the process by which a region of the DNA molecule, corresponding to a particular gene, is selected by an enzyme called RNA polymerase to synthesize a complementary single-stranded RNA using one of the strands of the DNA as a template. The process of transcription consists of five discrete stages: promoter recognition, local unwinding, chain initiation, chain elongation and chain termination (Fig. 1). RNA polymerase binds to double-stranded DNA within a specific base sequence (promoter, 20-40 bases long). After the initial binding step, the open promoter complex is formed by RNA polymerase and unwinding of DNA occurs. Then, the RNA polymerase selects a transcription start site, which is close to the initial binding site. The first nucleoside triphosphate is added at this site and synthesis begins. RNA polymerase then moves along the DNA in the 5' to 3' direction adding nucleotides to the 3'-OH of the growing RNA chain. When RNA polymerase reaches a termination sequence, both the newly synthesized RNA and the RNA polymerase are released.

Typically, prokaryotic promoters consist of two conserved regions, one localized about 10 base pairs (bp) upstream from the site of initiation (the socalled -10 box), the other at about 35 bp from the start site (-35 box). Also part of the promoter architecture is the spacing between these two elements, 17 bp being considered optimal. These recognition sequences instruct RNA polymerase as to where to start transcribing.

I.2.1.1. Alternative sigma factors

Bacterial RNA polymerase consists of five protein subunits, four of the subunits forming the core enzyme, which catalyzes the polymerization of nucleoside triphosphates into RNA. The fifth subunit, called the σ subunit, is required for promoter binding. Once polymerization begins, the σ subunit dissociates from the core enzyme, when transcription is completed, the core enzyme binds another σ subunit and is then ready to bind to a promoter again. Bacteria makes use of several different σ subunits, also called alternative σ factors, to regulate the coordinate expression of groups of genes (for a review, see [START_REF] Gruber | Multiple sigma subunits and the partitioning of bacterial transcription space[END_REF]). Most commonly, polymerase associates with σ 70 , the most abundant subunit, capable of recognizing the promoters of genes needed to maintain the main physiological processes of the growing cell (housekeeping genes). In addition, and in response to different environmental and internal stimuli, bacteria can use: σ 38 (also known as σ S ): stationary phase and starvation/stress factor; σ 24 (σ E ): extracytoplasmic stress sigma factor; σ 32 (σ H ): heat shock sigma factor; σ 54 (σ N ): Nitrogen-limitation sigma factor; σ 28 (σ F ) : Flagellar sigma factor; σ 19 : Ferric citrate sigma factor; regulates the fec gene for iron transport;

The genes under control of a given σ factor share a specific sequence in their promoter regions that allows recognition of the gene by the corresponding factor. σ will bring along the RNA core polymerase when it binds to the DNA and will promote transcription of the gene. In this way, bacteria can coordinately express groups of genes upon activating a specific σ factor.

Binding of σ factors to core RNA polymerase is thought to be mainly dependent on the relative concentration of the different factors. Thus, conditions that lead to increase in the concentration of a given σ factor, will entail the preferential transcription of the genes belonging to its regulon.

I.2.1.2. Transcriptional regulators

A second main strategy for regulating transcription is the use of specific transcription regulators, that is proteins that can either repress or activate transcription. Bacterial repressors typically bind to an "operator" locus, located downstream from the promoter of target genes (often comprised in the transcript) to prevent transcription of the structural gene(s). Activators facilitate the expression of specific genes by helping RNA polymerase to initiate transcription either through direct binding to the enzyme, thus increasing its affinity for the promoter, and/or by locally modifying the conformation/structure of the DNA to increase transcriptional rate [START_REF] Lee | Activating transcription in bacteria[END_REF]. There exists over 300 transcription regulators in E. coli; some of them work alone and others associate with ligands or auxiliary factors to perform their regulatory action. To illustrate the complexity of transcriptional regulatory networks, the number of transcriptional factors working on a single bacterial promoter can be as high as 30 [START_REF] Ishihama | Prokaryotic genome regulation: a revolutionary paradigm[END_REF]. Finally, DNA itself can modulate the transcription rate by its physical properties. Although more seldom than transcription factors, DNA bending and supercoiling can also affect the initiation step of transcription [START_REF] Bossi | Conformational change in the DNA associated with an unusual promoter mutation in a tRNA operon of Salmonella[END_REF][START_REF] Figueroa | Common sequence determinants of the response of a prokaryotic promoter to DNA bending and supercoiling[END_REF] reviewed by [START_REF] Travers | DNA supercoiling -a global transcriptional regulator for enterobacterial growth? Nature reviews[END_REF].

I.2.1.3. Transcription termination

Two kinds of termination events are well known; one is dependent on the DNA sequence only (intrinsic termination), the other requires t h e participation of a termination protein factor called Rho (Rho-dependent termination). Most intrinsic terminators (or Rho-independent terminators) contain an inverted repeated sequence, that can base pair with itself once it has been transcribed, to create a stem loop. Usually the hairpin loop is 7-20 bp long and is rich in G:C pairs, making a particularly stable structure that causes polymerase to temporarily pause at this point. Immediately following the stem-loop, there is a stretch of U residues (6 to 8) which form a weak RNA:DNA duplex. This destabilizes the elongation complex and causes its dissociation from the DNA template (reviewed in [START_REF] Peters | Bacterial transcription terminators: the RNA 3'-end chronicles[END_REF].

Rho-dependent termination is governed by sequences in the nascent RNA. The process leading to termination requires three steps: In the first step, Rho factor, an hexameric protein, binds to C-rich unstructured region in the RNA. This induces a conformational rearrangement that causes the RNA chain to enter in the central cavity of the hexamer (Fig. 2). The rearrangement triggers the RNA-dependent ATPase activity that powers translocation [START_REF] Richardson | Loading Rho to terminate transcription[END_REF], Boudvillain e t a l . , 2013). In the second step of Rho termination, RNA is translocated 5′ to 3′ through the central cavity of Rho. In the final step of Rho-dependent termination, an elongation complex that has been halted at a pause site is dissociated by Rho action. [START_REF] Boudvillain | Terminator still moving forward: expanding roles for Rho factor[END_REF]), which causes an RNA to form. 3. Rho reaches the elongation complex, causing its destabilization and its dissociation from the DNA template. RNAP, RNA polymerase.

I.3. Translation

Translation is most complexly regulated step of protein synthesis in all three domains (Archaea, Bacteria and Eukarya) of life. The translation cycle can be schematically divided into three parts: initiation, elongation and termination. The translation initiation is the process that brings together mRNA, initiator transfer RNA, and the ribosome (Fig. 3).

In bacteria, translation initiation proceeds through several steps involving the formation of three major intermediary initiation complexes [START_REF] Simonetti | A structural view of translation initiation in bacteria[END_REF], Myasnikov et al., 2009[START_REF] Laursen | Initiation of protein synthesis in bacteria[END_REF]. The first complex starts assembling when initiation factors IF2 and IF3 bind the 30S ribosomal subunit to form an unstable 30S-IF2-IF3 complex. Then there is the arrival of initiation factor IF1, which locks the factors in a stable 30S preinitiation complex (PIC). At this point, fMet-tRNAfMet will bind. Recent evidence indicates that binding of mRNA is independent of initiation factors and can take place any time during 30S PIC assembly [START_REF] Milon | Real-time assembly landscape of bacterial 30S translation initiation complex[END_REF].

The second step is the formation of the 30S initiation complex through a conformational change which brings the first codon of the mRNA, usually AUG, to physically interact with the anticodon of the initiator tRNA in the 30S pepetidyl (P) site. Interaction of this complex with the 50S subunit occurs and IF2 hydrolyzes GTP [START_REF] Grigoriadou | A quantitative kinetic scheme for 70 S translation initiation complex formation[END_REF]. The 70S initiation complex (70SIC) forms and initiation factors IF1 and IF3 are released. The third step is the release of IF2.GDP with 70S initiation complex now ready for the elongation step of protein synthesis. [START_REF] Simonetti | A structural view of translation initiation in bacteria[END_REF].

In bacteria, AUG is the most common translation initiator codon because of its perfect pairing with the CAU anticodon in fMet-tRNAf Met . However, weaker pairing initiator codons (where two rather than three bases can pair with fMet-tRNA anticodon) also exist in bacteria. For example, 14% of E. coli genes use GUG as the start codon and another 3% use UUG; only two E. coli genes use AUU as a start codon [START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF]. UUG as a start codon is more common in Gram-positive bacteria and in some bacteriophages [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF][START_REF] Lobocka | Genome of bacteriophage P1[END_REF]. Weaker pairing initiation codons can affect translation efficiency. For instance, in E. coli, when AUG changed by GUG or UUG, the translation efficiency in vivo was reduced.

Change to AUU had an even bigger effect than GUG or UUG [START_REF] Sussman | Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo[END_REF].

In prokaryotes, the mRNA sequence that directs the interaction between mRNA and the ribosome, upstream from start codon, called the Shine-Dalgarno (SD) sequence [START_REF] Shine | The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites[END_REF] plays the main role in initiation. This purine-rich sequence is complementary to, and base pairs with, a sequence near the 3' end of 16S rRNA, the RNA component in the 30S ribosomal subunit [START_REF] Steitz | How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli[END_REF][START_REF] Jacob | A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins[END_REF][START_REF] Brink | Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species[END_REF][START_REF] Yusupova | The path of messenger RNA through the ribosome[END_REF]. The SD sequence (GGAGG) is typically 4 or 5 nucleotides in length, and it is usually positioned 4-8 nt upstream from the start codon. The optimal spacing depends on exactly which bases at the 3' end of 16S rRNA (3'-AUUCCUCCAC5') participate in the interaction [START_REF] Chen | Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs[END_REF]. Many mRNAs contain pyrimidine-rich (AU-rich in E.coli) sequences in the region upstream from the SD sequence. These sequences binds to ribosomal protein S1 [START_REF] Boni | Ribosomemessenger recognition: mRNA target sites for ribosomal protein S1[END_REF][START_REF] Yusupova | The path of messenger RNA through the ribosome[END_REF][START_REF] Laursen | Initiation of protein synthesis in bacteria[END_REF], Komarova et al., 2005b), A direct interaction between protein S1 and mRNA has been confirmed by cryoelectron microscopy (EM) studies in 2001 [START_REF] Sengupta | Visualization of protein S1 within the 30S ribosomal subunit and its interaction with messenger RNA[END_REF].

I.3.1. S1 protein

S1 protein is an mRNA-binding protein associated with the translational machinery [START_REF] Sorensen | Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo[END_REF]. As a ribosomal component, it recognizes and binds to mRNAs affecting translation efficiency during initiation phase [START_REF] Boni | Ribosomemessenger recognition: mRNA target sites for ribosomal protein S1[END_REF][START_REF] Zhang | A uridine-rich sequence required for translation of prokaryotic mRNA[END_REF][START_REF] Nakagawa | Dynamic evolution of translation initiation mechanisms in prokaryotes[END_REF], this function being relatively independent of the "strength" of SD sequence [START_REF] Tzareva | Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions[END_REF][START_REF] Komarova | Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation[END_REF]. In contrast, in leaderless mRNA (mRNAs beginning directly with the AUG initiating codon), S1 has proven to be dispensable for translation initiation [START_REF] Tedin | Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence[END_REF], Moll et al., 2002).

Furthermore, because of the interaction between S1 and the AU-rich sequences in mRNA, it is thought that AU-rich sequences act as translational enhancers (Komarova e t a l . , 2005a[START_REF] O'connor | Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA[END_REF][START_REF] Hook-Barnard | Identification of an AU-rich translational enhancer within the Escherichia coli fepB leader RNA[END_REF]. By its association with these sequences, S1 could also help stabilize mRNA by protecting it against cleavage by RNase E, an endonuclease with similar sequence recognition patterns (Komarova et al., 2005b) in mRNA.

I.3.2. Translation initiation factors

As described above, three non-ribosomal proteins -the initiation factors (IFs) -play important roles during the translation. IFs are highly conserved and homologous proteins are present in all three domains of life [START_REF] Kyrpides | Universally conserved translation initiation factors[END_REF][START_REF] Sorensen | Remarkable conservation of translation initiation factors: IF1/eIF1A and IF2/eIF5B are universally distributed phylogenetic markers[END_REF]. IF1, encoded by the infA g e n e , i s t h e s m a l l e s t o f t h e i n i t i a t i o n factors [START_REF] Pon | Structure--function relationships in Escherichia coli initiation factors. II. Elucidation of the primary structure of initiation factor IF-1[END_REF]. IF1 stimulates IF2 and IF3 activities by increasing their binding efficiency to the 30S subunit [START_REF] Wintermeyer | Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study[END_REF][START_REF] Pon | Mechanism of protein biosynthesis in prokaryotic cells. Effect of initiation factor IF1 on the initial rate of 30 S initiation complex formation[END_REF] and can occupy the aminoacyl (A) side of ribosome to prevent tRNA binding [START_REF] Moazed | Specific protection of 16 S rRNA by translational initiation factors[END_REF].

IF2 is the product of the infB gene and largest of the initiation factors. It belongs to the family of the GTP-GDP binding protein(GTPase)[ [START_REF] Bourne | The GTPase superfamily: conserved structure and molecular mechanism[END_REF]. The presence of IF2 on the 30S subunit promotes the binding of initiator tRNA [START_REF] Canonaco | Mechanism of translational initiation in prokaryotes. Evidence for a direct effect of IF2 on the activity of the 30 S ribosomal subunit[END_REF][START_REF] Gualerzi | Initiation of mRNA translation in prokaryotes[END_REF].

IF3 is encoded by the infC gene [START_REF] Sacerdot | Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon[END_REF]. Several roles have been suggested for IF3 in translation initiation. For example, IF3 stimulates the rapid formation of codon-anticodon interaction at the ribosomal P-site [START_REF] Gualerzi | Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3[END_REF][START_REF] Wintermeyer | Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study[END_REF] and it is involved in the adjustment of the mRNA from the standby site to the decoding P-site of the 30S ribosomal subunit [START_REF] Teana | From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors[END_REF]. IF3 prevents the formation of 70S ribosome by binding to the 30S subunit [START_REF] Grunberg-Manago | Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes[END_REF][START_REF] Sacerdot | The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli[END_REF].

I.4. Global transcription regulatory networks: selected examples

I.4.1. The leucine response regulator Lrp

Lrp is a major bacterial global transcriptional regulator controlling at least 10% of all E.coli genes, according to transcriptomic data [START_REF] Tani | Adaptation to famine: a family of stationary-phase genes revealed by microarray analysis[END_REF]. In most instances, its regulatory action (negative or positive) operates at the level of transcription of genes involved in amino acid metabolism and transport. However, some genes participating in other cellular functions, particularly fimbrial operons [START_REF] Baek | Lrp acts as both a positive and negative regulator for type 1 fimbriae production in Salmonella enterica serovar Typhimurium[END_REF], and pilus assembly (van der [START_REF] Van Der Woude | Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap[END_REF] are also part of the regulon.

Lrp belongs to a family of regulators that is widely distributed among bacteria and Archea. Lrp is a relatively small protein (18.8 kD in E. coli) that can exist in the cell as a dimer but also further associate in higher order structures, especially octamers [START_REF] Chen | Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers[END_REF] and/or hexadodecamers. (de los [START_REF] De Los Rios | Structure of the Escherichia coli leucineresponsive regulatory protein Lrp reveals a novel octameric assembly[END_REF]. Alternation between the two latter forms is thought to correspond to the autotrophic versus heterotrophic mode, octamer formation being stimulated by leucine (see below; [START_REF] Kawashima | Transcription regulation by feast/famine regulatory proteins, FFRPs, in archaea and eubacteria[END_REF]). Although there is no strict consensus sequence for Lrp binding, a modified "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX) p r ocedure has identified a sequence YAGHAWATTWTDCTR, where Y = C or T, H = not G, W = A or T, D = not C, and R = A or G [START_REF] Cui | A consensus sequence for binding of Lrp to DNA[END_REF].

The biologically significance of the multimeric structure of Lrp is underlined by the fact that many promoters controlled by Lrp have multiple binding sites. The best characterized member of the Lrp family is the 164 amino acids Lrp protein from E. coli. Like most transcriptional regulators it has a DNA binding domain and a ligand binding site. The amino acid leucine is such a ligand, which modulates the action of Lrp. Since L-leucine is the most abundant amino acid in proteins (9% occurrence for leucine, followed by 7.5%

for alanine), it has been speculated that the bacterial cell could use this amino acid as a reporter for the availability of proteins, peptides and amino acids [START_REF] Brinkman | The Lrp family of transcriptional regulators[END_REF]. Upon binding to leucine, the efficiency of Lrp action, either negative or positive, is further stimulated or reduced (reviewed in [START_REF] Brinkman | The Lrp family of transcriptional regulators[END_REF]). Lrp, together with the regulator of asparagine synthase C gene product, AsnC, are called feast/famine regulatory proteins, by virtue of their implication in regulation of metabolic pathways as a function of amino acid and nitrogen bases availability in the environment (for a review, see [START_REF] Kawashima | Transcription regulation by feast/famine regulatory proteins, FFRPs, in archaea and eubacteria[END_REF].

Lrp can also function independently of leucine (as in its self-control mechanism) or it can associate with other regulators (CRP, H-NS or IHF).

Because of Lrp's relatively abundance (3000 Lrp dimers per E. coli cell;

(Willins e t a l . , 1991)), and since it appears to be involved in DNA condensation and DNA bending [START_REF] Wang | Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure[END_REF], it has been proposed that it can also function as non-specific DNA organizer, depending on nutritional availability and phase of growth.

In Salmonella enterica, the Lrp protein is 99% identical to that of E. coli at the aminoacid level and controls also several virulence genes. The expression of virulence plasmide spv operon [START_REF] Marshall | A role for the leucineresponsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium[END_REF] and conjugal transfer gene traJ [START_REF] Camacho | Regulation of traJ transcription in the Salmonella virulence plasmid by strand-specific DNA adenine hemimethylation[END_REF] are under Lrp control. In addition, virulence regulators hilA, invF, and ssrA have also been

shown to be controlled by Lrp at the transcriptional level [START_REF] Baek | Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium[END_REF] .

It has been shown that constitutive expression of the lrp gene attenuates Salmonella virulence, whereas deletion of the gene enhances Salmonella invasion of cultured cell lines and virulence on BALB/C mice [START_REF] Baek | Leucine-responsive regulatory protein (Lrp) acts as a virulence repressor in Salmonella enterica serovar Typhimurium[END_REF]. Because of the global nature of Lrp action and the complexity of the network, the precise role of the protein in several of the described mechanisms remains to be fully understood.

I.4.2. The glycine cleavage system

De novo biosynthesis of several cellular constituents, including nucleotides and amino acids, requires one-carbon units supplied directly as formyl tetraydrofolate (CH2-H4 folate). There are two pathways leading to CH2-H4 folate production. The major pathway involves the enzyme serinehydroxymethyl-transferase (the product of the glyA gene) which converts serine to glycine plus CH2-H4 folate (Stauffer, 1987). In bacteria growing in a glucose-supplemented minimal medium -conditions where the requirement for one-carbon units is the highest -as much as 15% of the carbon from glucose is thought to be channeled to the formation of serine and glycine [START_REF] Calvo | The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli[END_REF]. A second route for one-carbon unit production involves the oxidative cleavage of glycine to NH3 and CO2 and the transfer of a C-1 methylene unit to folate. This reaction, which is conserved in all domains of life, is carried out by the four enzymes of the glycine cleavage system: the P, T, L and H proteins. Since the serine-hydroxymethyltransferase reaction can run in both directions, the glycine cleavage pathway can also be a source of serine as CH2-H4 folate can donate a carbon to glycine to form serine [START_REF] Jourdan | Genetic analysis of the GcvA binding site in the gcvA control region[END_REF][START_REF] Lin | Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine[END_REF].

In E. coli and S. Typhimurium, the genes encoding the P,T and H components of the glycine cleavage systems are organized in an operon (gcvTHP) while the L prtein is encoded by a gene at a separate chromosomal location. Transcription of gcvTHP is activated in the presence of glycine Wilson, 1993 #1039} and repressed by purines [START_REF] Wilson | Roles of the GcvA and PurR proteins in negative regulation of the Escherichia coli glycine cleavage enzyme system[END_REF]. Key mediator of both responses is the GcvA protein, a LysR-type regulator that binds to three sites in the region upstream from the gcvTHP promoter. While

GcvA alone is required for glycine-dependent activation, purine-induced repression requires both GcvA and a second regulatory protein, GcvR [START_REF] Ghrist | Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression[END_REF]. Stauffer and coworkers have proposed that GcvA homocomplexes function as activators while GcvA-GcvR heterocomplexes repress gcvTHP transcription [START_REF] Ghrist | Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression[END_REF][START_REF] Jourdan | Genetic analysis of the GcvA binding site in the gcvA control region[END_REF]. In addition to these specific regulators, global regulatory protein Lrp is required for both activation and repression of the gcvTHP operon [START_REF] Lin | Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine[END_REF][START_REF] Stauffer | Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli[END_REF], Stauffer & Stauffer, 1999). Lrp is thought to play primarily a structural role by bending the DNA and allowing GcvA to make contact with separate sites on the DNA or with GcvR (Fig. 4).

This action (independent of leucine) is essential for GcvA-mediated activation of gcvTHP as this operon is essentially uninducible in a strain devoid of Lrp [START_REF] Lin | Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine[END_REF][START_REF] Stauffer | Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli[END_REF]. Since Lrp levels are significantly reduced in cells growing in rich medium, the gcvTHP operon is expected to be expressed at low levels under such conditions, consistent with the reduced demand from one-carbon units in rich notional environments [START_REF] Calvo | The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli[END_REF] .

I.5.1. Riboswitches

A riboswitch is a non-coding RNA element located in the 5' untranslated region (5'-UTR) of mRNA that regulates mRNA expression by directly sensing the presence of small metabolites. The mechanism of "riboswitching" was first discovered in 2002 and experimentally validated by a number of examples [START_REF] Nahvi | Genetic control by a metabolite binding mRNA[END_REF][START_REF] Mironov | Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria[END_REF], Winkler et al., 2002b, Winkler et al., 2002a). These data also confirmed the earlier idea that expression of some mRNAs might be directly related with concentration changes in small metabolites [START_REF] Gold | From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops[END_REF][START_REF] Gelfand | A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes[END_REF][START_REF] Miranda-Rios | A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria[END_REF].

Riboswitches comprise two domains: the aptamer (also called ligandbinding domains or sensor) and the expression platform. The sequence and structure of the aptamer component is highly conserved among different bacteria. Riboswitches can sense multifarious signals such as coenzymes [START_REF] Nahvi | Genetic control by a metabolite binding mRNA[END_REF], metal ions (Cromie e t a l . , 2006) amino acids, nucleobases and their derivatives [START_REF] Serganov | A decade of riboswitches[END_REF] including negatively charged fluoride anions (Baker e t a l . , 2012). After metabolite binding to the aptamer moiety, the conformation of the platform changes, leading to modulation of downstream events. Such events include transcription termination, antitermination, translation activation or activation (Fig. 5). Mechanisms of modulation of gene expression are highly diversified in prokaryotes and involve control of transcription, translation, and mRNA stability. From [START_REF] Serganov | A decade of riboswitches[END_REF].

I.5.2. CRISPR systems

Clustered regularly interspaced short palindromic repeat (CRISPR) are sequences composed of direct repeats that are separated by similarly sized non-repetitive spacers. In bacteria the CRISPR RNAs assemble with CRISPRassociated proteins (Cas) to large surveillance complexes that interfere with foreign nucleic acids such as bacteriophage and plasmid DNA.

The first report of what is now known as a CRISPR was made by Ishino and his colleagues in 1987 [START_REF] Ishino | Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[END_REF]. Further characterization of the locus revealed the existence of 14 repeats of 29 base pairs (bp) that were interspersed by 32-33 bp non-repeated spacer sequences [START_REF] Nakata | Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome[END_REF]. In 2005, G. Vergnaud and his coworkers identified the origin of the spacers in CRISPR elements in Yersinia pestis [START_REF] Pourcel | CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies[END_REF]. In recent years, computational analyses have revealed the presence of CRISPRs in almost 40% of the sequenced bacterial genomes [START_REF] Kunin | Evolutionary conservation of sequence and secondary structures in CRISPR repeats[END_REF].

The function of CRISPR systems is provide a defensive response against foreign nucleic acids in bacteria (Fig. 6). In CRISPR systems, foreign DNA is integrated into the CRISPR locus, and the host spacer sequence replaced by invading genetic elements (protospacers). Then the CRISPR loci are transcribed and yield a long primary transcript which is processed by a Cas or by RNase III family nucleases [START_REF] Carte | Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes[END_REF][START_REF] Haurwitz | Sequenceand structure-specific RNA processing by a CRISPR endonuclease[END_REF][START_REF] Deltcheva | CRISPR RNA maturation by transencoded small RNA and host factor RNase III[END_REF][START_REF] Sashital | An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3[END_REF] to form a library of short CRISPR-derived RNAs (crRNAs). Each crRNA contain a sequence complementary to a foreign invading nucleic acid (Fig. 6). After processing, the crRNA associates with one or more Cas proteins to form a surveillance complex that targets and destroys invading genetic material [START_REF] Brouns | Small CRISPR RNAs guide antiviral defense in prokaryotes[END_REF][START_REF] Hale | RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex[END_REF][START_REF] Garneau | The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[END_REF], Wiedenheft et al., 2011a, Wiedenheft et al., 2011b). In each system, the mature crRNA associates with one or more Cas proteins to form a surveillance complex (green rectangles). Type I systems encode a Cas3 nuclease (blue pacman), which may be recruited to the surveillance complex following target binding. A short high-affinity binding site called a seed-sequence has been identified in some type I systems27, 60, and genetic experiments suggest that type II systems have a seed sequence located at the 3′end of the crRNA spacer sequence. From: [START_REF] Wiedenheft | RNA-guided genetic silencing systems in bacteria and archaea[END_REF].

I.5.3. Small regulatory RNAs

Small RNAs (sRNAs) comprise an important class of regulators in all kingdoms of life. They participate in the cell responses to stress, changes in environmental conditions and nutrient availability. In doing so, sRNAs play a key role in cell adaptation [START_REF] Repoila | Small non-coding RNAs, coordinators of adaptation processes in Escherichia coli: the RpoS paradigm[END_REF].

The first sRNA capable of regulating gene expression was discovered in the 1980s [START_REF] Mizuno | A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA)[END_REF]. For some time this sRNA, MicF, remained an isolated example. But starting in the early 2000s, a number of different in silico and experimental strategies converged to uncover more than 70 sRNAs by [START_REF] Gottesman | Micros for microbes: non-coding regulatory RNAs in bacteria[END_REF]Vogel, 2005 #956}. #956}. in 2011 the number of sRNA has reached to 100 (Gottesman & Storz, 2011a). Based on the size of enterobacterial genomes, it has been estimated that the number of sRNA genes may be in the range of 200-300 (Vogel & Wagner, 2007). There appears to be a core of conserved sRNAs in different related pathogens, such as E.coli, S.Typhimurium and S.

flexneri [START_REF] Hershberg | A survey of small RNA-encoding genes in Escherichia coli[END_REF].

Regulatory sRNAs can be divided into two classes depending on whether they target a protein or an RNA.

I.5.3.1. Protein-targeting sRNAs

They can be further divided into two groups. In the first group, the sRNA itself has the essential biological activity or contributes functions to a protein. This is the case, for example, of M1, the RNA component of Ribonuclease P, (RNase P) [START_REF] Esakova | Of proteins and RNA: the RNase P/MRP family[END_REF] and of the 4.5S

RNA component of the signal recognition particle [START_REF] Ribes | E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle[END_REF]. In contrast, sRNAs of the second group can regulate the activities of their target proteins by mimicking the structures of other nucleic acids. For example, CsrB targets CsrA (Carbon Storage Regulator) protein, a global regulator of carbon metabolism which represses translation of a number of mRNAs by binding to their Shine-Dalgarno sequences [START_REF] Romeo | Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB[END_REF]. CsrB sRNA contains multiple copies of a sequence mimicking the CsrA binding site. When the level of CsrB is high, this sRNA can compete out CsrA from target mRNA, thus relieving the translational repression (Fig. 7) [START_REF] Majdalani | Bacterial small RNA regulators[END_REF].

I.5.3.2. RNA-targeting sRNAs

Undoubtedly, the largest and most extensively studied class of sRNAs acts through base paring with complementary sequence in target messenger RNAs (mRNAs), affecting translation and/or mRNA stability [START_REF] Masse | Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli[END_REF][START_REF] Caron | Small RNA-mediated regulation at the level of transcript stability[END_REF]. RNA-targeting sRNAs can be divided into two groups depending on the positions of their genes relative to the target genes.

I.5.3.2a. Cis-encoded sRNAs

These sRNAs that are encoded at the same locus of the target RNA but in opposite strand and, as a result, share extended regions of sequence complementarity with their target. Cis-encoded sRNAs have been mostly

found in plasmids, phages and transposons [START_REF] Brantl | Antisense-RNA regulation and RNA interference[END_REF] and only a few in the bacterial chromosome [START_REF] Kawano | Detection of 5'-and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli[END_REF]. They can function by several mechanisms depending of the position of the pairing region. They can inhibit translation initiation, promote transcription termination or induce RNA degradation (Fig. 8) [START_REF] Brantl | Antisense-RNA regulation and RNA interference[END_REF][START_REF] Waters | Regulatory RNAs in bacteria[END_REF]. [START_REF] Waters | Regulatory RNAs in bacteria[END_REF].

I.5.3.2b. Trans-encoded sRNAs

The largest class of regulatory sRNAs comprises molecules that are encoded at separate location from their target genes. They are characterized by a limited and often imperfect sequence complementarity with their mRNA targets and by the fact that they often have multiple targets [START_REF] Gottesman | Micros for microbes: non-coding regulatory RNAs in bacteria[END_REF], Prevost e t a l . , 2007). Pairing normally involves at least 6-8 contiguous nucleotides, which in multi-target sRNAs are generally located at the same positon of the molecule [START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF][START_REF] Bouvier | Small RNA binding to 5' mRNA coding region inhibits translational initiation[END_REF]. On the mRNA side, the pairing residues are usually part of the 5' UTR or of the initial portion of the coding sequence. An additional relevant feature of transencoded sRNA is their requirement for RNA chaperon protein Hfq for stability and function [START_REF] Valentin-Hansen | The bacterial Sm-like protein Hfq: a key player in RNA transactions[END_REF], Vogel & Luisi, 2011). The role of Hfq in sRNA function is described in more detail in section I. 5.3.3. Trans-encoded sRNAs can act as either positive or negative regulators (Fig. 9). In the case of positive regulation, the most common mechanism involves the sRNA activating translation by preventing the formation of the inhibitory secondary structure in the mRNA [START_REF] Majdalani | Bacterial small RNA regulators[END_REF][START_REF] Prevost | The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis[END_REF]. Annealing of the sRNA to the mRNA changes the mRNA structure, freeing the ribosome binding site (Shine-Dalgarno sequence) so that the ribosome can access the translation initiation region [START_REF] Hammer | Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae[END_REF][START_REF] Urban | Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation[END_REF]. It was proposed that an additional mechansim by which sRNA can activate gene expression is by increasing the stability of the mRNA [START_REF] Opdyke | GadY, a small-RNA regulator of acid response genes in Escherichia coli[END_REF][START_REF] Ramirez-Pena | The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript[END_REF]. This mechanism remained hypothetical until recently, when the group of Jörg Vogel showed that the small RNA SgrS activates the synthesis of YigL (a phospahtase involved in the detoxification of phosphosugars) by stabilizing a decay intermediate of the bicistronic pldB-yigL mRNA [START_REF] Papenfort | Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis[END_REF]. Still, some aspects of this mechanism remain elusive; in particular the surprising finding that RNase E, an enzyme that normally degrades mRNAs, is required for the activation step [START_REF] Papenfort | Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis[END_REF].

Negative regulation is by far the most outcome of sRNA activity. By base pairing with sequences in the 5'UTR of the mRNA, the sRNA occludes the ribosome binding site, thus inhibiting translation initiation (Fig. 9). No longer shielded by translating ribosomes, the mRNA becomes exposed to degradation by RNase E [START_REF] Masse | Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli[END_REF], Morita e t a l . , 2005).

Furthermore, in some cases, mRNA degradation is not just the consequence of translation inhibition but the formation of the sRNA:mRNA duplex directly recruits RNase E [START_REF] Pfeiffer | Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation[END_REF]. Interestingly, there are some sRNAs that inhibit translation through base pairing far upstream of the initiating AUG, for example: GcvB and RyhB and RybB (Sharma et al., 2007[START_REF] Vecerek | Control of Fur synthesis by the non-coding RNA RyhB and iron-responsive decoding[END_REF][START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF]. It was proposed that sRNA can act at such distant sites by sequestering a ribosome standby site (Darfeuille et al., 2007), or through direct RNase E recruitment [START_REF] Wagner | Kill the messenger: bacterial antisense RNA promotes mRNA decay[END_REF].

Figure 9. Functions of trans-encoded sRNAs. From [START_REF] Waters | Regulatory RNAs in bacteria[END_REF].

I.5.3.3. Hfq: mediator of sRNA activity

In enteric bacteria, Hfq is a protein that participates i n m R N A expression and stability and, as a chaperone, in sRNA-mediated regulation.

Hfq was first identified as a host factor for RNA phage Qß replication in vitro (also known as host factor 1)(Franze de Fernandez et al., 1968[START_REF] Franze De Fernandez | Bacterial proteins required for replication of phage Q ribonucleic acid. Pruification and properties of host factor I, a ribonucleic acid-binding protein[END_REF][START_REF] Miranda | Recognition of bacteriophage Qbeta plus strand RNA as a template by Qbeta replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor[END_REF][START_REF] Su | Strongly reduced phage Qbeta replication, but normal phage MS2 replication in an Escherichia coli K12 mutant with inactivated Qbeta host factor (hfq) gene[END_REF]. Phylogenetic analyses show that the hfq gene (or a related gene) is present in approximately half of all sequenced Gram-positive and Gram-negative genomes, with some bacteria harboring more then one hfq-like gene (Sun et al., 2002, Valentin-Hansen et al., 2004). Expression of these genes and functionality of their products remains to be demonstrated in most cases.

Proteins of the Hfq family range from 70 to 110 amino acids and are generally organized in a homohexameric structure [START_REF] Franze De Fernandez | Bacterial proteins required for replication of phage Q ribonucleic acid. Pruification and properties of host factor I, a ribonucleic acid-binding protein[END_REF], Moller et al., 2002[START_REF] Zhang | The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs[END_REF]. E. coli contains between 50,000 to 60,000 Hfq monomers (about 10,000 hexamers), the majority of which (80% to 90%) are found in the cytoplasmic fraction in association with ribosomes [START_REF] Vasil'eva Iu | The regulatory role of the Hfq protein in bacterial cells[END_REF]. A significant amount is also located in the close proximity to the cytoplasmic membrane [START_REF] Diestra | Cellular electron microscopy imaging reveals the localization of the Hfq protein close to the bacterial membrane[END_REF][START_REF] Vassilieva | Cloning, purification, and crystallization of a bacterial gene expression regulator--Hfq protein from Escherichia coli[END_REF].

A main role of Hfq is to associate with trans-encoded sRNA and participate in the mechanism of post-transcriptional regulation. Thus, the protein is a direct regulator of genes under sRNA control, which include genes involved in stress responses [START_REF] Muffler | The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli[END_REF], virulence [START_REF] Sittka | The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium[END_REF], Ding et al., 2004) and quorum-sensing regulation [START_REF] Meibom | Hfq, a novel pleiotropic regulator of virulenceassociated genes in Francisella tularensis[END_REF][START_REF] Lenz | The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae[END_REF]. Furthermore Hfq plays a key role in the cellular response to phosphosugar toxicity and low iron levels [START_REF] Fantappie | The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression[END_REF][START_REF] Gorke | Noncoding RNA control of the making and breaking of sugars[END_REF][START_REF] Vanderpool | Physiological consequences of small RNA-mediated regulation of glucose-phosphate stress[END_REF][START_REF] Masse | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF]. Hfq also functions in cell envelope homeostasis through its role in the activity of MicA and RybB. These two sRNAs coordinately downregulate the expression of outer membrane proteins in stationary phase and under stress conditions [START_REF] Bossi | Porin biogenesis activates the sigma(E) response in Salmonella hfq mutants[END_REF], Figueroa-Bossi et al., 2006a, Papenfort et al., 2006[START_REF] Rasmussen | Regulation of ompA mRNA stability: the role of a small regulatory RNA in growth phase-dependent control[END_REF].

I.5.3.3a. Hfq structure and RNA binding patterns

Hfq is composed of 6 identical 11.2 kDa subunits. The stable, ringshaped structure of the hexamer was initially visualized by electron microscopy [START_REF] Zhang | The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs[END_REF]. and subsequently resolved by x-ray crystallography, with or without synthetic RNA bound to it [START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF]. The ring, about 70 Å in diameter, has a positively charged central pore on one of its two faces (Fig. 10). Studies with synthetic oligomers suggest that the two surfaces of the protein have different binding specificities: sequences rich in U residues bind preferentially to the proximal face, whereas Acontaining sequences bind to the distal face [START_REF] Link | Structure of Escherichia coli Hfq bound to polyriboadenylate RNA[END_REF], Mikulecky et al., 2004). It should be mentioned that even though the global architecture of the hexamer is conserved among species, some differences exist, in particular in the charge distribution. For instance, E. coli Hfq has a positive electrostatic surface for the trough that connects the proximal and distal faces, which is in contrast to the same area on the S. aureus Hfq, which shows a negative electrostatic surface (Fig. 10). From (Brennan & Link, 2007).

The overall architecture of Hfq is strongly reminiscent of that of Sm proteins that participate in many different RNA-processing reactions in eukaryotes [START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF][START_REF] Arluison | Structural Modelling of the Sm-like Protein Hfq from Escherichia coli[END_REF][START_REF] Achsel | A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro[END_REF][START_REF] Salgado-Garrido | Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin[END_REF][START_REF] Tharun | Yeast Sm-like proteins function in mRNA decapping and decay[END_REF]. Further in vitro experiments show that besides poly(A) tails, Hfq can bind poly(A-R-N) triplets, such as (AGG) 8 , (AGC) 8 , and the shorter (A-R-N) 4 AACAACAAGAAG (Link et al., 2009). Binding takes place on the distal face of Hfq [START_REF] Sun | Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites[END_REF], Mikulecky et al., 2004, Brennan & Link, 2007). In contrast, the proximal face of the protein is thought to be involved in the binding of sRNAs. This occurs at the level of the U-rich 3' ends resulting from Rho-independent transcription termination in sRNA genes [START_REF] Sauer | Structural basis for RNA 3'-end recognition by Hfq[END_REF], Otaka e t a l . , 2011). Work from our laboratory showed that Hfq has a strong binding affinity for the sequence AAUAA found in regulatory sRNA ChiX (Figueroa-Bossi et al., 2009) in the 5' UTR of the ompC mRNA [START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF] and in the yifK mRNA studied here (see Results section). Furthermore Balbontin and coworkers showed that Hfq can bind to a short stretch of Us located internally in the RybB sRNA [START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF].

I.5.3.3b. Mechanism of action of Hfq

The main role of Hfq in sRNA reguation is to stimulate the base-pair interaction between sRNAs and mRNAs (Fig. 11). Hfq was shown to increase the rate of sRNA-mRNA binding [START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF][START_REF] Kawamoto | Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq[END_REF] and to remodel RNA secondary structures [START_REF] Geissmann | Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator[END_REF], Moller et al., 2002). For example Hfq binding to the 5' end of rpoS mRNA -to the sequence (AAN) 4 -was shown to facilitate pairing between rpoS mRNA and DsrA sRNA [START_REF] Soper | The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA[END_REF]. The (AAN) 4 sequence is also required in vivo for Hfq-dependent regulation of rpoS translation by rpoS activating sRNAs in E. coli [START_REF] Soper | Positive regulation by small RNAs and the role of Hfq[END_REF]. In vivo, the loss of Hfq leads to a rapid degradation of the majority of trans-encoded sRNA, indicating that Hfq binding protects these sRNA against the action of RNases, mostly RNaseE [START_REF] Zhang | Global analysis of small RNA and mRNA targets of Hfq[END_REF][START_REF] Gottesman | The small RNA regulators of Escherichia coli: roles and mechanisms*[END_REF].

This effect is opposite to that exerted on most sRNA-regulated mRNA, which are destabilized upon Hfq binding. A study by the Aiba group showed that the Hfq/sRNA complex can recruit RNase E and stimulate mRNA cleavage following sRNA pairing [START_REF] Morita | RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs[END_REF]. Furthermore, Hfq can affect mRNA stability directly by stimulating poly(A) adenylation (Fig. 12) [START_REF] Hajnsdorf | Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I[END_REF], Mohanty e t a l . , 2004) and by binding to poly(A) tails [START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF]. These latter activities are somewhat contradictory since stimulation of poly(A) adenylation is expected to stimulate mRNA degradation whereas poly(A) binding was shown to prevent degradation [START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF]. To explain the paradox, Folichon and coworkers proposed that Hfq prevents poly(A)-dependent degradation of mRNAs whose stability is mostly controlled by RNase E and favors poly(A)-dependent degradation of structured mRNA lacking RNase E sites [START_REF] Folichon | The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation[END_REF]. it is used as a cofactor in many enzymes, such as those involved in the tricarboxylic acid (TCA) cycle, respiration and DNA synthesis. However, iron is also potentially toxic since it can react with oxygen to form free radicals that can damage cell components. Therefore, the concentration of free ferrous ion in all cells is tightly regulated. In E. coli and other bacteria, this regulation involves two factors: the Fur protein and RyhB sRNA. Fur is an iron-binding protein, that when bound to iron, represses transcription of genes involved in iron uptake [START_REF] Hantke | Iron and metal regulation in bacteria[END_REF]. Among the genes repressed by Fur is the ryhB gene [START_REF] Masse | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF]. Like the other members of the Fur regulon, the ryhB gene is activated (derepressed) when iron becomes limiting. Upon accumulating, RybB downregulates the synthesis of a number of ironutilization and iron-storage proteins by pairing with sequences near the translation initiation sites of their mRNAs and inducing their degradation (Fig. 13) [START_REF] Masse | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF]. In doing so, the sRNA limits iron consumption when the metal is scarce. As iron levels return to normal, ryhB repression by Fur is restored. Therefore, indirectly Fur upregulates all of the RybB-repressed genes, as many as 18 of which have been identified in E. coli [START_REF] Masse | Effect of RyhB small RNA on global iron use in Escherichia coli[END_REF]. From [START_REF] Masse | Small RNAs controlling iron metabolism[END_REF].

I.5.3.4b. GcvB and amino acid management

GcvB is a 200 nucleotide sRNA that downregualtes expression of a large number of genes encoding the transporters for amino acids, dipeptides and oligopeptides (Urbanowski e t a l . , 2000, Sharma et al., 2007, Pulvermacher et al., 2009a[START_REF] Pulvermacher | The role of the small regulatory RNA GcvB in GcvB/mRNA posttranscriptional regulation of oppA and dppA in Escherichia coli[END_REF], Sharma et al., 2011).

In addition, GcvB downregulates some amino acid biosynthetic genes (Sharma et al., 2011, Sharma et al., 2007). Although the physiological role of this regulation is not fully understood, it appears to be an important part of amino acid metabolism during growth in nutrient-rich environments (Sharma et al., 2011) GcvB was discovered fortuitously during the study of the promoter region for the gcvA gene, which encodes the main transcriptional regulator of the glycine cleavage system (see section II.3.2.) This work revealed the presence of a small gene divergently transcribed from gcvA with its promoter overlapping the gcvA promoter (Urbanowski et al., 2000). Further analysis showed that GcvA activates gcvB transcription by binding to a region between positons -29 and -76 relative to the transcription start site (Urbanowski et al., 2000).

The sequence of GcvB is highly conserved in gram-negative bacteria, particularly in the region involved in base-pairing with most target mRNAs.

This region, called the R1 region, is characterized by the presence of a long stretch of alternating G and U residues (Fig. 14).

Figure 14. GcvB sequence and secondary strutcure. From (Sharma et al., 2007).

As a result, most GcvB regulated mRNAs contain CA-rich sequences at variable distances from the translation initiation sites. In some targets, these elements lie more than 40 nucleotides upstream from the initiator AUG (Sharma et al., 2007). Two exceptions to the R1 pairing rule have been reported. One exception is in the downregulation of the phoP m R N A (encoding the response regulator of the PhoP/PhoQ two-components system) by GcvB in E.coli. Pairing was shown to involve sequences in stem-loop 4

(see Fig. 14) (Coornaert et al., 2013). The second exception is the cycA mRNA (encoding a permease for various amino acids) which pairs with GcvB region R2 (Pulvermacher et al., 2009b). Recently GcvB was also found to negatively control the leucine response regulator Lrp (Sharma et al., 2011). In this case, more than one region of GcvB appears to participate in the interaction with lrp mRNA (Sharma et al., 2011). Overall, the number of GcvB targets is thought to be greater than 40, making the GcvB network the largest of its kind (Fig. 15). Several lines of evidence strongly suggest that GcvB is an Hfqdependent sRNA. GcvB coimmunoprecipitates with Hfq in extracts of E. coli [START_REF] Zhang | Global analysis of small RNA and mRNA targets of Hfq[END_REF] and is unstable in ∆hfq strains of E. coli [START_REF] Urban | Translational control and target recognition by Escherichia coli small RNAs in vivo[END_REF] and Salmonella (Sharma et al., 2007). In E. coli, the GcvB requirement for Hfq has been demonstrated in the regulation of dppA, oppA (Pulvermacher et al., 2009a[START_REF] Urban | Translational control and target recognition by Escherichia coli small RNAs in vivo[END_REF], sstT (Pulvermacher et al., 2009c) and cycA (Pulvermacher et al., 2009b). For dppA, this requirement has also been confirmed in S. Typhimurium (Sharma et al., 2007). It was demosntrated that

Hfq confers increased the stability of the sRNA and facilitated pairing between GcvB and target mRNA (Pulvermacher et al., 2009a).

II. RESULTS

II.1. Characterization of yifK, a Hfq-regulated locus in Salmonella

Some years ago, our laboratory developed a genetic screen for identifying genes regulated by sRNA chaperon protein Hfq in Salmonella (Figueroa-Bossi et al., 2006a). This approach was aimed at identifying novel genes regulated by Hfq-dependent small RNAs. The strategy employed a transposable element derived of phage Mu, MudK, which carries a lac operon with a promterless lacZ gene which also lacks the initiating AUG codon. MudK can give rise to translational fusion upon inserting randomly in the bacterial chromosome [START_REF] Hughes | Transitory cis complementation: a method for providing transposition functions to defective transposons[END_REF]. Presence of a kanamycin-resistance (Kan R ) marker within the transposon allows to positively select for the transposition events. The transposition experiment was performed in a strain with the normal copy of the hfq gene deleted and a second copy of hfq fused to the promoter of the arabinose operon. Therefore this strain is a conditional hfq mutant: Hfq -in the absence of arabinose and Hfq+ in the presence of arabinose. Colonies selected on LB plates supplemented with kanamycin were replica-plated on lactose indicator plates (either MacConkey-lactose or LB-Xgal) that contained or lacked arabinose. Colonies whose color differed in the presence or absence of arabinose where characterized further. This study led to the identification of 19 lac fusions showing increased expression in the absence of arabinose and 4 fusions expressed less efficiently under these conditions (Figueroa-Bossi et al., 2006a). The members of the first group were candidates for loci downregulated by Hfq-dependent sRNAs, whereas the second group might contain loci requiring sRNAs for activation. In fact, three of the four members of the second group are genes transcribed by alternative sigma factor σ S , previously known to be activated by sRNAs [START_REF] Repoila | Small non-coding RNAs, coordinators of adaptation processes in Escherichia coli: the RpoS paradigm[END_REF].

When I joined the laboratory to prepare my thesis, the involvement of small RNAs in the regulation of most of the loci identified above was not yet known. I was assigned the task of determining the sRNA involvement in two such loci. The first locus was yifK, which codes for a highly conserved protein annotated as "putative amino acid transporter" in DNA sequence databases.

The second locus was aphA, coding for a periplasmic acid phosphatase [START_REF] Grose | Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica[END_REF]. Encouraging results originating from the yifK project during the initial phase of my thesis pushed me to concentrate more of this part, which became my official thesis project. The preliminary data obtained in the aphA project are described in Appendix #1.

Two MudK insertions generating in-frame lacZ fusions to the yifK gene had been isolated in the laboratory, one at position 143, the other at position 284 of the coding sequence. Both fusions are activated 5 to 7-fold in a strain deleted for hfq. The fusion at position 143 expresses higher ß-galactosidase activity and was chosen in most of the experiments described in this thesis.

Fur simplicity, I will often call this fusion yifK-lacZY, although one should remember that there is also Kan R cassette downstream from it. It should be noticed that the presence of the entire lac operon in the MudK element allows one to follow the entire lactose utilization pathway -not just the lacZ gene product. This will be important in many experiments described here.

II.1.1. yifK is repressed by GcvB

In Salmonella and E. coli, the small RNA GcvB represses translation of several mRNAs encoding the transporters for amino acids and peptides (see section I. 5.3.4b). Since yifK belongs to this class (se above), GcvB appeared a likely candidate for a yifK regulator. To test this hypothesis, I constructed a deletion of the gcvB gene by the technique of "Lambda Red" recombination (Datsenko & Wanner, 2000). (Sharma et al., 2007). The sequence diagram is from [START_REF] Vogel | A rough guide to the non-coding RNA world of Salmonella[END_REF]. Mutants #4 and #5 were isolated and characterized as described in the text.

I found the gcvB deletion to cause a 3-to 5-fold increase in the expression of the yifK-lacZY fusion (see section II.2 below). Although this increase was less than that observed with the hfq deletion (see above), these findings confirmed that GcvB downregulates yifK. The greater effect of the hfq deletion might indicate that additional sRNAs participate in the regulation or the Hfq acts directly in the repression mechanism (section II.2). Presence of a sequence segment complementary to GcvB in the region immediately upstream of yifK Shine-Dalgarno motif suggested that GcvB represses yifK translation by basepairing. GcvB uses a GU-rich portion of its sequence -called the R1 region (Fig. 16) -for pairing with most of its targets. This also appears to be the case with yifK, even though the portion of the R1 region involved in yifK pairing differs from that used with other targets (see below) .

In the initial part of my project, we sought to obtain evidence for the GcvB:yifK mRNA interaction through mutational analysis. In a first approach, I randomly mutagenized the gcvB gene and searched for mutants expressing yifK-lacZY at higher levels than the wild-type strain. I followed a procedure that combines mutagenic PCR and Lambda red recombination. Performing the mutant selection on MacConkey-lactose indicator plates allowed the identification of isolates with higher ß-galactosidase activity (red colonies in Fig. 17). Analysis of a number of these mutants showed that most them carried mutations in the promoter of gcvB, or in the promoter of gcvA (the gene adjacent to gcvB on the 5' side), or within the portion of the gcvA gene included in the mutagenized fragment. The gcvA gene encodes the activator of gcvB transcription (Sharma et al., 2007, Urbanowski et al., 2000). Thus, these results suggested that derepression of yifK-lacZY in these mutants was the consequence of a decrease in GcvB concentration due to reduced transcription of the gcvB gene. The position and nature of some of these mutations are shown in Fig. S1 of the manuscript (section II.2.I). In addition, two mutations were found to affect the CG-rich stem of gcvB Rho-independent transcription terminator (see Fig. 16, a bove). Similar mutations were previously found in other regulatory sRNAs and were shown to cause a decrease in sRNA levels, presumably by destabilizing the stem and making the sRNA more susceptible increased 3' end degradation [START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF], Bossi & Figueroa-Bossi, 2007, Figueroa-Bossi et al., 2009). This possibility was not tested here.

In parallel to the above analysis, a procedure identical to that depicted in Fig. 17A was carried on a DNA segment spanning the 5' end of the yifK gene. In this case, most of the red colonies analyzed were found to contain two or more mutations at different positions. This prevented the interpretation of the sequence data and the mutants were discarded. Only three mutants containing a single base-changes were identified; one carried a mutation in the -35 box of the yifK promoter increasing its strength, and two carried mutations inside the yifK 5' UTR that appear to increase the stability of the mRNA. The characterization of these mutants can be found in section II.2 below.

II.1.2. Genetic analysis of GcvB:yifK interaction unveils a translational enhancer in the target sequence

No mutations affecting the presumptive pairing sequences of GcvB or yifK mRNA were found by the above analysis. We interpreted this finding as an indication that perhaps single base changes did not relieve repression enough to cause a detectable color change on MacConkey plates. We thus proceeded to introduce multiple mutations by site-directed mutagenesis. As an initial test, we changed a four-base segment in the pairing interval: the sequence UGUG in GcvB was changed to ACAC; the reciprocal change was introduced at the corresponding position of yifK (ACAC to UGUG). The effects of these modifications on the expression of the yifK-lacZY fusion were assessed measuring ß-galactosidase activity (Fig. 18). Fig. 18 shows that changing the UGUG segment in GcvB to ACAC causes yifK-lacZY expression to increase a little more than two-fold. This is less than the increase observed upon deleting the gcvB gene (presumably because the four-base change still does not completely abolish the interaction) but it is nonetheless indicative of the involvement of the sequence in yifK repression.

In contrast, analysis of the reciprocal change in yifK gave an unexpected result: when the ACAC sequence was changed to UGUG, yifK expression did not increase (as one expected due to reduced pairing with GcvB) but actually decreased. In addition, this decrease was not aggravated when we combined the ACAC → U G U G yifK mutant with the GcvB mutant carrying the complementary change, UGUG → ACAC (fourth bar in Fig. 18). These findings suggested that the ACAC sequence in yifK stimulated yifK expression and this effect superseded GcvB regulation. To analyze this phenomenon in more detail, the core of the presumptive pairing interval was arbitrarily divided in two parts that were mutagenized separately. The results of this analysis, described in detail in the manuscript in section II.2 provided convincing evidence that GcvB represses yifK through base-pairing and corroborated the existence of an enhancer element embedded in the yifK sequence recognized by GcvB.

II.1.3. Separate subdomains of GcvB R1 region pair with yifK and dppA

One of the gcvB mutants constructed in the course of this study (a three-base change at positions 86-88) causes a nearly complete relief of yifK repression (comparable to a gcvB deletion; Fig. 19). Interestingly, this triple mutant has absolutely no effect on repression of a dppA-lacZY (Fig. 19) This confirms that separate R1 domains are involved in the recognition of yifK and dppA targets. The gcvB sequence between nucleotides 86 and 88 was changed from UUU to ACA as described in section II,2. The dppA-lacZ fusion was constructed using the two-step procedure of [START_REF] Ellermeier | Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria[END_REF], using primers ppK77 and ppK78 (see supporting information in section II,2.1). Suicide plasmid pCE40 was used to obtain an in-frame lac operon fusion starting at codon 16 of dppA (see Materials and Methods).

II.1.4. Role of ACA motifs in translation enhancement

A major part of my thesis project was devoted the characterization of the enhancer element discovered in yifK 5' UTR. Experiments described in section II. DNA sequence analysis identified triplets which have a particular detrimental effect. For example, replacing the upstream ACA by GGG causes a 92% reduction in yifK translation efficiency (see section II.2 below).

Abstract

GcvB is an archetypal multi-target small RNA regulator of genes involved in amino acid uptake or metabolism in enteric bacteria. Included in the GcvB regulon is the yifK locus, encoding a conserved putative amino acid transporter. Here we show that GcvB inhibits yifK mRNA translation by pairing with a sequence immediately upstream from the Shine-Dalgarno motif. Surprisingly, we found that some target sequence mutations that disrupt pairing, and thus are expected to relieve repression, actually lower yifK expression and cause it not to respond to GcvB variants carrying the corresponding compensatory changes. Work prompted by these observations revealed that the GcvB target sequence in yifK m R N A i n c l u d e s elements that stimulate translation initiation. Replacing each base of an ACA triplet near the center of the target sequence, by any other base, leads to a decrease in yifK expression.

Effects are additive, with some triple replacements causing up to a 90% reduction. These findings identify the ACA triplet as a component of a translational enhancer and reveal that GcvB acts by countering the enhancer function. We show that the enhancer activity does not require the ACA motif to be located at a fixed distance from the Shine-Dalgarno sequence nor does it depend on the spacing between the latter and the initiating AUG. Finally, additional data show that besides the GcvB control, yifK is regulated at the transcriptional level by the leucine responsive regulator Lrp, which completely silences yifK e x p r e s s i o n i n l e u c i n edeprived minimal medium.

Author summary

The majority of small RNA (sRNA) regulators in bacteria act by inhibiting translation initiation in target messenger RNAs. The study of this regulatory mechanism not only allows a better understanding of sRNA function but it can also provide new insight into aspects of the translation initiation process that remain incompletely characterized. This was the case in the work described here. Analyzing the mechanism by which GcvB, a multi-target sRNA, downregulates a putative amino acid transporter in Salmonella, we discovered that the sequence base-pairing with GcvB in the target mRNA functions as a translational enhancer.

Replacing an ACA triplet near the center of the sequence with unrelated triplets leads to a decrease in translational initiation efficiency that can be as severe as more than 90%.

Interestingly, some of these replacements concomitantly render the mRNA insensitive to GcvB variants carrying the appropriate compensatory changes, suggesting that targeting the enhancer element is paramount for GcvB regulatory effectiveness. Overall the data presented in the paper unveil the role of the ACA motif in the translation initiation process and lay the grounds for further analysis of the mechanism involved. which represses expression of mRNA for dispensable iron-sequestering proteins when iron is limiting (Jacques et al., 2006[START_REF] Masse | Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli[END_REF][START_REF] Masse | A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli[END_REF][START_REF] Masse | Effect of RyhB small RNA on global iron use in Escherichia coli[END_REF], Prevost et al., 2011); RybB, which downregulates several outer membrane protein mRNAs under envelope stress conditions [START_REF] Balbontin | Recognition of heptameric seed sequence underlies multi-target regulation by RybB small RNA in Salmonella enterica[END_REF], Gogol et al., 2011, Johansen et al., 2006, Papenfort et al., 2006, Thompson et al., 2007), Spot 42, which amplifies the regulatory range of catabolite repression by targeting several mRNAs involved in sugar uptake and consumption (Beisel & Storz, 2011)and GcvB, which downregulates dozens of different mRNAs involved in amino acid uptake or metabolism in E. coli and Salmonella (Pulvermacher et al., 2009b, Sharma et al., 2007, Sharma et al., 2011, Urbanowski et al., 2000). GcvB, a 200 nucleotide-long sRNA, was identified serendipitously during a study of gcvA, the gene for the main transcriptional regulator of the glycine cleavage operon gcvTHP (Urbanowski et al., 2000). The latter encodes the enzymes of the glycine cleavage system, the pathway generating one-carbon units from the oxidative cleavage of glycine [START_REF] Stauffer | GcvA interacts with both the alpha and sigma subunits of RNA polymerase to activate the Escherichia coli gcvB gene and the gcvTHP operon[END_REF]. The gcvB g e n e i s l o c a t e d immediately adjacent to gcvA in the opposite orientation with its promoter partially overlapping the gcvA p r o m o t e r . I n t h e presence of excess glycine, the GcvA protein activates transcription of the gcvTHP operon as well as of gcvB (Urbanowski et al., 2000).

Initial characterization of GcvB showed this sRNA to downregulate the synthesis of DppA and OppA proteins, main components of dipeptide-and oligopeptide-transport systems, respectively (Sharma et al., 2007, Urbanowski et al., 2000). Since then, the number of genes whose LacZ levels changed (either increased or decreased) upon inactivating Hfq (Figueroa-Bossi et al., 2006b). Among the candidates that were found, two independent isolates upregulated in the hfq mutant background carried the lacZ insert translationally fused to the yifK gene (Figueroa-Bossi et al., 2006b).

Presumptive identification of this gene as an amino acid transporter suggested that yifK might be a GcvB target. We thus proceeded to

Results

Genetic identification of a GcvB-regulated locus

Our original screen for H f q -regulated genes yielded two isolates carrying the MudK Somewhat surprisingly, LacZ levels in the gcvB-deleted strain are not as high as the levels measured in a strain deleted for hfq (Figure 1).

This might reflect the existence of one or more additional sRNA(s) participating in yifK repression. Alternatively, Hfq could repress yifK directly [START_REF] Desnoyers | Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq[END_REF]. The data in Figure 1 show that loss of Hfq is epistatic to the gcvB deletion.

Mutations affecting of yifK expression

Primer extension experiments mapped the 5' end of yifK mRNA to 64 nucleotides (nt) upstream from the initiating AUG (Figure 2). promoter consensus match, TTGACA, Figure 2A), strongly suggest that the mutation increases the strength of the yifK promoter.

The mutation leads to a sharp rise in the intensity of the primer extension product (Figure 2B) and a more than 10-fold increase in ß-galactosidase activity (data not shown).

These findings confirmed that the 5' end identified by primer extension corresponds to yifK transcription initiation site. The remaining two mutations affect residues within the 5' UTR (Figure 2A). One allele, resulting in a U

to C change at position +21, falls within a AUrich segment (AUAACAAUAA) that might constitute a site for Hfq binding (Brennan & Link, 2007, Vogel & Luisi, 2011). Consistent with this interpretation, the mutation has no effect in ∆hfq background (Figure 2C). Finally the third allele (G to A at +27) affects the CGrich stem of a presumptive secondary structure immediately adjacent to the AU-rich segment.

The change causes yifK-lacZY expression to increase even in the hfq mutant (Figure 2C)

suggesting that it affects an Hfq-independent step. To date, however, this step remains unidentified.

GcvB inhibits yifK translation by pairing with an enhancer element

The above approach yielded no mutations affecting the presumptive pairing sequences of In vitro toeprint experiments, showing that GcvB inhibits the binding of ribosomal 30S subunit to yifK t r a n s l a t i o n i n i t i a t i o n s i t e , specifically and in a dose-dependent manner (Figure 4), provided independent support to this conclusion.

Again, however, changing the CA doublet on the 3' side of the AAA triplet to UC produced an unusual pattern: like in the quadruplet mutant above, yifK-lacZY expression decreased rather than increase, becoming insensitive to a GcvB variant carrying the compensatory change (Figure 3B)

as well as to gcvB or hfq deletions (Figure 3C).

We interpreted these findings to suggest that the CA to UC conversion affects translation and in that under such conditions, GcvB action is no longer rate-limiting for yifK expression.

The effects of the mutation on translational efficiency were examined in vitro u s i n g a reconstituted system. Results in Figure 5 showed an epitope-tagged Cat protein to accumulate at significantly greater levels when made from a gene fusion to the wt yifK 5' UTR than from an equivalent construct carrying the CA to UC change. These data confirm that the CA doublet stimulates translation and suggest that GcvB effectiveness in regulation reflects the targeting of an activating element.

Anatomy of a translational enhancer

To characterize the enhancer element, A peculiarity of the yifK t r a n s l a t i o n signal is the unusually short distance (four nucleotides) between the most conserved base of the SD motif [START_REF] Shultzaberger | Anatomy of Escherichia coli ribosome binding sites[END_REF] and the initiating AUG. We thus envisaged that the role of the enhancer could be to somehow compensate for such suboptimal arrangement. To test this possibility, we generated a 7 nt tandem direct duplication of the SD region and then inactivated either copy of the SD by changing the GAGG motif to GACG (Figure 7). Thus, the resulting 1984). The approximately 500 nt RNA accumulates in the RNase E mutant (Figure 7C). Previous work in E.coli, showed that this tRNA precursor is processed by the concerted actions RNase E and RNase P in a pathway that, intriguingly, also sees the participation of Hfq [START_REF] Zhang | Global analysis of small RNA and mRNA targets of Hfq[END_REF].

yifK is repressed by Lrp

Early on in this study, it became apparent that yifK expression was exquisitely sensitive to the growth medium and virtually silenced in minimal medium. As a result, a strain with the yifK-lacZY fusion is phenotypically Lac -when plated in minimal medium. We exploited this phenotype to positively select for spontaneous extent in minimal medium. This is not surprising as GcvB is transcribed at very low level under these conditions (Sharma et al., 2007) and inactivating Lrp does not reverse this pattern (Figure S3). The data in Figure S3 differ from those of Modi et al (Modi et al.) who reported an approximate 30-fold increase in GcvB levels in an lrp deletion mutant of in E.coli. This discrepancy might reflect differences in the organisms used or in media composition.

Discussion

In Translation initiation efficiencies have been known to vary greatly as a function of the sequence context of the initiation region [START_REF] Dreyfus | What constitutes the signal for the initiation of protein synthesis on Escherichia coli mRNAs?[END_REF], Yarchuk e t a l . , 1992).

Computational analysis of sequences

surrounding translation initiation sites of E.coli genes showed that the spacing between the SD and the initiation codon affects SD sequence conservation and its pattern. This study did not reveal significant biases outside these main elements [START_REF] Shultzaberger | Anatomy of Escherichia coli ribosome binding sites[END_REF]; however, conserved patterns occurring at variable positions might have been difficult to identify by the statistical analysis. Indeed, separates lines of evidence point to the role of the ACA motif in translation initiation. The motif is found in other translation enhancer sequences [START_REF] Komarova | Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation[END_REF][START_REF] Mccarthy | Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation[END_REF] and, as an ACAA repeat, was shown to promote translation initiation in the absence of a SD sequence [START_REF] Tzareva | Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions[END_REF]. ACA is also found in the loops of pseudoknots formed by RNA ligands to ribosomal protein S1, obtained through Systematic Evolution of

Ligands by Exponential Enrichment (SELEX) [START_REF] Ringquist | Highaffinity RNA ligands to Escherichia coli ribosomes and ribosomal protein S1: comparison of natural and unnatural binding sites[END_REF] a n d i s p a r t o f t h e SELEX-determined consensus sequence for binding of protein CsrA, a translational regulator [START_REF] Dubey | RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction[END_REF]. Finally, ACA is the recognition sequence of the MazF endonuclease that inactivates E.coli mRNAs by preferentially cleaving near the translation initiation codon [START_REF] Vesper | Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli[END_REF].

The lack of position requirements for the functioning of the enhancer suggests that its role is to provide an anchor point for the 30 S ribosomal subunit so as to facilitate subsequent recognition of the SD sequence. Some of the evidence reviewed above tentatively identifies protein S1 as the possible candidate for the interaction. In vitro S1-binding studies with some of the mutants constructed in the course of this work should allow testing of this idea.

Combined with the mutational analysis of other GcvB-regulated mRNAs, this approach might provide further insight into how the ACA motif participates in the translation initiation step.

Materials and Methods

Bacterial strains and culture conditions

Strains used in this study were derivatives of Salmonella enterica s e r o v a r Typhimurium strain LT2 [START_REF] Lilleengen | Typing of Salmonella typhimurium by means of bacteriophage[END_REF].

Strain SV4280 was a gift of J. Casadesús.

Except for the latter strain and for strain MA7224, all other strains were derived from MA3409, an LT2 derivative cured for the Gifsy-1 prophage (Figueroa-Bossi e t a l . , 1997). The genotypes of the relevant strains used are listed in Table 1. Bacteria were cultured at 37°C in liquid media or in media solidified by the addition of 1.5% Difco agar.

LB broth [START_REF] Bertani | Lysogeny at mid-twentieth century: P1, P2, and other experimental systems[END_REF]

Genetic techniques

Generalized transduction was performed using the high-frequency transducing mutant of phage P22, HT 105/1 int-201 (Schmieger, 1972) a s d e s c r i b e d (Lemire e t a l . , 2011).

Chromosomal engineering (recombineering)

was carried out by the λ red recombination method (Datsenko & Wanner, 2000, Murphy et al., 2000, Yu et al., 2000) implemented as in (Datsenko & Wanner, 2000). Donor DNA fragments were generated by PCR using plasmid DNA or chromosomal DNA or DNA oligonucleotides as templates. A m p l i f i e d fragments were electroporated into appropriate strains harboring the conditionally replicating plasmid pKD46, which carries the λ red operon under the control of the P BAD promoter (Datsenko & Wanner, 2000). Bacteria carrying pKD46 were grown at 30°C in the presence of ampicillin and exposed to arabinose (10 mM) for 3 hours prior to preparation of electrocompetent cells. Electroporation was carried out using a Bio-Rad MicroPulser under the conditions specified by the manufacturer.

Recombinant colonies were selected on LB plates containing the appropriate antibiotic.

Constructs were verified by PCR and DNA sequence analysis (performed by GATC company).

Random PCR mutagenesis

PCR amplification of DNA fragments under error-prone conditions was carried out as previously described (Bossi & Figueroa-Bossi, 2007). Typically, the DNA fragment in the second step was also obtained by PCR using oligonucleotides with complementary sequences at their 3' ends priming DNA synthesis on each other ("reciprocal priming").

In site-directed mutagenesis experiments, one of the two primers contained the desired nucleotide changes or randomized sequence stretches. All constructs were verified by DNA sequencing. Table S2 shows the list of alleles made by standard or scarless recombineering.

RNA extraction and analysis by primer extension and Northern blotting

RNA was prepared by the acid-hotphenol method from exponentially growing cells (OD 600 of 0.35) as previously described (Bossi & Figueroa-Bossi, 2007). R e v e r s e transcriptase reactions (enzyme Superscript II from Invitrogen) were carried out using 5 µg of bulk RNA and 32 P-labeled primer ppF49.

The same DNA primer was used for the Laemmli buffer and immediate freezing.

Aliquots were loaded on 12.5% Acrylamide gels and Western analysis performed as previously described (Uzzau et al., 2001).

Toeprinting assay

Toeprinting reactions were carried out as a Srains are derived from Salmonella enterica serovar Typhimurium strain MA3409 (Figueroa-Bossi et al., 1997) except for strains SV4280 (Camacho & Casadesus, 2002), MA3398 [START_REF] Figueroa-Bossi | Unsuspected prophagelike elements in Salmonella typhimurium[END_REF] and MA7224 (Uzzau et al., 2001). The term "scar" denotes the DNA sequence left following Flp-mediated excision of antibiotic-resistance cassettes introduced by the procedure of [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF]. Additional details on relevant alleles in this Table can be found in Table S2.

Figures

Figure 1. Effect of hfq and gcvB deletions on the expression of a yifK-lacZ fusion.

Deleting gcvB causes yifK expression to increase approximately 5-fold in exponential cultures (OD600 ≈ 0.4) and less than three-fold in stationary overnight cultures (OD600 ≈ 2.0). A greater increase is observed in the hfq deletion mutant, suggesting the involvement of a separate Hfq-dependent step in yifK regulation. Strains used were MA8020 (wt), MA8021

(∆hfq), MA10377 (∆gcvB) and MA10403 (∆hfq ∆gcvB). All strains carry the yifK::MudK lac fusion. Their full genotypes are listed in Table 1. ). This analysis identifies the 5' end of yifK mRNA and shows that all three mutations lead to higher mRNA levels. Measurements of ß-galactosidase activity (C) show that U21C, but not G27A, does not cause any further increase in yifK-lacZ expression in the ∆hfq background, suggesting that U21C affects Hfq binding to yifK mRNA.

ß-galactosidase activity was measured in exponentially growing cultures (OD600 ≈ 0.4). (primers used to generate the set of mutagenized fragments are listed in Table S2). this change in a strain with a single SD). The upstream ACA triplet was converted to GGG in the constructs carrying either SD sequence mutated (constructs n. 6 and 7). ß-galactosidase activity was measured as described in the legend to Fig. 6. The activity of the wild-type strain (construct n. 1) was set to 100. Standard deviations were less than 5% of the mean in all cases. The data (see also strains' phenotypes on MacConkey-lactose plates) show that the upstream ACA maintains its enhancer effect when placed further upstream from the initiation region, independent of the spacing between the SD sequence and the starting AUG. oligonucleotides complementary to a sequence near the 5' end of yifK mRNA (ppF16; probe 1 above) or to an internal portion of the argX tRNA (ppG42, probe 2), or to a sequence in the argX-hisR intercistronic region (ppH27, probe 3). Probing for the SsrA RNA (pp813) served as loading control. The strains used as source of RNA are listed in Table 1. DNA oligonucleotides used as hybridization probes are listed in Table S1. S1) and chromosomal DNA from strain MA1179 (Table 1) as template. The amplifed fragment was introduced into strain MA10280 (yifK-lacZY / pKD46) and recombinants were selected on MacConkey lactose plates supplemented with chloramphenicol as described in the text. Red colored colonies were picked and the region of the gcvB locus analyzed by DNA sequencing. Most of the isolates were found to harbor DNA sequence changes in the gcvA-gcvB intergenic region, which affected either the -35 or -10 box of the gcvB promoter, or the -10 box of the gcvA promoter [1]. Mutations within the initial portion of the gcvA coding sequence and a mutation affecting the CG-rich stem of gcvB's Rho-dependent terminator were also identified (data not shown). S1 andS2.

Figure S3. Comparing GcvB sRNA levels in wild-type and in an lrp insertion mutant as a function of the growth medium. Bacteria were grown in minimal medium (NCE [1]) supplemented with 0.2 % glycerol or in LB to an OD 600 ≈ 0.4. RNA was extracted, fractionated on an 8% polyacrylamide-8 M urea gel and subjected to Northern blot hybridization. Blot was hybridized to DNA oligonucleotides complementary to GcvB and to 5S RNA (for loading control). The probes used were ppI67 (GcvB) and ppB10 (5S)(Table S1).

II.3. An intriguing regulator of yifK expression

Some of yifK's translation enhancer mutations lower the expression of a yifK-lacZY fusion to the extent that the strain becomes Lac -even if deleted for the lrp gene (for Lrp effects on yifK see section II.2). We considered that possible factors mediating the enhancer activity could be identified through the analysis of suppressor mutations restoring a L a c + phenotype. Strain YQ114 (yifK C49U,A50C::MudK lrp::cat; was used to select spontaneous mutants capable of using lactose as sole carbon source (note: the MudK element in YQ114 includes the lacZY operon plus a kanamycin-resistance gene; see II.1). A total of 36 Lac + colonies were were picked and genetically characterized. Anticipating that a fraction of these mutants might contain from cis-acting alleles, a preliminary test was performed to identify mutations genetically linked to the yifK-lacZY fusion. For this, P22 transducing lysates were made on all mutants and used to transduce strain 8920 selecting kanamycin resistance (Kan R ). Transductant colonies were replica-plated onto NCE-lactose plates. With 9 of the 36 lysates more than 95% of Kan R transductants were Lac + indicating that the mutation conferring the Lac + phenotype was genetically linked to yifK. These mutants were characterized further by amplifying and sequencing the region at the 5' end of the yifK-lacZY fusion. As expected all of the mutants carried sequence changes in this region (Fig. 21). With the lysates from the remaining 27 mutants, the vast majority of the Kan R transductants remained Lac -, indicating that the mutation causing the Lac + phenotype in the donor strain was not near to the yifK locus. Still, a small fraction of transductants (bewteen 2 and 4 % of the total) were Lac + suggesting that the Lac + mutation was in the same transduction interval as the yifK::MudK insertion, albeit distant from this site. We therefore performed additional mapping experiments tests using other genetic markers in the region that were available in our laboratory. This work revealed the mutation to be approximately 50% linked to a Tn10 insertion in the ilvA gene. To map the mutation more precisely we sought additional markers from other laboratories. One such marker, obtained froim the laboratory of Prof.

Casadesús at the University of Seville, Spain, was a cat insertion in the yifA gene (also named hdfR) about 10 Kb counterclockwise from ilvA. Much to our surprise, when we used the hdfR::cat insertion in transduction experiments, we discovered that this insertion by itself conferred a Lac + phenotype to a strain with yifK C49U,A50C::MudK and ∆lrp. Postulating that our Lac+ mutations could be in this gene, we sequenced the entire hdfR gene from two of the mutants isolates (#2 and #14). This analyses showed no hdfR sequence changes in either strain. However, sequencing runs that extended to the region upstream from the hdfR promoter revealed the presence of mutations in the adjacent gene, yifE, oriented in the opposite direction from hdfR (Fig. 22). Isolate #2 carried a 10 bp deletion approximately in the middle of the 336 nucleotide yifE orf, while isolate #14 had a nonsense mutation in the 15 th codon (AAA to UAA). To confirm these findings, new hfdR (yifA) and yifE mutants were constructed by the lambda Red method. Introduction of either of these constructs (∆hdfR::tetAR or ∆yifE::tetAR) into strain YQ114 (yifK C49U,A50C::MudK lrp::cat) caused the strain to become Lac + .

The hdfR gene encodes a poorly characterized LysR-type transcriptional regulator originally identified in E.coli as a repressor of the flagellar master operon flhDC [START_REF] Ko | H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein[END_REF]. Purified HdfR protein was shown to bind to the flhDC promoter region and the hdfR gene to be itself negatively regulated by H-NS [START_REF] Ko | H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein[END_REF]. In Salmonella, hdfR was identifed a locus responsible for the high-level expression of the std fimbrial operon in dam methylase mutants [START_REF] Jakomin | Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR[END_REF]. In this case, HdfR functioned as an activator of transcripton, although it appeared to do so only when the std promoter DNA was unmethylated [START_REF] Jakomin | Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR[END_REF]. To date the environmental signals HdfR modulating expression and/or activity of HdfR remain unknown.

The yifE gene encodes a small protein (112 aa) highly conserved in the bacterial kingdom, but whose function is currently unknown.

The roles of HdfR and YifE in yifK expression were further characterized measuring the effects of the hdfR or yifE insertions on yifK expression in various genetic backgrounds in cells growing exponentially in LB medium (Fig. 23). The results in Fig. 22 allow some important points to be made: i) inactivation of either gene causes yifK-lacZY expression to increase regardless of whether yifK 5'UTR is normal or contains the C49U,A50C mutation. In other words, the effects of the hdfR or yifE are not specific to the C49U,A50C allele.

Although constructs with the C49U,A50C mutation have an about 10-fold lower overall activity, the derepression ratios in the presence or absence of HdfR or YifE are the same as in yifK w i l d -type; ii) H d f R / Y i f E -mediated regulation is active in cells growing in rich medium (LB). This is unlike what observed with Lrp regulation which only operates in minimal medium (note the absence of Lrp effects above). In fact, HdfR/YifE-mediated regulation appears to be completely independent of Lrp regulation.

In conclusion, these data suggest that hdfR and yifE gene products act in concert to repress yifK expression. The most likely scenario is that repression takes place at the transcriptional level and that the active repressor is a heterodimer containing HdfR and YifE polypeptides. This scenario can incorporate the finding that the majority of cis-acting mutations from the Lac + selection (above) map on the 5' side of yifK promoter -35 box (Fig. 21, above). This could be the site where the repressor binds and the mutations in Fig. 21 could affect the binding. However, alternative scenarios are also possible. For example, either HdfR or YifE could act alone in repressing yifK and the other protein be required for expression of the repressor. Since YifE shows no similarity to known transcriptional regulators, this includes the possibility that YifE acts post-transcriptionally, and HfdR is required for yifE transcription. Work aimed at characterizing this system is currently under way in the laboratory.

II.4. A specialized mutant ribosome improves initiation from a UUG start codon

One of the MudK inserts identified in the original screen for Hfqreguated lac fusions was in the aphA gene (Figueroa-Bossi et al., 2006a).

This gene encodes a periplasmic acid phosphatase involved in the uptake of nicotinamide mononucleotide [START_REF] Grose | Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica[END_REF]. The MudK insert position is unusual as it is inside the AUG initiation codon of aphA, between the second and third nucleotide position. The insert generates a UUG codon in-frame with the lacZ coding sequence, which appears to function as initiation codon (Fig. 23). The aphA-lacZ fusion is poorly expressed -about 20 Miller units of ßgalactosidase in exponentially growing cells -and the expression levels increase 3.5-fold upon inactivating hfq (Figueroa-Bossi et al., 2006a). These findings, together with the presence of a long AU-rich sequence that could constitute a Hfq binding site upstream from the Shine-Dalgarno motif (Fig. 23), suggested that a Hfq-dependent regulatory sRNA might contribute to the poor expression of the aphA-lacZY fusion.

The low ß-galactosidase activity does not allow growth on lactose as sole carbon source (strain is phenotypically Lac -). However, the 3.5-fold increase that results from the inactivation of Hfq is sufficient to restore growth on lactose (Lac + ). This suggested that a lac-based selection could be used to analyze the possible participation of a small RNA in aphA repression. We postulated that mutations inactivating the hypothetical sRNA regulator would have effects similar to that of Hfq inactivation and confer a Lac + phenotype. I therefore used the strain carrying aphA-lacZY to select spontaneous mutants forming colonies on NCE-lactose minimal plates. A total of 36 mutants were picked and genetically characterized. In all of them, except one, the mutation conferring Lac + mapped very close to aphA. The unique unlinked mutation appeared as the best candidate for an sRNA mutant. I therefore undertook its characterization. Using a P22 transducing lysate made on a pool of random T-POP (Tet R ) transposon insertions in the Salmonella chromosme [START_REF] Rappleye | A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes[END_REF], I was able to isolate an insertion closely linked to the site of the Lac + mutation. I then used the technique of inverse PCR [START_REF] Hartl | Inverse polymerase chain reaction[END_REF] to amplify and sequence the region adjacent to the insertion site. This analysis showed the T-POP element to be in close proximity of the 3' end of a 16 S ribosomal RNA gene. The sequencing revealed that the Lac + mutation caused the deletion of the U residue in the middle of anti-Shine-Dalgarno sequence of 16S RNA, changing the sequence from CCUCCU to CCCCU. To confirm that this mutation was responsible for the Lac + phenotype, I prepared a P22 lysate on the Lac + mutant with the T-POP insertion and transduced the aphA-lacZY strain (Lac -) selecting Tet R . More than 95% of the Tet R transductants became Lac + , conclusively demonstrating the role of the 16 S RNA change in this phenotype.

In conclusion, this analysis did not allowed me to confirm the existence of aphA-regulating sRNA(s). Nonetheless, the study provided an interesting example of ribosomal "specialisation" [START_REF] Hui | Specialized ribosome system: preferential translation of a single mRNA species by a subpopulation of mutated ribosomes in Escherichia coli[END_REF]. We interpret our findings postulating that the poor expression of the aphA-lacZY fusion is due to an imperfect interaction between the 3' end of the 16 RNA and the Shine-Dalgarno sequence (see Fig. 24), combined to the presence of a weak initiation codon. Deleting a U residue in the anti-Shine Dalgarno sequence results in the replacement of a G-U base pair by a G:C base pair (Fig. 24).

This could strenghten the interaction enough to positively affect translation intiation rates and increase ß-galactosidase synthesis. Like Escherichia coli, Salmonella enterica contains 7 copies of the 16 S RNA gene. From examination of the sequence adjacent to the mutant site in our Lac + strain, we can conclude that the mutation is located in either rrsA or rrsB loci.

characterized structurally, the levels of expression of the hfq gene and the role of S aureus Hfq in bacterial physiology have remained poorly defined .

The Bossi laboratory has been interested in Hfq function as part of the sRNA regulatory function from the very beginning of their studies on sRNA regulation in Salmonella enterica. Indeed, the first experiments designed in the laboratory were aimed at identifying sRNA target genes using the dependence of target gene expression on Hfq activity as an indicator of sRNA-mediated regulation. This genetic approach yielded dozens of reporter gene fusions whose level of expression depend on Hfq function (Figueroa-Bossi et al., 2006a). Using these tools, the functional properties of Staphylococcus aureus Hfq protein were studied, when the protein is expressed from the Salmonella chromosome, in replacement of the endogenous protein. To further extened the work, the B. burgdorferi Hfq protein was also included in the project. By the same experimental approach we could examine this Hfq protein, a recent report having indicated the functionality of Borrelia's Hfq and its ability to complement an E. coli null mutant [START_REF] Lybecker | Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi[END_REF].

The experimental design also took advantage of the "no-mark" recombineering approach based on Lambda red mediated gene replacement.

The coding sequence of S. Typhimurium Hfq (Hfq STM ) was replaced by the respective coding sequences of S. aureous (Hfq SA ) or B burgdorferi (Hfq BB ).

The work in which I participated examined three main aspects related to Hfq in these strains: first, expression of the heterologous proteins under S.

Typhimurium promoter control was confirmed by Western analysis of epitope tagged proteins; second, reporter fusions to sRNA controlled genes known to be dependent on Hfq function served to evaluate the relative activity of the proteins; and third, Northern analysis of selected sRNAs, whose stability is known to depend on the presence of Hfq, were performed in the different strains. The ensemble of the results indicates that, even though the heterologous proteins were expressed to detectable levels, comparable to those of the endogenous protein, no functional complementation could be detected. Finally, results from Northern blot analysis showed the sRNA patterns in the heterologous hfq background to be close to those of the S.

Typhimurium hfq deletion strain.

Introduction

Base pairing between regulatory RNAs and complementary sequences in messenger RNAs is a highly conserved mechanism controlling gene expression at the post-transcriptional level in all forms of life. In bacteria, best-studied regulatory small

RNAs (sRNAs) are encoded at separate locations from their target genes and interact with target mRNAs through short and imperfect stretches of complementarity (reviewed in [1]). In some bacterial species, like Escherichia coli, Salmonella enterica and Vibrio species, these trans-encoded sRNAs require chaperon protein Hfq for activity. Discovered in the late nineteen sixties as a host factor needed for in vitro replication of RNA phage Qß [2], Hfq was later found to be a key player in a number of RNA transactions (reviewed in [3][4][5]). In particular, Hfq participates in sRNAmediated regulation by binding both sRNAs and cognate mRNAs and stimulating their association. For many sRNAs, Hfq binding is also essential to confer protection against degradation by ribonucleases [6]. Hfq and Hfq-like proteins belong to the Smlike (Lsm) family of RNA-binding proteins characterized by a ring-shaped multimeric architecture. X-ray crystal structure analysis of the Hfq-like protein from

Staphylococcus aureus (Hfq SA ) and of E. coli Hfq (Hfq EC ) showed that both proteins assemble in homohexameric rings approximately 70 Å in diameter [7][8][9]. A short Urich synthetic RNA co-crystallized with Hfq SA was found to circle around the positively charged central pore of the torus on the so-called proximal face [9]. In contrast, A-containing RNA oligomers bind to the distal face of Hfq EC [7]. The notion of opposite Hfq surfaces having different ligand specificities is independently supported by mutational studies [10]. It should be noticed, however, that while similar on the proximal face, Hfq from S. aureus and E. coli differ sharply in their charge distribution on the distal face and in the trough that connects proximal and distal faces. This latter region has a positively charged surface in Hfq EC and a negatively charged surface in Hfq SA [3]. Finally, while Hfq-like proteins have an evolutionarily conserved core of 65 amino acids, the C-terminus is variable in length, leading to a controversy about its function [11,12]. The Hfq extended C-terminus is found in γand β-proteobacteria whereas in the case of Gram-positive bacteria such as in Hfq SA , extensions are short.

In most bacteria, loss of Hfq function, albeit not a lethal event, causes a variety of pleiotropic defects and renders strains particularly susceptible to environmental stress [13]. Many of these phenotypes result from the loss of sRNA activities. For example, the σ S -dependent stress response is poorly induced in hfq mutants due to the lack of sRNA-mediated activation of rpoS mRNA translation (reviewed in [14]). At the same time, the σ E -dependent envelope stress response is chronically induced due to overaccumulation of outer membrane proteins that are normally downregulated by sRNAs [15][16][17][18][19][20].

Genes encoding Hfq-like proteins are found in about half of the sequenced genomes [START_REF] Sun | Predicted structure and phyletic distribution of the RNA-binding protein Hfq[END_REF]. In many pathogens, they are required for virulence and were shown to participate in sRNA-mediated regulatory processes (reviewed in [START_REF] Chao | The role of Hfq in bacterial pathogens[END_REF]). The functional proficiency of Hfq homologues was inferred from their ability to complement the loss of Hfq in E. coli. For example, Pseudomonas aeruginosa Hfq, which shares 92% identity with Hfq EC in the initial 68 amino acids, fully replaced Hfq Ec in terms of its requirement from Qß replication and rpoS expression [START_REF] Sonnleitner | Functional replacement of the Escherichia coli hfq gene by the homologue of Pseudomonas aeruginosa[END_REF]. Likewise, the Moraxella catarrhali hfq-like gene, in spite of being twice the size of the hfq EC (but highly similar in the N-terminal encoding domain) partially complemented the growth defect and the stress sensitivity of an E. coli hfq mutant [START_REF] Attia | Moraxella catarrhalis expresses an unusual Hfq protein[END_REF]. Intriguingly, even a protein with very limited sequence relatedness to Hfq (only 12% identity), encoded by Borrelia burgdorferi (Hfq BB ), was recently reported to complement an E. coli hfq mutant [START_REF] Lybecker | Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi[END_REF]. On the other hand, Hfq-like proteins from Neisseria meningitides and Aquifex aeolicus, and from archaeon Methanocaldococcus jannaschii did not reverse the chronic σ E induction of a Salmonella strain lacking Hfq [START_REF] Sittka | Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes[END_REF]. However, these proteins were capable of binding some Salmonella sRNAs and also caused specific RNA processing defects [START_REF] Sittka | Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes[END_REF].

The function of Hfq-like proteins remains unclear for some bacterial species. For example, deletion of the Bacillus subtilis hfq-like gene (ymaH) does not affect growth rate, stress adaptation, or activities of all sRNAs tested [START_REF] Gaballa | The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins[END_REF][START_REF] Heidrich | The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism[END_REF][START_REF] Silvaggi | Small untranslated RNA antitoxin in Bacillus subtilis[END_REF]. Similar results were reported for S. aureus. Deletion of the hfq SA gene in several pathogenic isolates did not impair or in any way impact their physiology [START_REF] Bohn | No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus[END_REF] possibly because hfq SA is poorly expressed or not expressed in the strains used for these studies [START_REF] Bohn | No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus[END_REF][START_REF] Geisinger | Inhibition of rot translation by RNAIII, a key feature of agr function[END_REF](see also [START_REF] Felden | The Staphylococcus aureus RNome and its commitment to virulence[END_REF]).

On the other hand, in S. aureus strains where Hfq is detected, deletion of its coding gene reportedly resulted in decreased toxicity and virulence, suggesting that Hfq is a global regulator [START_REF] Liu | Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus[END_REF].

To gather insight into the functional status and regulatory properties of S. aureus

Hfq, in the present study, we introduced the sequence encoding this protein in place of the endogenous hfq gene in the Salmonella c h romosome. (Hfq STM is 100% identical to Hfq EC in the initial 78 amino acids). In parallel with the above work, a similar exchange was performed using a DNA fragment spanning the Borrelia hfqlike gene. Both constructs were made with surgical precision replacing only protein coding portionsi.e., the segment between initiation and termination codons -to allow the heterologous sequences to fall under the control of signals normally devoted to hfq STM expression. We show below that Hfq SA and Hfq BB in spite of being made at levels comparable to Hfq STM , fail to replace the latter in sRNA-mediated regulation as well as in protecting representative sRNAs from degradation.

Materials and methods

Bacterial strains and growth conditions

Strains used in this study, listed in Table 1, were all derived from Salmonella enterica serovar Typhimurium strain MA3409, a derivative of strain LT2 cured for the Gifsy-1 prophage [START_REF] Figueroa-Bossi | Unsuspected prophage-like elements in Salmonella typhimurium[END_REF]. Bacteria were cultured at 37°C in liquid media or in media solidified by the addition of 1.5% (w/v) Difco agar. LB broth [1% bacto tryptone (w/v), 0.5% Difco yeast extract (w/v), 0.5% NaCl (w/v)] was used as complex medium. When needed, LB medium was supplemented with 0.2% (w/v) arabinose.

Antibiotics (Sigma-Aldrich) were included at the following final concentrations: chloramphenicol, 10 µg/ml; kanamycin monosulphate, 50 µg/ml; sodium ampicillin 100 µg/ml; tetracycline hydrochloride, 25 µg/ml. Liquid cultures were grown in New Brunswick gyratory shakers and growth was monitored by measuring the optical density at 600 nm with a Shimazu UV-mini 1240 spectrophotometer.

Enzymes and chemicals

Restriction enzymes, T4 polynucleotide kinase and Taq DNA polymerase were from New England Biolabs, Pfu-Turbo DNA polymerase was from Stratagene. DNA oligonucleotides were obtained from Sigma Aldrich. Acrylamide-bisacrylamide (30%, 29:1) and other electrophoresis reagents were from BioRad. Hybond-N + membranes and hybridization buffer used for Northern blot analysis were from GE Healthcare and from Applied Biosystems-Ambion, respectively.

Genetic techniques

Generalized transduction was carried out using the high frequency transducing mutant of phage P22, HT 105/1 int-201 [START_REF] Schmieger | Phage P22-mutants with increased or decreased transduction abilities[END_REF]. "λ Red"-mediated chromosomal recombineering was carried out by the method of Datsenko and Wanner [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF] implemented as in [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF]. Donor DNA fragments were generated by the polymerase chain reaction (PCR) using plasmid or chromosomal DNA templates. DNA oligonucleotides used as primers for PCR amplification are listed in Table 2.

Amplified fragments were electroporated into the appropriate strains using a Bio-Rad

MicroPulser under the conditions specified by the manufacturer. Constructs were verified by PCR and DNA sequence analysis (performed by GATC company).

Construction of relevant strains

Salmonella strains carrying the structural portions of the hfq-like genes from

Staphylococcus aureus or Borrelia burgdorferi were constructed with a two-step recombineering procedure as described [START_REF] Karlinsey | λ-Red genetic engineering in Salmonella enterica serovar Typhimurium[END_REF]. Firstly, a tetAR module (amplified with primers ppH71 and ppH72) was inserted in the hfq gene in the Salmonella chromosome. Subsequently, the entire hfq::tetAR was crossed out selecting for the loss of tetracycline resistance [START_REF] Bochner | Positive selection for loss of tetracycline resistance[END_REF] using DNA fragments amplified from chromosomal DNA of S. aureus RN4220 and B. burgdorferi clinical isolate 28354 with primer pairs ppI06/ppI07 and ppJ44/ppJ45, respectively (Table 2). Introduction of the 3xFLAG epitfope at the 3' ends of the coding sequence of hfq STM , hfq SA and hfq BB was carried out using DNA fragments amplified from plasmid pSUB11 [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF] with primer pairs pp913/pp914, ppJ41/pp914 and ppJ40/pp914, respectively, as previously described [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF].

RNA extraction and Northern blot analysis

RNA was prepared by the acid-hot-phenol method from exponentially growing cells (OD 600 of 0.35) as previously described [START_REF] Bossi | A small RNA downregulates LamB maltoporin in Salmonella[END_REF]. RNA was separated on an 8%

polyacrylamide-8 M urea gel and electro-blotted onto a nylon membrane. Blots were hybridized to 5' end-labelled DNA oligonucleotide probes specific for the sRNAs under study (listed in Table 3). Hybridization signals were analyzed by Phosphorimaging using ImageQuant software.

Western blot analysis

Bacterial cells harvested by centrifugation were resuspended in 250 µl of Laemmli protein gel loading buffer. Whole-cell extracts were fractioned in a 15% polyacrylamide-SDS gel. Proteins were transferred to poly-vinylidene difluoride (PVDF) membranes and probed with anti-FLAG M2 monoclonal antibodies from Sigma. Horse Radish Peroxidase (HRP) conjugated goat anti-mouse IgG (KPL) was used as a secondary antibody. Detection was performed by Enhanced Chemioluminescence (ECL, Amersham) with ECL films (Amersham).

ß-galactosidase assays

Activity of ß-galactosidase was measured in toluene-permeabilized cells as described [START_REF] Miller | A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria[END_REF] and is expressed in Miller units. Reported values were the average of at least two independent determinations, each involving duplicate or triplicate samples.

Results and discussion

Exchanging hfq coding sequences

Replacement of the structural portion of the Salmonella hfq gene with the corresponding regions from the hfq genes of Staphylococcus aureus and Borrelia burgdorferi was achieved by a two-step recombineering procedure (see Materials and Methods). DNA sequence analysis of the constructs (strains MA10741 and MA11042) showed that they carry no extra material or scars and that the hfq SA and hfq BB coding sequences are precisely positioned in replacement of the endogenous hfq coding sequence in the Salmonella chromosome (Figs. 1 andS1). We therefore expected that hfq SA and hfq BB would be expressed from the transcription/translation initiation signals of hfq STM . To confirm this, strain derivatives carrying 3xFLAG epitope fusions to the 3' ends of all three hfq genes were constructed, and expression of the carboxy-terminally tagged proteins assessed by Western blot analysis. As shown in Fig. 2, all three proteins accumulate at significant levels. Some differences in the intensity of the hybridization signals are apparent; however, the fact that they correlate with the molecular weights of the proteins suggest that they are intrinsic to the Western protocol (increasingly less quantitative with smaller molecules) and do not reflect a difference in expression rates. Overall, the data in Fig. 2 strongly suggest that hfq SA and hfq BB are expressed at levels closely comparable to that of hfq STM .

Hfq SA and Hfq BB fail to replace Hfq STM in sRNA-mediated regulation

Genes regulated directly or indirectly by small RNA constitute reporter systems for Hfq activity. Three representative loci were chosen for this analysis. The chiP gene of Salmonella encodes a chitoporin whose synthesis is repressed by a constitutively made sRNA, ChiX, which blocks chiP mRNA translation under most laboratory growth conditions. ChiX is the tightest Hfq-binding RNA known to date [START_REF] Moon | Competition among Hfq-binding small RNAs in Escherichia coli[END_REF] and strongly dependent on Hfq binding for stability [START_REF] Figueroa-Bossi | Caught at its own game: regulatory small RNA inactivated by an inducible transcript mimicking its target[END_REF]. Thus, a chromosomal translational chiP-lacZ fusion is expressed at very low level when Hfq is functional, but becomes derepressed nearly 40-fold in a strain deleted for hfq (Fig3A). As shown in Fig3A, strains carrying hfq SA or hfq BB in place of hfq STM have ß-galactosidase activities similar that of the hfq deleted strain. Consistent with these findings, Northern blot analysis reveals that ChiX sRNA is nearly undetectable in the hfq SA or hfq BB expressing strains, like what observed in the absence of Hfq (Fig. 4). Failure of Hfq SA or Hfq BB to protect ChiX from degradation suggests that neither of the two proteins binds to this sRNA in vivo.

The second reporter system used in this study is the eptB gene, which encodes phosphoethanolamine transferase, an enzyme involved in lipopolysaccharide modification. A previous study identified eptB as one of the genes most dramatically upregulated in a ∆hfq mutant [18]. This results from the combined effect of two apparently independent mechanisms: activation of the σ E regulon [18] and loss of repression by MgrR sRNA [START_REF] Moon | A PhoQ/P-regulated small RNA regulates sensitivity of Escherichia coli to antimicrobial peptides[END_REF]. To examine whether Hfq SA or Hfq BB corrected these regulatory defects in any way, a translational eptB-lacZ fusion was introduced into in the hfq SA -or hfq BB -expressing strains and ß-galactosidase activity measured. Again, the results in Fig. 3B show that the two strains are indistinguishable from the hfq null mutant in this test.

Both the above systems represent examples of negative regulation. We thus included a third reporter system in which Hfq activity is needed for gene activation.

Due to the requirement of H f q -dependent sRNAs for optimal translation of σ S mRNA, genes controlled by σ S are typically less expressed in the absence of Hfq [18].

One such gene encodes the KatE catalase. A further set of strains was therefore constructed carrying a katE-lacZ fusion in the hfq SA or hfq BB genetic background. As shown in Fig. 3C neither of the heterologous Hfq proteins can compensate for the loss of the endogenous Hfq as far as katE-lacZ expression is concerned.

Hfq SA and Hfq Bb fail to protect sRNAs from degradation

As described above, ChiX sRNA is almost undetectable in the strains carrying hfq SA or hfq BB (Fig. 4). This analysis included additional representative sRNAs:

GcvB, MicA, MicC and MicF. As shown in Fig. 4, the Northern hybridization patterns from the hfq SA -and hfq BB -carrying strains in all cases are indistinguishable from that of hfq deleted strain: MicC and MicF are both undetectable, while GcvB decreases significantly. In the case of MicA, the slight increase in sRNA levels is ascribable to σ E activation since the micA gene is a member of the σ E regulon [18,20]. The broadening of the MicA band, on the other hand, reflects the increased decay resulting from the Hfq defect (see [18]). These findings are reminiscent of those by Sittka and co-workers who found Methanococcus Hfq incapable of reversing the chronic σ E activation of a Salmonella strain lacking Hfq [START_REF] Sittka | Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes[END_REF]. Altogether, the above data suggest that neither Hfq SA nor Hfq BB are capable of binding Salmonella sRNAs in vivo.

Staphylococcus aureus Hfq is representative of bacterial Sm-like proteins and an inspirational reference in the small RNA field. Somewhat ironically, however, the function of this protein remains enigmatic. Although the protein was shown to bind RNAIII, a major RNA regulator in S. aureus [START_REF] Huntzinger | Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression[END_REF], it does not appear to facilitate the RNAIII's interaction with any of the mRNA targets that have been looked at (reviewed in [46]). Data on the effects of hfq deletions on various aspects of S. aureus physiology are contradictory, possibly due to strain variability in the expression levels of hfq [START_REF] Bohn | No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus[END_REF][START_REF] Liu | Hfq is a global regulator that controls the pathogenicity of Staphylococcus aureus[END_REF]. Considering that a possible redundancy in Hfq-like functions might underlie such variability in S. aureus, we chose to examine the functional status of Hfq SA in Salmonella enterica, where the role of the unique Hfq protein is well established. Our results showed that Hfq SA does not participate in any of the sRNAmediated regulatory mechanisms that were tested. Furthermore, we found no evidence of Hfq SA binding to sRNAs in vivo. Altogether, these data indicate that Hfq SA is functionally very different from its homologues in enteric bacteria. Such functional divergence might be in relation with the structural differences noted earlier on the distal face of the protein.

A second set data from this study shows that the Hfq-like protein from Borrelia burgdorferi is also unable to replace Hfq STM in sRNA-mediated regulation and sRNA stabilization. These findings are surprising as a recent publication reported Hfq BB to complement the defect in rpoS mRNA regulation of an E. coli hfq mutant [START_REF] Lybecker | Identification and function of the RNA chaperone Hfq in the Lyme disease spirochete Borrelia burgdorferi[END_REF]. The discrepancy is difficult to reconcile. The authors of the above report performed their analysis using a plasmid-born hfq BB gene; thus, in principle, the discrepancy could arise from gene dosage differences. Somehow, however, this explanation seems

unlikely. An alternative explanation might lie in the nature of the E.coli hfq allelehfq-1 -used in the above study. The hfq-1 allele was originally constructed by inserting an Ω (KnR) cassette into the BclI site spanning positions 117-122 of the hfq EC coding sequence [13]. Therefore, the mutant is expected to express a truncated Hfq fragment of about 40 amino acids, which would include the N-terminal α1 helix and two of the β strands of the Sm1 motif. Perhaps this fragment could somehow oligomerize with the Hfq BB monomers to reconstitute a functional Hfq protein.

Testing complementation in an E. coli strain in which the hfq gene is completely deleted would help remove this doubt.

Concluding remarks

The data presented here convincingly show that the Hfq proteins from S. aureus and B. burgdorferi are not interchangeable with their Salmonella counterpart as far as sRNA-mediated regulation is concerned. Clearly, more work is needed to elucidate the basis and biological rationale for the differences. Some of the strains constructed in the course of this work might prove useful to this end. The 3xFLAG-tagged derivative of Hfq SA and Hfq BB could be used to test whether there exist RNAs that bind either of these proteins in Salmonella and possibly identify them [START_REF] Sittka | Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes[END_REF]. As a complementary strategy, one could take advantage of the wide array of Salmonella genetic tools to search for genes whose expression might change in the presence or Salmonella extracts. Bacteria harvested from stationary cultures were lysed [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF] and crude extracts were fractionated by electrophoresis in a 15% SDS-polyacrylamide gel.

Legends to the Figures

The gel was processed for the immunodetection of epitope-tagged proteins [START_REF] Uzzau | Epitope tagging of chromosomal genes in Salmonella[END_REF]. The strains used were (from left to right): MA9132 (wt, no FLAG), MA11054 (hfq STM -3xFLAG), MA11055 (∆hfq::hfq SA -3xFLAG) and MA11056 (∆hfq::hfq BB -3xFLAG)

The identity and origin of the non-specific, low molecular weight band present in all strains analyzed is unknown. MA11057 and MA11058 (see Table 1 for full genotypes). Blots were hybridized to 5' end-labeled DNA oligonucleotide probes (Table 3).

Salmonella strains used were: MA8028 (hfq STM wt), MA8029 (∆hfq STM ::cat), MA10746 (∆hfq STM ::hfq SA ), MA11044 (∆hfq STM ::hfq BB ) and MA9132 (hfq STM wt). TATACAGTAGAAACTGAAGGTCAAGCATCTACTGAAAGTGAAGAAGACTACAAAGACCATGACGG above a Primers are defined as "forward" (fw) or "reverse" (rv) depending on whether they have same or opposite orientation (5'-3') relative to the gene being modified. The portions of primers annealing to template DNA are in red italics.

III. DISCUSSION AND PERSPECTIVES

The work described in this thesis can be regarded from different angles.

On one hand, the study deals with a basic topic like the initiation of translation and its regulation. On the other hand, the study contributes to a better characterization of processes related to bacterial physiology and metabolic regulation.

III.1. The role of ACA motifs in translation initiation

A major finding in this work was the discovery that GcvB sRNA represses expression of the yifK gene by countering the action of a translation enhancer embedded in its the target sequence. Having the recognition sequence of a regulatory sRNA coincide with a translation enhancer seems a particular effective way to achieve regulation and we might predict that this feature will be shared by other targets of GcvB and possibly by targets of other sRNAs.

Two ACA triplets were identified as major contributors to the translational enhancer activity. ACA motifs are found in multiple copies at other GcvB targets. We do not yet know if the enhancing effects are a general property of these motifs or if their effects somehow depend on the local sequence context. In this respect, it is important to notice that the yifK translation initiation signal remains relatively weak -in spite of including the enhancer element -as compared to other mRNAs studied in the laboratory. This raises the possibility that the function of the enhancer is to compensate for some weakening elements elsewhere in the initiation region. If that is the case, in other contexts, ACA motifs could play a much less important role.

Experiments addressing this point are currently being performed in the laboratory. In one approach, we are testing the effects of progressively removing the four ACA triplets found at the GcvB target site in the dppA mRNA (in the region immediately upstream of the Shine-Dalgarno sequence) on the translation efficiency of this mRNA. In a complementary approach, we are testing if introducing ACA triplets near the Shine-Dalgarno sequence of an mRNA that does not normally contain ACA will increase its translation efficiency. We have chosen for this analysis the mRNA of the chiP gene (Figueroa-Bossi et al., 2009). To produce minimal perturbation, the ACA triplets will be derived from preexisting triplets, namely UCA and AUA found within a 10 nt segment immediately upstream of chiP S h i n e -Dalgarno sequence.

The other important question that remains open concerns the mechanism by which the ACA motif improves translation initiation. Results described in this thesis (section II. 2), together with data from the Vogel's laboratory (Sharma et al., 2007) and other data in the literature [START_REF] Martin-Farmer | A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in Escherichia coli[END_REF], indicate that the enhancer effects do not require a precise positioning of ACA relative to the translation start site. This suggests that ACA does not directly participate in the architecture of the initiation complex between the mRNA and the 30 S ribosomal subunit. Rather, the ACA motif could serve as an anchoring point for some component of the 30 S subunit and stimulate the initial recognition of the initiation region. Presence of the ACA sequence in RNA aptamers selected by the SELEX procedure against to ribosomal protein S1 [START_REF] Ringquist | Highaffinity RNA ligands to Escherichia coli ribosomes and ribosomal protein S1: comparison of natural and unnatural binding sites[END_REF] makes this protein a candidate to be such component. A study comparing the binding affinities of wild-type yifK 5' UTR and of the mutants constructed in this study to purified S1 protein in vitro should allow it to verify this hypothesis.

III. 2. Role of GcvB in bacterial physiology.

In spite of extensive work, some basic questions regarding the physiological role of GcvB are still unanswered: why does GcvB inhibit amino acid uptake when bacteria grow at fast rates in rich medium and amino acids are maximally required for protein synthesis? What is the relationship between GcvB function and the glycine cleavage pathway?

A tentative answer to the former question is that GcvB activity is required to optimize the intracellular concentrations of amino acids to their ratios of occurrence in bacterial proteins. Alternatively, GcvB could balance amino acid levels with those of other macromolecualr precursors such nucleotides or carbohydrates. Most GcvB studies, including those involving transcriptomic analyses (Sharma et al., 2011[START_REF] Modi | Functional characterization of bacterial sRNAs using a network biology approach[END_REF] have been performed using bacteria growing in Luria-Bertani (LB) medium. This medium contains tryptone (a pancreatic digest of casein from cow milk) and yeast extract (autodigest of the yeast Saccharomyces cerevisiae). Although permitting fast growth rates, this medium may not be optimized for bacterial metabolism. For example, a study by R. D'Ari group showed that LB is limiting for carbohydrates, which causes a metabolic switch to take place during the exponential phase when sugars are exhausted and bacteria begin using amino acids as carbon sources (Sezonov e t a l . , 2007). It would be interesting to study gcvB expression and activity under more defined growth conditions, such as a synthetic medium supplemented with all amino acids, nucleotide precursors and a carbon source. This would allow varying the ratios of some of these supplements and studying the effects on gcvB expression.

The fact that both the gcvB gene and the gcvTHP operon encoding the enzymes of the glycine cleavage pathway are under the control of the GcvA regulatory protein strongly suggests the existence of a physiological link between GcvB activity and the glycine cleavage system. However, this link is not obvious given the gcvTHP operon is maximally transcribed under conditions -glycine-supplemented minimal-glucose medium -in which the gcvB gene is expressed at very low level. The situation is likely reversed in LB medium where gcvB is maximally transcribed. Although we have been unable to find data on gcvTHP expression in LB in the literature, such levels are likely to be quite low since gcvTHP activation by GcvA is completely dependent on Lrp and the levels of this protein are drastically reduced in LB [START_REF] Newman | Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli[END_REF]. Interestingly, GcvB activity contributes to the Lrp decrease (Sharma et al., 2011) raising the intriguing possibility that a role of GcvB is to act as an indirect negative regulator of gcvTHP expression. This would make physiological sense since the need for one-carbon units (products of the glycine cleavage pathway) is greatly reduced in rich medium as compared to minimal medium (see [START_REF] Calvo | The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli[END_REF] and Discussion of article in section II.2).

Hoping to gather insight onto the physiological role of GcvB, in the course of this thesis, I randomly mutagenized a strain carrying a lacZ transcriptional fusion to the gcvB promoter with a tetracycline-resistance transposon and screened for insertion mutants showing increased or decreased ß-galactosidase activity. I found a number of such mutants.

Unfortunately, their initial characterization gave inconclusive and confusing results that discoraged further analysis as well as their description in this report.

III. 3. The intriguing HdfR(YifA)-YifE regulatory locus

In the course of this work, I found that inactivation of either hdfR (yifA) or yifE, two adjacent genes in opposite orientation results in a significant increase in yifK expression (see section II.3). These findings suggest that the products of these genes directly or indirectly inhibit yifK expression. The HdfR protein is a poorly known transcriptional regulator previously shown to repress the flagellar master operon flhDC (acting in concert with H-NS) in E. coli [START_REF] Ko | H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein[END_REF] and to activate the fimbrial operon std in Salmonella [START_REF] Jakomin | Regulation of the Salmonella enterica std fimbrial operon by DNA adenine methylation, SeqA, and HdfR[END_REF]. It therefore seems possible that HdfR represses yifK transcription by binding to the yifK promoter. The independent identification of several mutations causing increased yifK expression, which map in the region upstream from the -35 box of the yifK promoter suggest that this could be the site of HdfR binding.

In contrast, nothing can be said of YifE since no reports on this protein exist and its sequence, although highly conserved, does not show similarity to any known protein in databases. The YifE implication in yifK regulation is therefore intriguing and encourages further investigation. In particular, it will be important to determine if YifE acts on yifK directly, perhaps as part of a complex with HdfR, or indirectly; for example if required for hfdR expression.

An alternative possibility is that YifE is the sole regulator of yifK and that the role of HdfR is to activate yifE expression. The construction of lacZ fusions to either of these genes and the measurement of ß-galactosidase activity in cells carrying the hfdR-lacZ fusion combined with wild-type or mutant yifE, or, viceversa, carrying yifE-lacZ combined with wild-type or mutant hdfR, should help distinguishing between these hypotheses. Finally, the construction of epitope-tagged derivatives of both proteins can allow testing the possible interaction of the two proteins by co-immuno precipitation techniques as already done in the laboratory to study repressor-antirepressor interactions [START_REF] Lemire | Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors[END_REF]. These experiments should not take too long to be completed and the results could lead to an additional publication from my thesis work.

IV. MATERIALS AND METHODS

All strains used in this study are derivatives of S. enterica serovar Typhimurium. Strains that were used for the article in section II.2 are listed in Table 1 in that section (page). Additional strains are listed in Table 2. The bacteria were cultured in LB broth [START_REF] Bertani | Lysogeny at mid-twentieth century: P1, P2, and other experimental systems[END_REF] solidified by the addition of 1.5% Difco agar when needed. When appropriate, the LB medium was supplemented with 0.2% arabinose. Antibiotics were included at the following final concentrations: chloramphenicol, 10 µg mL -1 ; kanamycin monosulfate, 50 µg mL -1 ; sodium ampicillin, 75 µg mL -1 ; spectinomycin dihydrochloride, 80 µg mL -1 ; and tetracycline hydrochloride, 25 µg mL -1 . LB plates containing 40 µg mL -1 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (Sigma-Aldrich) or MacConkey agar plates containing 1% lactose [START_REF] Macconkey | Lactose-Fermenting Bacteria in Faeces[END_REF] were used to monitor lacZ expression in bacterial colonies. Liquid cultures were grown in New Brunswick gyratory shakers and growth was monitored by measuring the optical density at 600 nm with a Shimazu UV-mini 1240 spectrophotometer.

Genetic Techniques

Generalized Transduction

P22 is a temperate phage that infects Salmonella by binding to the Oantigen, part of the lipopolysaccharide on the outer membrane. After infection, P22 circularizes by recombination between terminal redundancies at each end of the phage DNA. During lytic growth, the circular genome of P22 initially undergoes several rounds of θ (tetha) replication, then changes to rollingcircle replication. Rolling circle replication produces long concatemers of double stranded P22 DNA. These concatemers are packaged into phage heads by a "headful" mechanism: packaging is initiated at a specific sequence on the DNA called a pac site, then a nuclease moves down the concatemer, cutting every 48 Kb [START_REF] Casjens | Analysis in vivo of the bacteriophage P22 headful nuclease[END_REF]. Since the P22 genome is only 44 Kb, this yields the terminal redundancy at the ends of P22 (Susskind and Botstein,197). This linear double stranded DNA is packaged into new phage particles. When the cell lyses, it releases 50-100 new phage.

There are sequences in the Salmonella chromosome that are homologous to the P22 pac site. When the phage infects bacteria, occasionally the P22 nuclease cuts one of these chromosomal sites and packages 48 Kb [START_REF] Casjens | Analysis in vivo of the bacteriophage P22 headful nuclease[END_REF] fragments of chromosomal DNA into P22 phage heads. The P22 particles carrying chromosomal DNA (transducing particles) can inject this DNA into a new host. The DNA can then recombine into the chromosome by homologous recombination. In order to increase the efficiency of the process, most laboratories doing P22 transduction use P22 HT105/1 int-201, a P22 mutant that is very useful for generalized transduction (Schmieger, 1972). This phage has a high transducing (HT) frequency due to a nuclease with less specificity for the pac sequence. About 50% of the P22 HT phage heads carry random transducing fragments of chromosomal DNA ( (Schmieger, 1972)). The int m u t a t i o n prevents formation of stable lysogens.

Transduction Procedure

Typically, P22 lysates were used at a 1:50 dilution, mixed with aliquots from overnight cultures of recipient bacteria in a 1:2 ratio, and incubated for 30 min at 37°C prior to being plated on selective media. Transductant colonies were purified by two sequential passages on selective plates and verified to be free of phage by streaking on Evans Blue Uranine plates [START_REF] Bochner | Curing bacterial cells of lysogenic viruses by using UCB indicator plates[END_REF].

Plasmids and DNA templates.

Plasmids used as PCR templates for λ Red-mediated recombination included pKD3, pKD4 and pKD13 (Datsenko & Wanner, 2000). Plasmid pCP20, carrying Flp recombinase [START_REF] Cherepanov | Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant[END_REF], was used as needed, to remove antibiotic resistance cassettes flanked by FRT sites or to promote integration of plasmid pCE40. The latter was used to create chromosomal lacZ fusions to genes of interest [START_REF] Ellermeier | Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria[END_REF].

Chromosomal DNA from strain MA3397 [START_REF] Figueroa-Bossi | Unsuspected prophagelike elements in Salmonella typhimurium[END_REF] was used as template for amplification of the tetAR module in the first step of the scarless recombineering procedure (see below).

Chromosomal engineering: λ Red recombination method

Chromosomal engineering was carried out by the λ Red recombination method according to the procedure of [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF]. Donor DNA fragments were generated by PCR using plasmid or chromosomal DNA templates. Oligonucleotides used as primers in these experiments are presented in Table S1 (page 76) and in Table 3. Amplified fragments were electroporated into appropriate strains harboring the conditionally replicating plasmid pKD46, which carries a λ red operon under the control of the P BAD promoter (Datsenko & Wanner, 2000). Bacteria carrying pKD46 were grown at 30°C in the presence of ampicillin and exposed to arabinose (10 mM) for 3 hours prior to preparation of electrocompetent cells. Electroporation was carried out using a Bio-Rad MicroPulser under the conditions specified by the manufacturer. Recombinant colonies were selected on LB plates containing the appropriate antibiotic. Constructs were verifed by PCR and/or DNA sequencing. When needed, the antibiotic resistance cassette was excised by transforming strains with plasmid pCP20, which encodes the Flp recombinase [START_REF] Cherepanov | Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant[END_REF].

Scarless recombination experiments were performed as described in the Materials and Methods of the Article in section II.2.

Variations on λ Red Construction of dppA-lacZY fusions

The procedure involved the insertion of the aph (kan R ) module of plasmid pKD13 in the dppA gene, and Flp recombinase-mediated conversion of the insert to a lacZY fusion with plasmid pCE40 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]. Primer extension sequences were chosen so as to place the fusion boundary within signal peptide-encoding segments. Disruption of the signal sequence was intended to prevent deleterious effects that might result from translocation of the ß-galactosidase moiety of the hybrid proteins across cell membranes. Two fusions were constructed with the lacZY sequence at slightly different positions within dppA: dppA147::lacZY carrying lacZ fused to the 16 th codon of dppA (primers ppK77/ppK79; Table 3) and dppA148::lacZY carrying lacZ fused to the 9 th codon of dppA (ppK78/ppK79; Table 3). The two fusions behaved similarly and were used interchangeably throughout this study.

Random PCR mutagenesis

Chromosomal DNAs encompassing the gcvB gene, or the promoter proximal region of the yifK-lacZ fusion, were amplified by PCR under error-prone conditions. In both cases the amplified region included a suitably positioned cat marker for selection (Fig. 25 and26). S1) and plasmid pKD3 as template.

Figure 25. PCR mutagenesis of the promoter-proximal region of the yifK gene. Scheme of the mutagenic procedure ( s e e t e x t f o r d e t a i l s ) . T h e y i f K-linked cat insertion was

constructed by λ Red recombineering using primers ppF43 and ppF44 (Table S1) and plasmid pKD3 as template.

Briefly, amplification was carried out with Taq polymerase (New England Biolabs), in the absence of Pfu polymerase, in 10mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl 2 0.1% Triton X-100, 0.2 mg/ml BSA (Qbiogene). The amplification program included 10 cycles of differential temperature decrease (0.1°C/sec) from 95°C to 64°C (annealing temperature) followed by 15 cycles with annealing at 62°C (no ramp step). The elongation step was set at 72°C for 45 seconds in all cycles. For gcvB mutagenesis, the template was chromosomal DNA from strain MA10307 and the primers ppF19 and ppF20 (Table S1, page 76). For mutagenesis of yifK 5'UTR, template was chromosomal DNA from strain MA10308 and the primers were ppF45 and ppF47. In both amplification experiments, the priming sites were chosen so as to have approximately 0.5 Kb at both ends of the DNA products providing homology for recombination. The fragments obtained (1969 bp and 2034 bp, respectively) were used to transform strain MA10280 (yifK::MudK / pKD46) and Cm R recombinants selected on MacConkey-lactose plate supplemented with chloramphenicol. Deep red colonies were picked and purified by streaking on selective plates. The mutagenized region was PCR-amplified ppF19/pp595, GcvB; ppF45/ppF62 yifK 5' UTR) with a 3:1 Taq/Pfu polymerase mixture and the resulting fragment subjected to DNA sequence analysis.

"Scarless" DNA recombineering.

Scarless modification of chromosomal DNA sequences at the single base-pair level was achieved with a two-step recombineering procedure adapted from (Karlinsey, 2007). The procedure involves: i) inserting a tetAR module (produced by PCR) at the chromosomal site to be modified and ii) replacement of the tetAR module by a DNA fragment carrying the desired changed through positive selection tetracycline-sensitive recombinants (Bochner et al., 1980). Typically, the DNA fragment in the second step was also obtained by PCR using oligonucleotides with complementary sequences at their 3' ends priming DNA synthesis on each other ("reciprocal priming") ß-Galactosidase Assay (adapted from Miller (Miller, 1992))

For exponential growth measurements, an overnight culture was diluted The reverse transcriptase reaction (Superscript II from Invitrogen) was carried out using 5 μg of RNA from strains MA8020, MA11781, MA11782 and MA11783 with primer ppF49 labeled at the 5 end with 32 P. The same primer was used to generate the DNA sequence ladder. The sequencing template was a DNA fragment obtained by PCR amplification of chromosomal DNA from strain MA11780 (Table S1) with primers ppF84 and ppF85. (For primer sequences see Table 3). Reactions were performed with the fmol DNA Cycle Sequencing System from Promega, according to the manufacturer's protocol.

RNA extraction and Northern analysis

RNA was prepared by the acid-hot-phenol method from exponentially growing cells (OD 600 of 0.35) as previously described [START_REF] Figueroa | Common sequence determinants of the response of a prokaryotic promoter to DNA bending and supercoiling[END_REF].

Total RNA was estimated from the value of the OD at 260 nm. For Northern analysis, 7.5 mg of total RNA were separated under denaturating conditions either by 8M urea-8% polyacrylamide in TBE (Tris-borate-EDTA pH 8. S1) were 5' end-labelled using 10 U of T4 polynucleotide kinase (New England Biolabs) and 30 µCi of [γ-32 P]ATP (3,000 mCi mmol -1 , Amersham). Unincorporated radioactivity was eliminated by passage through Micro-Bio Spin 6 chromatography columns (BioRad).

Hybridization was carried out in Ambion Oligonucleotide Hybridization Buffer at 45-50°C following Ambion's protocol. RNA was analysed by Phosphorimaging using ImageQuant software.

In vitro RNA synthesis

Templates for in vitro transcription with T7 polymerase were generated by PCR from genomic DNA. To produce the yifK 5' UTR template, DNA was amplified with primers ppI22 and ppI23 from strain MA8020. To make the wild-type GcvB template, primers were ppJ01 and ppJ03. In vitro transcription was performed with the MegashortscriptT7 kit (Ambion AM1354) according to the manufacturer's protocol. Transcription products were an 137 nucleotide RNA corresponding to the first 134 residues of yifK mRNA plus a 3-nucleotide extension from the T7 promoter (GGG) at the 5' end, and full-length GcvB with an extra G at its 5' end. After incubation for 2 hrs at 37°C, DNAse was added and incubation continued for further 15 min. The samples were treated with phenol and the RNA precipitated at -20°C overnight with sodium acetateethanol-glycogen. RNA was recovered by centrifugation, resuspended in water and quantified by nanodrop reading.

5'-end mapping of yifK mRNA by primer extension.

The reverse transcriptase reaction (Superscript II from Invitrogen) was carried out using 5 μg of RNA with primer ppF49 labeled at the 5'end with 32P. The same primer was used to generate the DNA sequence ladder. The sequencing template was a DNA fragment obtained by PCR amplification of chromosomal DNA with primers ppI22 and ppF62 (Table S1). Reactions were performed with the fmol DNA Cycle Sequencing System from Promega, according to the manufacturer's protocol.

In vitro translation

In vitro coupled transcription/translation was performed using New England Biolabs' PURExpress In vitro Protein Synthesis kit (NEB #E6800) according to the manufacturer instructions, as described in the article in section II.2 of this thesis manuscript. DNA templates were constructed using the backbone of plasmid DHFR provided with the kit. To this end, plasmid DNA was digested with XbaI and PstI. PCR amplification from chromosome of strains MA11798 a Template DNA was from chromosome ("MA" numbers identify the Salmonella strains used as source; described in the text) or from plasmids pNK2883. [START_REF] Kleckner | Uses of transposons with emphasis on Tn10[END_REF] or pKD3 (Datsenko & Wanner, 2000).

  II. RESULTS...................................................................................................36 II.1. Characterization of yifK, a Hfq-regulated locus in Salmonella……....36 II.1.1. yifK is repressed by GcvB……….……….…….……….…...……37 II.1.2. Genetic analysis of GcvB:yifK interaction unveils a translational enhancer in the target sequence.........................40 II.1.3. Separate subdomains of GcvB R1 region pair with yifK and dppA..................................................................................41 II.1.4. Role of ACA motifs in translation enhancement………………..42 II.2. Manuscript submitted to PloS Genetics…….…….…………………….….44 Table 1................................................................................................60 II.2.1 Supporting information of manuscript........................................72 Table S1.............................................................................................75 Table S2.............................................................................................77 II.3. An intriguing regulator of yifK expression.........................................78 II.4. A specialized mutant ribosome improves initiation from a UUG start codon ..............................................................................83 II.5. Article II.............................................................................................86 III. DISCUSSION AND PERSPECTIVES ...................................................110 IV. MATERIALS AND METHODS...............................................................115 Table 2.............................................................................................124 Table 3.............................................................................................125 Table 4.............................................................................................126 V. REFERENCES.......................................................................................127

Figure 1 .

 1 Figure 1. Five steps of bacterial transcription. 1. RNA polymerase (light blue oval) binds to the promoter sequence (double green bars) through its σ subunit (yellow circle). 2. The DNA is locally melted. 3. σ is released. 4. RNA polymerase moves along the gene (double yellow bars) copying one DNA strand into an RNA sequence (blue ribbon). 5. RNA polymerase reaches a termination signal (double red bars) at which point the elongation complex dissociates.

Figure 2 .

 2 Figure 2. Model for Rho-dependent termination. 1. The Rho hexamer, in an open ring configuration, binds a C-rich unstructured sequence in nascent RNA. Binding causes the ring to close, trapping the RNA chain inside the central cavity. 2. Ring closure t r i g g e r s t h e t r a n s l ocase activity. Rho travels along the nascent RNA, maintaining the contacts with the C-rich segment (tethered tracking;[START_REF] Boudvillain | Terminator still moving forward: expanding roles for Rho factor[END_REF]), which causes an RNA to form. 3. Rho reaches the elongation complex, causing its destabilization and its dissociation from the DNA template. RNAP, RNA polymerase.

Figure 3 .

 3 Figure 3. Schematic view of the translation initiation process. Formation of 30S (30SIC) and 70S (70SIC) translation initiation complexes, containing ribosomes (30S subunit in orange, 50S in brown), initiator fMet-tRNAfMet,mRNAand initiation factors IF1 (in blue), IF2 (in green) and IF3 (in light blue). View of 30S ribosomal subunit and ribosome from the top. The platform of the 30S is in red with the anti-Shine-Dalgarno (aSD) sequence in cyan. From[START_REF] Simonetti | A structural view of translation initiation in bacteria[END_REF].

Figure 4 .

 4 Figure 4 . Lrp (black circles) bends the DNA upstream from gcvTHP promoter., allowing different GcvA subunits to contact a distal DNA site and RNA polymerase (activation) or separate DNA sites and the GcvR repressor (repression); from[START_REF] Stauffer | Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli[END_REF].

Figure 5 .

 5 Figure 5. Diversity of riboswitches and mechanisms of gene control in bacteria.Mechanisms of modulation of gene expression are highly diversified in prokaryotes and involve control of transcription, translation, and mRNA stability. From[START_REF] Serganov | A decade of riboswitches[END_REF].

Figure 6 .

 6 Figure 6. A diverse set of CRISPR-associated (cas) genes (grey arrows) encode proteins required for new spacer sequence acquisition (Stage 1), CRISPR RNA biogenesis (Stage 2) and target interference (Stage 3). Each CRISPR locus consists of a series of direct repeats separated by unique spacer sequences acquired from invading genetic elements (protospacers). Protospacers are flanked by a short motif called the protospacer adjacent motif (PAM) that is located on the 5′ (type I) or 3′ (type II) side in foreign DNA. Long CRISPR transcripts are processed into short crRNAs by distinct mechanisms. In type I and III systems, a CRISPR-specific endoribonuclease (yellow ovals and green circles, respectively) cleaves 8 nucleotides upstream of each spacer sequence. In type III systems, the repeat sequence on the 3′ end of the crRNA is trimmed by an unknown mechanism (green pacman,right). In type II systems, a trans-acting antisense RNA (tracrRNA) with complementarity to the CRISPR RNA repeat sequence forms an RNA duplex that is recognized and cleaved by cellular RNase III (brown ovals). This cleavage intermediate is further processed at the 5′ end resulting in a mature, approximately 40-nucleotide crRNA with an approximately 20-nucleotide 3′-handle. In each system, the mature crRNA associates with one or more Cas proteins to form a surveillance complex (green rectangles). Type I systems encode a Cas3 nuclease (blue pacman), which may be recruited to the surveillance complex following target binding. A short high-affinity binding site called a seed-sequence has been identified in some type I systems27, 60, and genetic experiments suggest that type II systems have a seed sequence located at the 3′end of the crRNA spacer sequence. From:[START_REF] Wiedenheft | RNA-guided genetic silencing systems in bacteria and archaea[END_REF].

Figure 7 .

 7 Figure 7. Model of CsrB and its action to inhibit CsrA. The regulatory RNA CsrB has many binding sites for the CsrA protein; the protein normally represses translation by binding to the Shine-Dalgarno sequence, CsrB has the ability to titrate out CsrA, thus relieving translational repression. From[START_REF] Majdalani | Bacterial small RNA regulators[END_REF].

Figure 8 .

 8 Figure 8. Functions of cis-encoded sRNAs. Two possible configurations of cisencoded antisense sRNAs (red) and their target RNAs (blue), which share extensive complementarity. (Left panel) An sRNA encoded opposite to the 5′UTR of its target mRNA. Base pairing inhibits ribosome binding and often leads to target mRNA degradation. (Right panels) An sRNA encoded opposite to the sequence separating two genes in an operon. Base pairing of the sRNA can target RNases to the region and cause mRNA cleavage, with various regulatory effects, or the sRNA can cause premature transcriptional termination. From[START_REF] Waters | Regulatory RNAs in bacteria[END_REF].

Figure 10 .

 10 Figure 10. Electrostatic potential energy surfaces of the known and proposed RNA binding sites of the S.aureus and E.coli Hfq proteins. (a-c) Views of the proximal side and distal electrostatic surfaces of S. aureus Hfq, respectively.(d-f) Views of the proximal, side and distal electrostatic surfaces of E. coli Hfq, respectively (blue is electropositive and red is electronegative). The side view of E. coli Hfq includes a plausible RNA binding cleft that would enable A27 to bind to both the Proximal and Distal Sites. The view of the Distal Site of the E. coli Hfq shows a possible binding site for A18 (i.e. a poly(A) tail). The bound RNAs are shown as solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored white, blue, red and orange, respectively. The electrostatic potential energy surfaces were created byPyMol and the APBS plug-in (Delano Scientific LLC, Palo Alto, CA). From(Brennan & Link, 2007).

Figure 11 .

 11 Figure 11. Model for Hfq action. Hfq protect sRNA against degradation, stimulates sRNA:mRNA pairing and in some systems stimulates the degradation of the sRNA:mRNA complex (see text for details).

Figure 12 .

 12 Figure 12. Model for Hfq bind 3' end and activity RNA decay. From[START_REF] Wilusz | Lsm proteins and Hfq: Life at the 3' end[END_REF].

Figure 13 .

 13 Figure 13. Model for RyhB-mediated reguation (see text). From[START_REF] Masse | Small RNAs controlling iron metabolism[END_REF].

Figure 15 .

 15 Figure 15. Schematic representation of the GcvB network. GcvB sRNA represses mRNAs of many ABC transporters involved in amino acid uptake and of amino acid biosynthetic enzymes. From :(Sharma et al., 2011).

Figure 16 .

 16 Figure 16. Sequence and secondary structure of GcvB sRNA. The secondary strcture of GcvB is based on in vitro chemical probing(Sharma et al., 2007). The sequence diagram is from[START_REF] Vogel | A rough guide to the non-coding RNA world of Salmonella[END_REF]. Mutants #4 and #5 were isolated and characterized as described in the text.

Figure 17 .

 17 Figure 17. PCR mutagenesis of the gcvB gene. A DNA fragment spanning the gcvB gene and an adjacent cat cassette was amplified by PCR under error-prone conditions (see Materials and Methods) and introduced into a strain carrying a yifK-lacZY f u s i o n a n d l a m b d a r e d p l a s m i d p K D 4 6(Datsenko & Wanner, 2000). Recombinants were selected on MacConkey-lactose indicator plates supplemented with chloramphenicol. B. Red colonies (indicated by arrows) express yifK-lacZY at higher level.

Figure 18 .

 18 Figure 18. Isolation of pairing mutants of yifK and gcvB. See text for details.

Figure 19 .

 19 Figure19. Effect of a three-base pair gcvB mutation on repression of yifK-and dppA-lac fusions. The gcvB sequence between nucleotides 86 and 88 was changed from UUU to ACA as described in section II,2. The dppA-lacZ fusion was constructed using the two-step procedure of[START_REF] Ellermeier | Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria[END_REF], using primers ppK77 and ppK78 (see supporting information in section II,2.1). Suicide plasmid pCE40 was used to obtain an in-frame lac operon fusion starting at codon 16 of dppA (see Materials and Methods).

  2 indicated that the enhancer acted at the level of translation initiation. These experiments correlated the enhancer activity with two ACA triplets in the yifK target sequence. Initial evidence for of the importance of these motifs in yifK expression was obtained in an experiment in which either ACA triplet was replaced by random triplets (in a strain carrying yifK-lacZY and deleted for gcvB) and mutants were screened on MacConkey-lactose indicator plates. As one can see in Fig. 20 the mutant colonies exhibited a whole range of red color intensities, from light pink to deep red, indicative of a wide range of expression levels.

Figure 20 .

 20 Figure 20. R a n d o m m u t a g e n e s i s o f A C A t r i p l e t s u p s t r e a m o f yifK t r a n s l a t i o n initiation site.The two ACA triplets (underlined in grey) are located at -17 to -15 and at -12 to -10 relative to the initiating AUG (underlined in green). Random mutagenesis of the -17 to -15 and -12 to -10 intervals was achieved using primer pairs ppL95/ppL96 and ppL97/ppL98, respectively (TableS1in section II.2.1) as described in Materials and Methods. Mutagenized fragments were introduced into strain carrying yifK-lacZY and deleted for gcvB (in addition to a tetAR insert in yifK 5' UTR for counter selection). Colonies selected on Bochner plates were picked and patched on MacConkey lactose plates. C1 and C2 denote patches made from yifK-lacZY ∆gcvB control strain. Brown overlining denote the Shine-Dalgarno motif.

A

  relevant chapter in the expanding field of RNA-mediated gene regulation is devoted to the activities of multi-target trans-encoded small RNAs in bacteria. Acting in concert with chaperon protein Hfq, these RNA regulators function by base-pairing with short, often imperfectly complementary sequences in the 5' untranslated regions (UTR) of target messenger RNAs. They can affect translation and turnover of several mRNAs simultaneously thus reprogramming gene expression of whole gene families in a coordinate manner in response toenvironmental cues (reviewed in(Gottesman & Storz, 2011b, Storz et al., 2011, Vogel & Luisi, 2011). Archetypal examples of this class of regulators are the RybB small RNA (sRNA)

  found to be regulated by GcvB has increased exponentially. A recent transcriptomic study in Salmonella enterica set this number to more than 40, making the GcvB regulon the largest of its kind(Sharma et al., 2011). The vast majority of these loci are linked directly or indirectly to amino acid metabolism and are negatively controlled by GcvB. Typically, regulation is exerted during exponential growth in nutrient rich environments and possibly aimed at coordinating the expression of interconnected metabolic pathways(Sharma et al., 2007, Sharma et al., 2011); however, its precise role remains poorly defined.GcvB uses a specific sequence region to pair with most, although not all(Coornaert et al., 2013) of its mRNA targets. This pairing domain -named the R1 region(Sharma et al., 2007) -is characterized by its high degree of sequence conservation, the lack of secondary structure and a typical GU-rich sequence bias. Hence, most sequences targeted by GcvB include CA-rich repeats. They are typically found in 5' UTRs of target mRNAs where GcvB pairing inhibits translation initiation causing the destabilization of the mRNA. In one of these targets -the gltI mRNA for a glutamate-aspartate transport protein -the CArich element is located 45 nucleotides (nt) upstream from the translation initiation codon. Removal of this sequence (as part of a 27 nt deletion), besides causing the loss GcvB regulation, affected gltI translation, suggesting that the CA-rich element acts as a translational enhancer. Consistent with this interpretation, crafting the 27 nt segment at the corresponding position of an unrelated mRNA conferred simultaneously GcvB control and increased translational efficiency (Sharma et al., 2007). Some years ago, our laboratory performed a lac-based genetic screen aimed at identifying genes controlled by trans-encoded small RNAs in Salmonella. A random library of lacZ f u s i o n s t o c h r o m o s o m a l g e n e s w a s generated using a phage Mu-derived transposon (MudK) and screened for isolates

  test this hypothesis and characterize yifK regulation. While this work was underway, Sharma and coworkers identified yifK mRNA as a member of the gcvB r e g u l o n b y microarray analysis; however, these authors could not confirm direct regulation by GcvB due to low reporter fluorescence of the yifKgfp fusion used in the study (Sharma et al., 2011). Here we present in vivo a n d in vitro evidence that GcvB downregulates yifK directly by pairing with a sequence immediately preceding the Shine-Dalgarno (SD) motif in yifK m R N A . A surprising observation in the course of this study was that some target sequence mutations that disrupted pairing did not cause yifK e x p r e s s i o n t o increase -as expected for the relief of GcvB repression -but had the opposite effect. The drop in expression was not suppressed by deleting gcvB nor was it accentuated in a GcvB mutant carrying the appropriate compensatory changes. Closer analysis revealed that GcvB target sequence includes elements that stimulate yifK m R N A t r a n s l a t i o n . I n t h e absence of such elements, the role of GcvB pairing in regulation becomes marginal.

(

  lac) transposon in the yifK g e n e ; o n e predicted to produce a LacZ protein fusion to the 48 th amino acid (aa) of the 461 aa YifK (yifK87::MudK); the other with LacZ inserted after the 95 th a a o f Y ifK (yifK88::MudK) (Figueroa-Bossi et al., 2006b). Preliminary tests showed both fusions to be regulated in a closely similar manner; however, yifK87::MudK produced significantly higher ßgalactosidase activity and was chosen for the present study. A survey of protein sequence databases showed YifK to be a highly conserved protein with the characteristic signature of amino acid transporters. The known role of GcvB in the regulation of some members of this family made this small RNA the likeliest candidate to control yifK expression. This was confirmed by deleting the gcvB g e n e a n d t e s t i n g t h e e f f e c t s o f t h e deletion on the expression of the yifK87::MudK fusion (hereafter referred to as yifK-lacZY). As shown in Figure 1, the gcvB deletion causes a nearly 5-fold increase of ßgalactosidase activity in exponentially growing cells, while effects decline in stationary phase.

This 5 '

 5 untranslated region (UTR) includes a 14-nucleotide sequence stretch complementary to the 3' half of GcvB's R1 region. As an initial step to characterize GcvB involvement in yifK r e g u l a t i o n , w e t e s t e d w h e t h e r p o i n t mutations in the gcvB gene or in the promoterproximal portion of yifK r e l i e v e d G c v Bmediated repression. For this, DNA fragments spanning either of these two regions were randomly mutagenized by the polymerase chain reaction (PCR) under error-prone conditions and introduced into the chromosome of a strain harboring the yifK-lacZY r e p o r t e r f u s i o n v i a l a m b d a red recombination. Selection of recombinants on MacConkey lactose indicator plates allowed distinguishing derepressed mutant candidates (red colonies) from the background of non mutagenized clones (pink colonies). Most of the isolates originating from the gcvB mutagenesis carried changes in the gcvB promoter or in the promoter of the adjacent gcvA gene (Figure S1). Thus, these mutations appeared to lower the levels rather than the activity of GcvB and were not further considered. No mutations likely to affect GcvB's ability to pair with yifK mRNA were found. Mutagenesis of yifK p r o mo t e r -proximal segment yielded three mutants with elevated yifK-lacZY expression. One isolate carried a C:G to A:T change 33 base-pairs upstream from the 5' end of yifK mRNA. The position and the nature of the change (producing a -35

  GcvB or yifK. Reasoning that single base changes might not disrupt regulation enough to be revealed by the MacConkey plate screen, we resorted to introducing multiple changes by site-directed mutagenesis. An initial test involved changing a UGUG quadruplet in the GcvB segment thought to pair with yifK mRNA. The alteration caused expression of this sequence in yifK repression. Unexpectedly, however, when the ACAC sequence at the corresponding position in yifK m R N A w a s c h a n g e d , yifK-lacZ expression did not increase but actually declined (data not shown). Trying to clarify this observation, portions of the region of interest were mutagenized separately. As shown in Figure 3A, converting an AAA triplet in the middle of the yifK target sequence to UGU, or making the opposite change (UGU to AAA) in GcvB, s i m i l a r l y r e l i e v e s yifK-lacZY repression. Repression is restored upon combining the compensatory alleles. Thus, this portion of the target sequence behaves as expected, and the compensatory mutant's phenotype strongly suggests that GcvB represses yifK through a base-pair interaction.

  selected portions of the ribosome binding sequence of yifK w e r e m o d i f i e d b y systematically changing individual residues to each of the three alternative bases. In particular, we concentrated on two ACA triplets, one corresponding to the site described above (positions -17 to -15 relative to the initiating AUG) the other nearly adjacent to the Shine-Dalgarno sequence (-12 to -10). This analysis was performed entirely in a strain deleted for the gcvB gene. As shown in Figure 6A, any nucleotide change in the upstream ACA lowers yifK-lacZY expression. Variations range between 23% and 62%, with G residues exerting the most adverse effects at any position. The effects appear to be additive since a separate experiment in which all three bases in the ACA triplet were randomized yielded alleles undergoing as much as 92% reduction in yifK-lacZY e x p r e s s i o n ( F i g u r e 6B). Alteration of the downstream ACA produced a somewhat different pattern. Changes in the central C are either neutral or stimulatory; in contrast, having a C at the first triplet position is highly deleterious resulting in nearly 95% reduction of ß-galactosidase activity (Figure 6A). Although the physical basis for these effects remains elusive, the data in Figure 6 clearly point to a role of ACA motifs in yifK translation.

  constructs have the functional SD sequence positioned either 4 or 11 nt from the AUG. As shown inFig 7, these two variants (n. 4 and n. 5) express ß-galactosidase levels that are similar to each other and to the strain in which both SD are functional (n. 1). However, when the upstream ACA motif is replaced by the GGG triplet, yifK-lacZ e x p r e s s i o n d r o p s sharply in both constructs(Figure 5, compare n. 4 and n. 6, and n. 5 and n. 7). Thus, these results indicate that the enhancer activity does not require the ACA motif to be positioned at a fixed distance from the initiation site and it does not depend on the spacing between the SD and the initiation codon. In an experiment carried out in parallel to that in Figure7, the upstream ACA triplet was replaced by random sequences and the mutants obtained screened directly on MacConkey-lactose indicator plates. The color of colonies spanned the whole range of shades between white and red (Figure S2), confirming that the data in Figure 7 do not reflect a specific inhibitory effect of the triple G, and providing a snapshot of the nuances in the enhancer-dependent effects on yifK-lacZ expression. GcvB activity stimulates RNase Edependent yifK mRNA decay Northern blot analysis was used to assess the effects of GcvB regulation on yifK mRNA levels. This study critically benefited from the availability of the -33 promoter mutant (see above), yifK m R N A b e i n g o t h e r w i s e undetectable when expressed from the wildtype promoter (compare Figs 7A and 7B). The analysis identified two yifK mRNA species, a 1.4 kilobase (Kb) mRNA covering just the yifK coding portion and a longer, 2.0 Kb RNA extending into the adjacent argX-hisR-leuT-proM tRNA operon. As shown in Figure 7, both RNAs accumulate upon RNase E inactivation, whereas only the shorter species accumulates in cells lacking GcvB or Hfq. We interpret these results to indicate that derepression of yifK translation in ∆gcvB or ∆hfq cells protects the 1.4 Kb RNA against RNase E cleavage, and that this protection does not extend to the untranslated, tRNA coding portion of the 2.0 Kb RNA. Absence of any obvious transcription termination signals in the intercistronic region between yifK and the tRNA operon suggests that the 1.4 Kb RNA originates from processing of the longer form. Likely, under normal conditions (i.e., wt yifK promoter) yifK transcription contributes to only a small fraction of the four tRNAs, as most the tRNA operon transcription results from a strong promoter located immediately upstream from the argX gene (Bossi & Smith,

  Figure 8 also show that GcvB does not contribute to yifK repression to any significant

  the present work, we have characterized the regulation of Salmonella's yifK l o c u s e n c o d i n g a p u t a t i v e a m i n o a c i d transporter highly conserved in Enterobacteriaceae. Our analysis showed yifK to be negatively controlled at the transcriptional level by the leucine response regulator Lrp, and at the post-transcriptional level by GcvB sRNA. These findings place yifK a t t h e i n t e r s e c t i o n o f t w o g l o b a lregulatory networks devoted to amino acid management[START_REF] Calvo | The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli[END_REF], Sharma et al., 2011). The relative impacts of two systems on yifK e x p r e s s i o n v a r y a s a function of growth conditions, with the Lrp control predominating in leucine-deprived poor media and the GcvB control operating when amino acids are plentiful, possibly in excess. The sole condition where yifK appears to escape negative control is leucinesupplemented minimal medium, where Lrp repression is relieved. This response closely parallels that of the oligopeptide permease operon, oppABCDF[START_REF] Calvo | The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli[END_REF] whose transcript is also a target of GcvB repression(Sharma et al., 2007, Urbanowski et al., 2000). Likely, the overlap of Lrp and GcvB networks reflects the link between amino acid metabolism and one-carbon units production; however, the precise physiological role and the implications of the above responses remain incompletely understood. Genetic analysis of GcvB:yifK m R N A interactions revealed that the GcvB target sequence in yifK mRNA contains a enhancer element. Intriguingly, mutations that disrupt the enhancer -and lower yifK expression as a result -r e n d e r yifK e x p r e s s i o n t o t a l l y insensitive to GcvB repression. This suggest that the effectiveness of GcvB regulation is dependent on the enhancer function and that when this component is removed, GcvBmediated repression no longer constitutes a rate-limiting step in yifK expression. Sharma and coworkers (2007) previously showed that GcvB's target sequence in the gltI ge n e of Salmonella a c t s a s t r a n s f e r a b l e t r a n s l a t i o n enhancer (see Introduction). Unlike in our study, the effects of GcvB as a translational repressor were much greater than the effects of removing the enhancer, leading the authors to conclude that GcvB did not simply block the enhancer effect (Sharma et al., 2007). It seems possible that the plasmid-borne nature of the gcvB g e n e i n t h e s t u d y b y S h a r m a a n d coworkers made the GcvB repression tighter than when the sRNA is expressed from the chromosome. Alternatively, the enhancer activity might be less conspicuous in gltI than in yifK. The gltI e n h a n c e r , l o c a t e d 4 5 n t upstream from the i n i t i a t i o n c o d o n , w a s characterized as part of a 27 nt segment andnot analyzed in any further detail(Sharma et al., 2007). Here we found that nucleotide replacement in either of two ACA triplets within GcvB target site in yifK can result in more than 90% reduction in yifK expression.Although our data do not allow defining the contours of the enhancer element, they unequivocally identify the ACA motif as a determinant of its activity. We also found that the enhancer activity is maintained following a 7 nt shift in the position of the initiation site, suggesting the absence of strict spatial requirements for the functioning of the element. This is consistent with the data from the gltI system and with a report showing CA repeats to stimulate translation even when placed downstream from the start codon[START_REF] Martin-Farmer | A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in Escherichia coli[END_REF].

"

  Scarless" DNA recombineering Scarless modification of chromosomal DNA sequences at the single base-pair level was achieved with a two-step recombineering procedure as previously described (Bossi et al., 2012). Briefly, this involved: 1) inserting a tetAR m o d u l e ( p r o d u c e d b y P C R ) a t t h e chromosomal site to be modified and: 2) replacing the tetAR m o d u l e b y a D N A fragment carrying the desired changed through positive selection tetracycline-sensitive recombinants (Bochner e t a l . , 1980).

  fmol D N A C y c l e Sequencing System from Promega, according to the manufacturer's protocol. Reaction products were fractionated on a 10% polyacrylamide-8 M urea gel. For Northern blot analysis, RNA was fractionated on a 1 % agarose-formaldehyde gel, blotted onto a nylon membrane, and hybridized to the appropriate 32 P-labeled DNA oligonucleotide probes. In vitro translation In vitro coupled transcription/translation was performed using New England Biolabs' PURExpress In vitro Protein Synthesis kit (NEB #E6800) according to the manufacturer instructions. Genes to be analyzed were cloned under T7 promoter control in the DFRH plasmid provided with the kit. The hybrid genes carried yifK w t o r m u t a n t 5' UTR sequences fused to the cat-3xFLAG coding sequence (chloramphenicol acetyl transferase in-frame fusion to the 3xFLAG epitope). Final volume of the transcription/translation reaction was 25 µl in all cases. In addition to kit solutions A and B, reaction mix contained, 10 U of RNase inhibitor SUPERase (Ambion) and template plasmid DNA added to either 0.5 or 5 pM final concentration. Incubation times at 37 °C varied from 15 to 90 min. Reactions were stopped by addition of equal volume of 2X

  described by Darfeuille et al(Darfeuille et al., 2007) w i t h m i n o r m o d i f i c a t i o n s . RNA fragments spanning positions +1 to +135 of yifK mRNA were synthesized in vitro from T7 DNA templates generated by PCR amplification of chromosomal DNA (from strains MA8020 or MA11793) with primers ppI22 and ppI23. 2 pmol of RNA were annealed with 5'end-labeled primer ppI23 (1 pmol) in 10 mM Tris-acetate [pH7.6], 0.1 M potassium acetate, and 1 mM DTT for 1 min at 90°C and chilled in ice for 5 min. Then, all dNTPs (final concentration 1 mM), Mg Acetate (10mM final) were added; this was followed by preincubation with 2 pmol of 30S ribosomal subunit (a gift of Dominique Fourmy and Satoko Yoshizawa) at 37°C for 5 min. In experiments involving GcvB, 5, 1 or 0.5 pmol of sRNA were added prior to both, addition of the 30S ribosomal subunit and the preincubation step. After the 5-min period, 2 pmol of tRNA fMet w e r e a d d e d a

Figure 2 .

 2 Figure 2. Characterization and mutational analysis of the leader region of the yifK gene.The promoter-proximal region of the yifK gene was randomly mutagenized by error-prone PCR using primers ppF45 and ppF47 and chromosomal DNA from a strain carrying a cat cassette 78 bp upstream from the yifK promoter (in opposite orientation; strain MA11780) as template. The mutagenized fragment was introduced into strain MA10280 (yifK-lacZY / pKD46) and recombinants were selected as described in the text. Three mutants expressing higher ß-galactosidase activity were identified: one carrying a yifK promoter change that

Figure 3 .

 3 Figure 3. Differential effects of yifK target sequence mutations on yifK-lacZ expression and on its response to compensatory changes in GcvB. Adjacent portions of the sequence presumed to base-pair with GcvB in yifK mRNA were randomly mutagenized by a "scarless" λ red recombineering procedure (see Materials and Methods). Briefly, DNA fragments amplified by "reciprocal priming" with oligonucleotide pairs ppG48/ppG49 (mutagenesis of the 46-48 interval) and ppH12/ppH13 (mutagenesis of the 49-50 interval) were introduced into strain MA11526 and tetracycline-sensitive recombinants selected one plates supplemented with fusaric acid (12 µg/ml). Two of the mutants obtained were chosen for further study. Compensatory changes in GcvB were obtained by standard recombineering using fragments amplified from the chromosome of strain MA11779 with primer pairs

Figure 4 .

 4 Figure 4. Toeprinting analysis of yifK mRNA. 30S ribosomal toeprinting was carried out in the absence or in the presence of GcvB RNA or of RyhB RNA as described in the Materials and Methods. "+" and "-" signs denote the presence of absence of indicated components. The decline and disappearance of the toeprint (shown by arrows) at increasing GcvB concentrations (50 nM, 100 nM and 500 nM), is indicative of interference with the 30S subunit binding to yifK mRNA. Failure of RyhB to do so at the concentration of 6.0 µM (lane "R") confirms the specificity of the effect.

Figure 5 .

 5 Figure 5. Effect of yifK 5' UTR's changes on mRNA translation. In vitro translation was performed using the coupled transcription/translation PURExpress kit (see Materials andMethods). DNA templates were from plasmids carrying the entire yifK 5' UTR from wildtype, or from the C49U,A50C mutant, fused to the coding sequence of a 3xFLAG epitopetagged version of the cat gene. Fusions were initially obtained as chromosomal constructs using DNA fragments amplified from strain MA7224 with primer pairs ppL50/ppL52 (wt) and ppL51/ppL52 (C49U,A50C). Subsequently, the fusions were cloned into plasmid DHFR following amplification (ppM29 / ppM30) and double Xba I / Pst I digestion. Transcription / translation reactions were carried out at a template DNA concentration of 0.5 pM for 90 min or 5 pM for 30 min and products analyzed by Western blotting using anti-FLAG monoclonal antibodies(Uzzau et al., 2001). Under both conditions, higher amounts of cat-3xFLAG protein were synthesized from the construct with the wild-type yifK sequence than from the construct harboring the double CA to UC change.

Figure 6 .

 6 Figure 6. Mutational analysis of ACA motifs. Each base position in two ACA triplets upstream of the yifK's SD sequence was randomized and changes were introduced in the chromosome of strain MA11594 (yifK-lacZY ∆gcvB) as described in the legend to Fig. 3

  Mutantsobtained were screened by PCR (primers ppF45/ppF62) and DNA sequencing. All possible replacements (9 variants for each triplet) were identified. These strains were assayed for ßgalactosidase activity (A). Typically, duplicate or triplicate ß-galactosidase measurements were carried out in parallel for all variants of any given position and the wild-type strain, whose value was set to 100. Standard deviations were less than 5% of the mean in all cases. Data in (A) show that any change in the upstream ACA causes a decrease in yifK expression, with G residues exerting the most negative effects. Replacements at the downstream ACA have contrasting effects: replacing the central C with A or U increases expression while having a C at the first position is highly deleterious. Representative examples of triple substitutions in the upstream ACA are also shown (B). ß-galactosidase measurements were as in (A).

Figure 7 .

 7 Figure 7. Increasing the distance between the enhancer and the translation initiation region. Constructs were made as described in the legends to Figs. 3 and 6 using strain MA11594 (yifK-lacZY ∆gcvB) as recipient and fragments amplified by reciprocal priming of oligonucleotides described in Table S2. A 7-nucleotide segment (boxed in light green), duplicating the SD sequence (overlined in brown), was inserted between the ACA triplets (underlined in grey) and the SD sequence (construct n. 3). A G to C change was then introduced in either copy of the SD (constructs n. 4 and 5; construct n. 2 shows the effect of

Figure 8 .

 8 Figure 8. Northern blot analysis of yifK transcription. RNA was extracted from strains carrying the wild-type yifK promoter (A) or the -33 promoter "up" mutation (B and C). RNA was separated on a 1% agarose-formaldehyde gel a n d p r o b e d w i t h 32 P-labeled DNA

Figure 9 .

 9 Figure 9. Lrp control of yifK-lacZY expression. The yifK gene is expressed at very low levels in minimal medium. As a result, strains carrying the yifK-lacZY f u s i o n a r e phenotypically Lac -in this medium, regardless of the gcvB allele (A, left panel). Inactivation of Lrp confers a Lac + phenotype (A, left panel). This suggests that low yifK expression results from Lrp repression. The Lac + phenotype is also restored upon addition of leucine (0.3 mM) (A, right panel), indicating that leucine relieves Lrp repression. ß-galactosidase measurements (B) confirm that GcvB plays no significant role in yifK regulation in minimal medium and provide a quantitative estimate of the Lrp effects. NCE medium[START_REF] Maloy | Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions[END_REF] supplemented with 0.2% lactose (A) or 0.2% glycerol (B) was used as minimal medium.

Figure S2 .

 S2 Figure S2. Effects of replacing the ACA motif at positions 48-50 by random triplets. The sequence of the 48-50 interval was randomized in strains carrying a 7 nt tandem direct duplication of the SD region with either the AUG-distal SD sequence inactivated (A) or the AUG-proximal SD sequence i n a c t i v a t e d ( B ) . M u t a g e n e s i s involved introducing DNA fragments amplified by reciprocal priming of DNA oligonucleotides ppN76 and ppN77 (A) or ppN78 and ppN79 (B) into strain MA11594 (yifK::tetAR yifK-lacZY ∆gcvB) selecting fusaric acid resistance (see text) and patching recombinants onto MacConkey lactose plates. A sample of 50 recombinants from each cross is shown. For more details on alleles and primer used, see TablesS1 and S2.

Figure 21 .

 21 Figure 21. DNA and RNA sequence changes increasing yifK lacZY expression in a strain carrying the yifK C49U,A50C mutation. A inverted triagle (red, AA) indicates a two-bp insertion.

Figure 22 .

 22 Figure 22. Gene organization in the yifA-yifB region of the Salmonella chromosome.

Figure 22 .

 22 Figure 22. Effect of hdfR (yifA) or yifE inactivation on the expression of the yifK-lacZY fusion (left panel) and of the yifK[C49U,A50C]-lacZY fusion (right panel) in Lrp + (blue bars) and Lrp -(red bars) genetic backgrounds .

Figure 23 .

 23 Figure 23. Sequence of aphA mRNA in wild-type and in a strain carrying the aphA::MudK insertion. The aphA sequence is boxed in red; the MudK sequence is boxed in cyan.

Figure 24 .

 24 Figure 24. Sequence of 3' end of 16 S ribosomal RNA in wild-type Salmonella and in a mutant selected for increased expression of an aphA-lacZY transaltional fusion

Fig. 1 .Fig. 2 .

 12 Fig. 1. Schematic representation of recombineering events replacing the coding segment of the hfq gene in the Salmonella chromosome (309 bp) with the 234 bp DNA fragment and the 480 bp DNA fragment encoding the structural portions of the hfq-like gene from S. aureus and B. burgdorferi, respectively. The construction was carried out as described in Materials and methods.

Fig. 3 .

 3 Fig. 3. Expression of translational lacZ fusions to chromosomal genes sensitive to Hfq inactivation in Salmonella enterica. ß-galactosidase activity was measured in exponentially growing LB cultures (OD 600 ≈ 0.3) (A and B) or in stationary phase cultures (OD 600 ≈ 2) (C). Strain used were: A. MA9132, MA10744, MA10747 and MA11044; B. MA8028, MA8029, MA10746 and MA11043; C. MA8149, MA8679,

Fig. 4 .

 4 Fig. 4. Northern blot analysis of small RNAs. Total RNA extracted from exponentially growing cells (OD 600 = 0.35) was fractionated on an 8% polyacrylamide-8 M urea gel and electrotransferred onto a Hybond-N + membrane.

Figure 25 .

 25 Figure 25. PCR mutagenesis of the gcvB gene. Scheme of the mutagenic procedure (see text for details). T h e g c v B -linked cat insertion was constructed by λ Red recombineering using primers ppF17 and ppF18 (TableS1) and plasmid pKD3 as template.

1 :

 1 100 and grown until O.D.600 around 0.4. From this same 1 ml culture, 0.1 ml (V C ) were taken and placed in glass tube with Z buffer to make up 1ml final. One drop of Toluene was added with a Pasteur pipette and each tube was immediately vortexed for 30 seconds. Toluene was evaporated at 42°C for 2h. Tubes were placed in a water bath at 28°C for 5min. Reaction was started b y a d d i n g 0 . 2 m l ortho-Nitrophenyl-β-galactoside (ONPG, 4mg/ml, freshly made). Start time (t 0 ) for each tube was recorded. After sufficient yellow color developed, the reaction was stopped with 0.5 ml of 1M Na 2 CO 3 and final time (t f ) recorded. In order to avoid the contribution of bacteria to the O.D.420, the bacteria were pelleted and the supernatant used to read OD420. β-galactosidase activity was calculated as as: 1000X O.D.420 (t f -t 0 )x V C x O.D.600 Molecular Biology and RNA structural techniques 5'-end mapping of yifK mRNA by primer extension.

  3) buffer or by 2.2 M formaldehyde-1.3% agarose gel electrophoresis in MOPS ([Nmorpholino] propanesulfonic acid-sodium acetate-EDTA pH7.0) buffer. For acrylamide gels, transfer of the RNA onto Hybond-N + membrane (Amersham), was performed with a semidry electrotransfer apparatus (Transblot SD; BioRad); for agarose gels, transfer to the same support was done using a vacuum blotter (Boekel/Appligene) after mild denaturation treatment in 50 mM NaOH, 10 mM NaCl. RNA was crosslinked to membrane by UV irradiation (Statagene UV Stratalinker 2400). Membranes were hybridized to probes as follows: 5 pmol of oligonucleotides ppF16, ppG42, ppH27 and pp813 (Table

  Thanks to the ability to regulate multiple targets, trans-encoded sRNAs can mediate global regulatory responses. A paradigm for t h i s t y p e o f response is provided by the RyhB sRNA, which controls iron homeostasis in E.coli and Salmonella. Iron is one of most important metal ions for bacteria, as

Table 1 .

 1 Relevant Salmonella enterica strains used in this work

	Region of GcvB. PLoS Genet 9:	extended Shine-Dalgarno sequence
	e1003156.				on translation. RNA 8: 1137-1147.
	21. Figueroa-Bossi N, Lemire S, Maloriol D,	33. McCarthy JE, Schairer HU, Sebald W
	Balbontín R, Casadesús J, et al.	(1985)	Translational	initiation
	(2006) Loss of Hfq activates the σ E -	frequency of atp genes from
	dependent envelope stress response in	Escherichia coli: identification of an
	Salmonella enterica. Mol M i c r o b i o l	intercistronic sequence that enhances
	62: 838-852.				translation. EMBO J 4: 519-526.
	22. Desnoyers G, Masse E (2012)	34. Tzareva NV, Makhno VI, Boni IV (1994)
	Noncanonical	repression	of	Ribosome-messenger recognition in
	translation initiation through small	the absence of the Shine-Dalgarno
	RNA recruitment of the RNA	interactions. FEBS Lett 337: 189-194.
	chaperone Hfq. Genes Dev 26: 726-	35. Ringquist S, Jones T, Snyder EE, Gibson
	739.				T, Boni I, et al. (1995) High-affinity
	23. Brennan RG, Link TM (2007) Hfq	RNA ligands to Escherichia coli
	structure, function and ligand binding.	ribosomes and ribosomal protein S1:
	Curr Opin Microbiol 10: 125-133.		comparison of natural and unnatural
	24. Shultzaberger RK, Bucheimer RE, Rudd	binding sites. Biochemistry 34: 3640-
	KE, Schneider TD (2001) Anatomy	3648.
	of Escherichia coli ribosome binding	36. Dubey AK, Baker CS, Romeo T, Babitzke
	sites. J Mol Biol 313: 215-228.		P (2005) RNA sequence and
	25. Bossi L, Smith DM (1984) Conformational	secondary structure participate in
	change in the DNA associated with an	high-affinity CsrA-RNA interaction.
	unusual promoter mutation in a tRNA	RNA 11: 1579-1587.
	operon of Salmonella. Cell 39: 643-	37. Vesper O, Amitai S, Belitsky M, Byrgazov
	652.				K, Kaberdina AC, et al. ( 2 0 1 1 )
	26. Zhang A, Wassarman KM, Rosenow C,	Selective translation of leaderless
	Tjaden BC, Storz G, et al. (2003)	mRNAs by specialized ribosomes
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	mRNA targets of Hfq. Mol Microbiol	coli. Cell 147: 147-157.
	50: 1111-1124.			38. Lilleengen K (1948) Typing of Salmonella
	27. Modi SR, Camacho DM, Kohanski MA,	typhimurium	b y	m e a n s	o f
	Walker GC, Collins JJ Functional	bacteriophage. Acta Pathol Microbiol
	characterization of bacterial sRNAs	Scand 77: 2-125.
	using a network biology approach.	39. Figueroa-Bossi N, Coissac E, Netter P,
	Proc Natl Acad Sci U S A 108:	Bossi	L	(1997)	Unsuspected
	15522-15527.			prophage-like elements in Salmonella
	28. Calvo JM, Matthews RG (1994) The	typhimurium. Mol Microbiol 25: 161-
	leucine-responsive regulatory protein,	173.
	a global regulator of metabolism in	40. Bertani G (2004) Lysogeny at mid-
	Escherichia coli. Microbiol Rev 58:	twentieth century: P1, P2, and other
	466-490.				experimental systems. J Bacteriol
	29. Martin-Farmer J, Janssen GR (1999) A	186: 595-600.
	downstream CA repeat sequence	41. Maloy SR, Roth JR (1983) Regulation of
	increases translation from leadered	proline utilization in Salmonella
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	coli. Mol Microbiol 31: 1025-1038.		put::Mu d(Ap, lac) operon fusions. J
	30. Dreyfus M (1988) What constitutes the	Bacteriol 154: 561-568.
	signal for the initiation of protein	42. Macconkey A (1905) Lactose-Fermenting
	synthesis	on	Escherichia coli	Bacteria in Faeces. J Hyg (Lond) 5:
	mRNAs? J Mol Biol 204: 79-94.		333-379.
	31. Yarchuk O, Jacques N, Guillerez J,	43. Schmieger H (1972) Phage P22-mutants
	Dreyfus M (1992) Interdependence of	with	increased	or	decreased
	translation, transcription and mRNA	transduction abilities. Mol Gen Genet
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	Biol 226: 581-596.			44. Lemire S, Figueroa-Bossi N, Bossi L
	32. Komarova AV, Tchufistova LS, Supina	(2011)	Bacteriophage	crosstalk:
	EV, Boni IV (2002) Protein S1	coordination of prophage induction
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Table S1

 S1

	, continued
	name	Sequence (5'-3')	a
	ppL97 AATCGATGTACCCATAACAATAACCGGTACTACCGGAACCGTTGCAAACACG
	ppL98 CACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCANNNCGTGTTTGCAACGGTTCCGGT
	ppM29 AATTTATATTTTAACTATTCTAGACAATCGATGTACCCATAACAATAACC
	ppM30 ATTTATATTTCGGCCGCTCGAGCTGCAGTGGCGCTTACTATTTATCGTCG
	ppM80 ACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGTCGTGVTTGCAACGGTTCCGGTAGTAC
	ppM81 ACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGTCGTHTTTGCAACGGTTCCGGTAGTAC
	ppM82 ACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGTCGVGTTTGCAACGGTTCCGGTAGTAC
	ppM83 CACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGVCGTGTTTGCAACGGTTCCGGT
	ppM84 CACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATHTCGTGTTTGCAACGGTTCCGGT
	ppM85 CACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCAVGTCGTGTTTGCAACGGTTCCGGT
	ppN58 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGCCTCATGTCGTGTTTGCAACGGTTCCG
	ppN59 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCGTCATGCCTCATGTCGTGTTTGCAACGGTTCCG
	ppN60 AATCGATGTACCCATAACAATAACCGGTACTACCGGAACCGTTGCAAACACGACA
	ppN63 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCGTCATGTCGTGTTTGCAACGGTTCCG
	ppN64 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGCGTCATGTCGTGTTTGCAACGGTTCCG
	ppN73 AATCGATGTACCCATAACAATAACCGGTACTACCGGAACCGTTGCAAGGGCGACATG
	ppN74 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGCGTCATGTCGCCCTTGCAACGGTTC
	ppN75 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCGTCATGCCTCATGTCGCCCTTGCAACGGTTC
	ppN76 TGTACCCATAACAATAACCGGTACTACCGGAACCGTTGCAANNNCGACATGACGCATGAGGATTTATGG
	ppN77 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGCGTCATGTCG
	ppN78 TGTACCCATAACAATAACCGGTACTACCGGAACCGTTGCAANNNCGACATGAGGCATGACGATTTATGG
	ppN79 CAGCCCACGCTGTAGCTCCGGTTTTTTCTCTGCCATAAATCGTCATGCCTCATGTCG

a Changes from the wild-type sequence are in red. Nucleotide insertions are in bold and underlined. "N" denotes an equimolar mixture of all four nucleotides, "V" denotes an equimolar mixture of A, C and G; "H" denotes an equimolar mixture of A, T and C.

Table S2 .

 S2 Relevant alleles constructed in this work

	Primer pair	Template a	Allele	Description
	ppF08 / ppF09	pKD3	∆gcvB141::cat	cat gene in place of gcvB
	ppF10 / ppF12	pSEB5	∆gcvB142::aadA	Spec	R gene in place of gcvB
	ppF17 / ppF18	pKD3	ygdI143::cat	cat on 3' side of gcvB (parallel orientation)
	ppF43 / ppF44	pKD3	wecG144::cat	cat on 5' side of yifK (opposite orientation)
	ppG44 / ppG45	MA3397	yifK145::tetAR	tetAR insertion 10 bp upstream of yifK
				AUG
	ppG48 / ppG49	self	yifK[46-48 UGU]	Replacement of yifK mRNA segment
				between +46 and +48 (AAA to UGU)
	ppG63 / ppF18	MA11779	gcvB[86-88 ACA]	Replacement of GcvB segment between
				+86 and +88 (UUU to ACA)
	ppH12/ ppH13	self	yifK[49-50 UC]	Replacement of yifK mRNA segment
				between +49 and +50 (CA to UC)
	ppH61 / ppF18	MA11779	gcvB[84-85 GA]	Replacement of GcvB segment between
				+84 and +85 (UG to GA)
	ppH81 / ppG51	MA3409	yifK[-33A]	yifK promoter "up" mutation (G to A at -33)
				in wild-type background
	ppL50 / ppL52	MA7224	yifK::cat-3xFLAG	Replacement of yifK orf by cat-3xFLAG orf
	ppL51 / ppL52	MA7224	yifK[49-50 UC]::cat-3xFLAG	Replacement of yifK orf by cat-3xFLAG orf
				in the C49U,A50C mutant
	ppL95 / ppL96	self	yifK[48-50 NNN]	Randomized sequence in 48-50 segment
				of yifK mRNA (AUG-distal ACA]
	ppL97 / ppL98	self	yifK[53-55 NNN]	Randomized sequence in 53-55 segment
				of yifK mRNA (AUG-proximal ACA)
	ppL95 / ppM80	self	yifK[48 V]	C, U, or G at position 48 of yifK mRNA
	ppL95 / ppM81	self	yifK[49 H]	A, U, or G at position 49 of yifK mRNA
	ppL95 / ppM82	self	yifK[50 V]	C, U, or G at position 50 of yifK mRNA

Table 1 .

 1 Salmonella enterica serovar Typhimurium strains used in this work.

	Strain a	Genotype

Table 2 .

 2 DNA oligonucleotides used as PCR primers for λ Red-mediated recombination.

	Primer a	Sequence (5'-3')
	ppH71(fw) ppH72 (rv)	ATTGCGTCGGGAACGTGTTCCAGTTTCTATTTATTTGGTGTTAAGACCCACTTTCACATT AACTGATCAAAGGACTCGATTTGACCTTGCAGCTTAATACCCTAAGCACTTGTCTCCTG
	ppI06 (fw) ppI07 (rv)	AGGTTCAAAGTACAAATAAGCATATAAGGAAAAGAGAATGATTGCAAACGAAAACATCCA ATTATCCGACGCCCCCGACATGGATAAACAGCGCGTGAACTTATTCTTCACTTTCAGTAGATGC
	ppJ44 (fw) ppJ45 (rv)	AAAGGTTCAAAGTACAAATAAGCATATAAGGAAAAGAGAATGTTTATAAGCAGGGAATTGAAG ATTATCCGACGCCCCCGACATGGATAAACAGCGCGTGAACTTATTCCTTCTTGCTCATTAAAG
	pp913 (fw) pp914 (rv)	GCAGGGGTCTACTGCGCAACAGGACAGCGAAGAGACTGAAGACTACAAAGACCATGACGG ATTATCCGACGCCCCCGACATGGATAAACAGCGCGTGAACCATATGAATATCCTCCTTAG
	ppJ40 (fw) pp914 (rv)	AAAGTTGTTGCAGATGCTATTAAAACTTTAATGAGCAAGAAGGAAGACTACAAAGACCATGACGG above
	ppJ41 (fw)
	pp914 (rv)

Table 3 .

 3 DNA oligonucleotides used as probes for Northern analysis.

	Name	Sequence (5'-3')	Specificity
	pp814	ATGATGATAACAAATGCGCG	MicA
	pp831	AGGTTAACGCAATGGCCCAG	MicC
	pp832	AGGGGTAAACAGACATTCAG	MicF
	ppB07	CGTCAAAGAGGAATTTCATCGTTATTATTATCCCGACGCTTTCGCTTC	ChiX
	ppB10	ACACTACCATCGGCGCTACG	5 S
	ppI67

AGACCAATTGCAAACACAACAACACAACATCACAACCGTAAGCCA

GcvB

Table 2 .

 2 Salmonella enterica strains used in this work (complements Table 1, page 61).

	Strain a	Genotype

Table 3 .

 3 Oligonucleotides used in this work (complements TableS1, page 76).

	name	Sequence (5'-3')
	pp813	GCGGAGGCTAGGGAGAGAGG
	pp595	ACCAGCTCACCGTCTTTC
	pp615	GCGGGCAAGAATGTGAAT
	ppF16	GGCAATCAATTCAATATGACGAGCTTCCAGCCCACGC
	ppF19	TGCTCGCGTAAAGCTCAAAT
	ppF20	CGCAAACTGGCTACGATGAA
	ppF45	CGTGGTGCGTTCAATACGTA
	ppF47	CGCCATCCACATAAACCAGT
	ppF62	CGCCCATTTCAGCGTACT
	ppF84	CGCCCGGTAGTGATCTTATT
	ppF85	CGAGGGCAATCAATTCAATA
	ppF90	CTGTAGCTCCGGTTTTTTCTCTGCCATAAATCCTCATGTCCACATTGCAACGGTTCCGGTAGTC
	ppF91	CTGATGGGCTTTTGGCTTACGGTTGTGATGTTGTGTTGTTCACATTGCAATTGGTCTGCGATTA
	ppG42	AGGGCAGCGCTCTATCCAGCTGAGCTACGGGCGCTTAG
	ppH27	TGAAACTTTTTACGCGGTATTAAACCACCGCAGCTCAAGCACCTAAATAAA
	ppI22	GGTAATACGACTCACTATAGGGCAATCGATGTACCCATAACAATAACCG
	ppI23	CCCGAGGGCAATCAATTCAA
	ppJ01	GAAATTAATACGACTCACTATAGGACTTCCTGAGCCGGAACG
	ppJ03	AGACAGGGTAAATGTACAGGAAGTG
	ppJ12	TGAGCTTTCGTATCAGGCAACTGGAGAATCAACTGGGCGTTTAAGACCCACTTTCACATT
	ppJ13	TTTTGCAGCCAAATAGCGTATAGTGGCCGTGAAAGCGTTGCGCTAAGCACTTGTCTCCT
	ppJ14	AATCATCAGCTTCGGTGTAGTCTTCTGCGCCTTCAACTTGCTAAGCACTTGTCTCCTG
	ppJ15	TTCACGATTAAAGAGGCACAACTGCTTGAGCGTCATGGTCTTAAGACCCACTTTCACATT
	ppJ16	GGGCGATAGACAGGTTGTTAATTC
	ppJ17	CGCCTGCCACGTATTCATCA
	ppK77	TATTTCCTTGAAGAAGTCAGGGATGCTGAAGCTTGGTTTGAGGATCCGTCGACCTGCAGTTC
	ppK78	ATTGGAGCAGAAGAATGAGTATTTCCTTGAAGAAGTCAGGGATCCGTCGACCTGCAGTTC
	ppK79	TTTATAGAGCTCAATACGCTTGTTGTGGTCATCGGTCGCATGTGTAGGCTGGAGCTGCTT
	ppM29	AATTTATATTTTAACTATTCTAGACAATCGATGTACCCATAACAATAACC
	ppM30	ATTTATATTTCGGCCGCTCGAGCTGCAGTGGCGCTTACTATTTATCGTCG

Table 4 .

 4 Relevant alleles constructed in this work (complements TableS2, page 78).

	Primer pair	Template	a	Allele	Description
	ppF18 / ppF91	MA11779	gcvB [83-86 CACA]	Replacement of GcvB segment between
					+86 and +88 (GUGU to CACA)
	ppF43 / ppF90	MA11780	yifK[48-51 UGUG]	Replacement of yifk segment between
					+48 and +51 (CACA to GUGU)
	ppJ12 / ppJ13	pNK2883	∆hdfR::tetAR	tetAR module replaces hdfR gene
	ppJ14 / ppJ15	pNK2883	∆yifE::tetAR	tetAR module replaces yifE gene
	ppK77 / ppK79	pKD13		dppA147::aph	aph gene (Kan R ) replaces dppA (starting at
					codon 16)
	ppK78 / ppK79	pKD13		dppA148::aph	aph gene (Kan R ) replaces dppA(starting at
					codon 9)

Urbanowski ML, Stauffer LT, Stauffer GV (2000) Mol

Acknowledgements

We are grateful to Dominique Fourmy and Satoko Yoshizawa for the generous gift of purified 30 S ribosomal subunits and to Josep Casadesús for strain SV4280. We thank Fabien Darfeuille for advice on the toeprinting protocol. Q. Yang was the recipient of a China Scholarship Council fellowship. This work was supported by grant BLAN07-1_187785 from the French National Research Agency (ANR) to LB. Acknowledgements This work was supported by a grant from the IFR 115, Genomes, Transcriptomes, Proteomes to NFB and a grant from the Agence Nationale pour la Recherche grant (ANR-2010-BLAN-1602-01 'Duplex-Omics') to PB. We thank Christine Pourcel (Orsay, France) for proving B. burgdorferi genomic DNA, and S. Yoshizawa and D. Fourmy (CGM, Gif-sur-Yvette) for helpful discussions. this work MA10740 ∆hfq116::tetAR / pKD46 this work MA10741 ∆hfq STM ::hfq SA this work MA10744 chiP91::pCE40(lac) ∆hfq116::tetAR this work MA10746 eptB115::MudK ∆hfq STM ::hfq SA this work MA10747 chiP91::pCE40(lac) ∆hfq STM ::hfq SA this work MA11042 ∆hfq STM ::hfq BB this work MA11043 eptB115::MudK ∆hfq STM ::hfq BB this work MA11044 chiP91::pCE40(lac) ∆hfq STM ::hfq BB this work MA11054 hfq-3xFLAG-aph (KnR) this work MA11055 ∆hfq STM ::hfq SA -3xFLAG-aph (KnR) this work MA11056 ∆hfq STM ::hfq BB -3xFLAG-aph (KnR) this work MA11057 katE561::MudK ∆hfq STM ::hfq SA this work MA11058 katE561::MudK ∆hfq STM ::hfq BB this work a All strains are derived from Salmonella enterica serovar Typhimurium strain

MA3409. The latter is a derivative of strain LT2 cured for the Gifsy-1 prophage [34].

and MA11799 with primers ppH29 and ppH30, yielded fragments carrying a XbaI and a PstI site at the ends. These fragments, as well as DHFR plasmid DNA, were digested with XbaI and PstI. Following appropriate ligation, two plasmids carrying wt or mutant yifK 5' UTR-cat-3XFLAG chimeric constructs under the control of the T7 promoter were obtained and used for in vitro transcription/translation.

MA10242 ∆gcvB142:: aadA (Spec R ) this work MA10280 yifK87::MudK / pKD46 this work MA10377 yifK87::MudK ∆gcvB141::cat this work MA10403 yifK87::MudK ∆gcvB141::cat ∆hfq67::cat this work MA11526 yifK145::tetAR / pKD46 this work MA11594 yifK145::tetAR ∆gcvB141::cat / pKD46 this work MA11779 ygdI143::cat this work MA11780 wecG144::cat this work MA11781 yifK[-33A] yifK87::MudK wecG144::cat this work MA11782 yifK[U21C] yifK87::MudK wecG144::cat this work MA11783 yifK[G27A] yifK87::MudK wecG144::cat this work MA11784 yifK[-33A] yifK87::MudK this work MA11785 yifK[-33A] this work MA11786 yifK[U21C] yifK87::MudK wecG144::cat ∆gcvB141::scar this work MA11787 yifK[G27A] yifK87::MudK wecG144::cat ∆gcvB141::scar this work MA11788 yifK[U21C] yifK87::MudK wecG144::cat hfq13::Tn5-T-POP this work MA11789 yifK[G27A] yifK87::MudK wecG144::cat hfq13::Tn5-T-POP this work MA11790 yifK[A46U,A47G,A48U] yifK87::MudK this work MA11791 yifK87::MudK gcvB[U86A,U87C,U88A] ygdI143::cat this work MA11792 yifK[A46U,A47G,A48U] yifK87::MudK gcvB[U86A,U87C,U88A] ygdI143::cat this work MA11793 yifK[C49U,A50C] yifK87::MudK this work MA11794 yifK87::MudK gcvB[U84G,G85A] ygdI143::cat this work MA11795 yifK[C49U,A50C] yifK87::MudK gcvB[U84G,G85A] ygdI143::cat this work MA11796 yifK[C49U,A50C] yifK87::MudK ∆gcvB141::cat this work MA11797 yifK[C49U,A50C] yifK87::MudK ∆hfq67::cat this work MA11798 yifK[-33A]∆yifK146::cat-3xFLAG this work MA11799 yifK[-33A] yifK[C49U,A50C] ∆yifK146::cat-3xFLAG this work MA11800 ∆gcvB141::scar [rluC-rne]IG::cat rne-3071 (ts) this work MA11801 yifK[-33A] ∆gcvB141::cat this work MA11802 yifK[-33A] [rluC-rne]IG::cat rne-3071 (ts) this work

II.2. Manuscript submitted to PloS Genetics (under revision

Brief presentation of the article:

As discussed in the Introduction section of this manuscript, the Sm-like Hfq protein is a key player in the regulatory mechanism involving small noncoding RNAs and target mRNAs. This is particularly the case for the most common bacterial models, namely, Gram-negative enteric bacteria such as Salmonella and E. coli. Indeed, the stability of the sRNAs and their successful interaction with the target sequences in mRNAs are largely dependent on the availability of Hfq see [START_REF] De Lay | Bacterial small RNA-based negative regulation: Hfq and its accomplices[END_REF]. The current view is that the different RNAs could compete with each other for Hfq binding. Thus, the cellular concentration of Hfq and the differential affinity of the sRNA for the chaperon protein could play a role in the regulation itself [START_REF] Wagner | Cycling of RNAs on Hfq[END_REF]. In Gram-positive bacteria, the precise role of Hfq remains ill defined in many instances (Jousselin e t a l . , 2009). Paradoxically however, the first highresolution crystallographic data were obtained with Staphylococcus aureus Hfq [START_REF] Schumacher | Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein[END_REF]. and subsequently, S. aureus Hfq structure together with E. coli's have played a determinant role in helping understand the mechanistic aspects of Hfq:RNA interactions [START_REF] Link | Structure of Escherichia coli Hfq bound to polyriboadenylate RNA[END_REF], Sauter et al., 2003, Schumacher et al., 2002). In spite of being so the well

Abstract

Hfq is an RNA-binding protein that participates in the regulatory activity of small non-coding RNAs (sRNAs) in many species of bacteria. Hfq protein was first crystallized from Staphylococcus aureus and this crystal structure constitutes a hallmark for bacterial Sm-like proteins. Paradoxically, however, the functional relevance/role of S. aureus Hfq (Hfq SA ) remains uncertain, as growing evidence suggests that the hfq SA gene is expressed at very low levels or unexpressed in many S. aureus strains. To gather further insight, in the present work we exchanged the structural portion of the hfq gene of Salmonella enterica serovar Typhimurium (hfq STM ) with hfq SA and analyzed the effects of the replacement on various Hfq-related phenotypes. Our results show that the replacement strain -in spite of expressing Hfq SA at levels comparable to Hfq STM in wild-type Salmonella -behaves as an hfq null mutant in three discrete small RNA-mediated regulatory responses. These defects correlate with an abrupt reduction in the intracellular concentration of sRNAs, as observed in an hfq null mutant. Failure of Hfq SA to protect Salmonella sRNAs from degradation suggests that Hfq Sa does not bind to these sRNAs. A parallel study with the Borrelia burgdorferi hfq gene (hfq BB ) gave essentially identical results: when made from a single copy chromosomal gene, Hfq BB fails to substitute for Hfq STM in sRNA-mediated regulation.

Keywords: Hfq, Salmonella, small RNA, Staphylococcus aureus, Borrelia burgdorferi absence of Hfq SA or Hfq BB . These studies should help improve our understanding of the function, mode of action and ligand specificity of Sm-like proteins in bacteria.

Staphylococcus Salmonella

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