Figure index

The aim of this PhD project is to solve the molecular structure of ComFA and ComFC, but also of the complex between ComFA and ComFC. The role at a molecular level of each of those components during DNA translocation is studied as well. The determination of the molecular architecture of the ComFA/ComFC complex will help to find compounds that could block natural transformation in Gram-positive pathogens.

For example, during pneumococcal infection, the use of such compounds could limit bacterial virulence by inhibiting the ability of bacteria to evade the host immune response or to acquire antibiotic resistance genes.

The structural biology work consists of the over-expression in Escherichia coli and the purification of isolated components and protein complexes. Proteins from S. pneumoniae and the thermophile Geobacillus thermodenitrificans were studied.

The first structural approach focuses on the identification of the interactions between the proteins of the DNA uptake apparatus using the bacterial two-hybrid method.

The second structural approach focuses on individual components by X-ray crystallography to obtain high-resolution structural models. The primary aim is to produce protein mono-crystals able to diffract an X-ray beam. An alternative technique is used due to the absence of crystal. A cross-linking mass spectrometry analysis is performed to obtain structural infomation about ComFA.

A third structural approach involves the structure determination of the sub-complexes of ComFA hexamer and ComFA/ComFC using electron microscopy in order to provide low-resolution (between 8Å and 20 Å) structural information. In fact, electron microscopy is used when it is not possible to crystallize proteins. These proteins are coexpressed and the complex formed is purified. This complex is visualized using negative stain electron microscopy, and the structure is determined using single particle analysis. This structure is extremely important to understand the interaction of the two proteins.

In parallel, functional analyses are performed in vivo on both proteins and in vitro on ComFA to understand its unique role in the uptake of single stranded DNA (ssDNA).

Null mutant strains of S. pneumoniae were engineered to determine if the proteins of interest are required for natural transformation. Functional analyses in vitro were performed to determine the contribution of ComFA during bacterial transformation.

Biochemical approaches are used to identify DNA binding, ATPase activity, the potential helicase activity of ComFA and the translocation process.

This manuscript presents an introduction to current knowledge of the DNA translocase machinery and the need to study the natural transformation. The results obtained are divided into four chapters: the identification of different complexes, the structural study, the functional study and the study of the shape of the transformation pilus. Finally, the impact of this research on the field is evaluated in the final discussion.

Introduction I. DNA is the support of the genetic information

The twentieth century witnessed the emergence of a large number of biological discoveries and especially one of the most important discoveries in life sciences: the discovery of the deoxyribonucleic acid polymer (DNA), the molecule that carries the genetic information of all living organisms. This discovery occurred in successive steps.

The first person to identify genetic information exchange between bacteria was Frederic Griffith in 1928. He demonstrated that a lysate from a virulent strain of Streptococcus pneumoniae contains information that transforms a non-virulent strain into a virulent strain [START_REF] Griffith | The Significance of Pneumococcal Types[END_REF]. However he was not able to identify the transforming principle that was present in the lysed mixture from the virulent strain. About one decade later, three scientists, Oswald Avery, Colin MacLeod and Maclyn

McCarty carried on his study and identified the molecule involved in the transformation process: DNA [START_REF] Avery | Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii[END_REF]. This seminal result led to an intense debate in the field because, at that time, proteins were proposed to be the support of the genetic information. In fact, the activity of some proteins was already demonstrated whereas the DNA seemed to be an inert and rather simple molecule.

In 1953, James Watson and Francis Crick revealed the double helix structure of DNA. They discovered that nucleotides are arranged in a specific base pair association, which allows a proper replication of DNA that is crucial for the heredity of the genetic information [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF]. They also proposed that specific sequences of DNA were encoding the amino acid sequences of proteins. This assumption was demonstrated few years later, when Crick showed that a triplet of nucleotides corresponds to one amino acid [START_REF] Crick | General nature of the genetic code for proteins[END_REF]. Then, Marshall Nirenberg and Philip Leder deciphered the genetic code and proposed that RNA would be the link between the genetic information present in the nucleus and the proteins mostly synthesized in the cytosol [START_REF] Nirenberg | Rna Codewords and Protein Synthesis. The Effect of Trinucleotides Upon the Binding of Srna to Ribosomes[END_REF]. Meanwhile, the regulation of gene expression was demonstrated by Jacques Monod, François Jacob and André Lwoff, who highlighted the presence of promoter and repressor regions required for the expression of operons in bacteria [START_REF] Jacob | Genetic regulatory mechanisms in the synthesis of proteins[END_REF].

After more than eighty years of research in genetics, scientists are now able to genetically modify living organisms but also to completely invent species as performed at the Craig Venter Institute. Usually transformations only recombine partial fragments of DNA, but Lartigue and colleagues recently inserted the whole genome of M. mycoides into M. capricolum, which leads to converting a species into another [START_REF] Lartigue | Genome transplantation in bacteria: changing one species to another[END_REF]. This work showed that the new species developed all phonotypical information of the donor strain. Another step was passed a few years later following the same path when a new bacterial strain was created from a synthetic chromosome. The genomic DNA of M. mycoides was synthesized in vitro then transferred into M. capricolum, to create a new species fully encoded by this synthetic DNA [START_REF] Gibson | Creation of a bacterial cell controlled by a chemically synthesized genome[END_REF].

The latest discovery has extended the abilities of scientists to create new species.

Romesberg and colleagues designed a semi-synthetic organism with an expanded genetic alphabet involving the unnatural base pairs d5SICS and dNaM [START_REF] Malyshev | A semi-synthetic organism with an expanded genetic alphabet[END_REF]. At present synthetic biology is not only able to create new organisms but also to modify the support of the genetic information.

II. Horizontal gene transfer is a source of genetic variability

Genetic variability is the source of biodiversity in living organisms to adapt to environmental changes over time. In eukaryotes, this genetic diversity commonly happens every generation by sexual reproduction when two germinal cells are fused, a process called vertical gene transfer. During the meiosis, gene multiplications and punctual mutations can occur and will be accumulated over generations. However, prokaryotes such as bacteria reproduce through asexual reproduction and cannot generate variability by mixing two parental genomes, but genetic exchange still occurs to cope with their extracellular habitat. They use a process called horizontal gene transfer, which allows genetic information exchange from one bacterium to another. The three main mechanisms to perform that transfer are described below. [START_REF] Appelbaum | Streptococcus pneumoniae resistant to penicillin and chloramphenicol[END_REF]. In the last decade, a new phenomenon has been observed. The emergence of multi-resistant bacteria, which are resistant to several treatments, has led to increase mortality caused by common infections such as pneumonia [START_REF] Mera | Increasing prevalence of multidrug-resistant Streptococcus pneumoniae in the United States over a 10-year period: Alexander Project[END_REF]. The emergence of these "superbugs" has led to the research for new class of antibiotics [START_REF] Rask-Andersen | Trends in the exploitation of novel drug targets[END_REF]. The 2014 report on antimicrobial resistance from the World Health Organization warns against a "post-antibiotic era". The WHO initiated an international surveillance of antibacterial resistance in order to evaluate the public health threat of a wide range of pathogens in multiple sectors such as human health, food-production and the food chain. The current state of resistance of bacteria causing common infections, such as S. pneumoniae, was analysed. Reduced susceptibility to penicillin was detected in S. pneumoniae worldwide up to 50 % of the pneumococcal population in some countries (WHO, 2014).

Horizontal gene transfer mechanisms are at the heart of these resistance acquisition processes. In general they contribute to bacterial fitness and adaptability, especially during infection. Therefore, they need to be better understood in order to identify specific components that could be used as drug targets to block these mechanims and limit their beneficial effects on bacterial populations during infection.

III. Natural transformation in S. pneumoniae

A. S. pneumoniae, a human pathogen

Streptococcus pneumoniae is a Gram-positive diplococcus bacterium living in the human nasopharynx. This bacterium, discovered by Louis Pasteur in 1881, is harmless in healthy subjects but can become pathogenic in susceptible individuals such as young children, elderly or immune-compromised people. Most causes of death are due to meningitis or pneumonia, but other invasive pneumococcal diseases like bronchitis or otitis are observed.

S. pneumoniae infection relies on the polysaccharide shell capsule and cell-surface proteins [START_REF] Alonsodevelasco | Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines[END_REF]. This capsule is 200 to 400 nm thick and is synthetized by glycosyltransferases that assemble the oligosaccharide repeats on the

i. Competence induction

External signals such as stress and antibiotics are sensed by two membrane complexes, CiaTH and StkP. Both systems control the transcription of the "early gene" promoters [START_REF] Claverys | Induction of competence regulons as a general response to stress in gram-positive bacteria[END_REF][START_REF] Johnsborg | Natural genetic transformation: prevalence, mechanisms and function[END_REF]. Another soluble protein regulates competence by measuring the amount of misfolded proteins in the bacteria, this serine protease HtrA is a widely conserved protein involved in protein quality control [START_REF] Stevens | Competence in Streptococcus pneumoniae is regulated by the rate of ribosomal decoding errors[END_REF]. In case of stress, promoters of twenty early genes are activated including .ComC which encodes a precursor peptide called pre-CSP (CSP for Competence Stimulating Peptide) that is further cleaved and secreted through the ComAB transporter. This mature pheromone is detected by the membrane histidine kinase, ComD, which phosphorylates ComE. This protein is a sensor regulator that induces a positive feedback stimulating the expression of the early competence genes in the competent cells but also induces competence in non-competent neighbor cells [START_REF] Martin | Expression and maintenance of ComD-ComE, the two-component signal-transduction system that controls competence of Streptococcus pneumoniae[END_REF][START_REF] Martin | Crossregulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae[END_REF]. This activation loop requires the presence of comC for the CSP synthesis [START_REF] Cheng | The com locus controls genetic transformation in Streptococcus pneumoniae[END_REF], but a comC-null mutant strain is used in the laboratory in order to trigger competence by adding CSP to culture media. The CSP is a way of communication between pneumococcal strains in order to synchronize the induction of the competence state.

In addition to CSP induction, it appears that the pneumococcal competence is activated by high cell densities such as biofilm formation [START_REF] Hakansson | Chapter 7 -Pneumococcal genetic transformation during colonization and biofilm formation. Streptococcus Pneumoniae: Molecular Mechanisms of Host-Pathogen Interactions[END_REF]. In fact, biofilm formation upregulates comD and comX transcription [START_REF] Marks | High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae[END_REF]. In parallel, competence influences biofilm formation since competence mutants failed to form biofilm [START_REF] Vidal | Quorumsensing systems LuxS/autoinducer 2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells[END_REF] and CSP is observed to increase biofilm stability [START_REF] Oggioni | Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis[END_REF]. Moreover, gene transfer is found in biofilm models [START_REF] Wei | Fratricide is essential for efficient gene transfer between pneumococci in biofilms[END_REF]. These data suggest interlaced pathways of competence activation and biofilm formation.

ii. Activation of the late genes

Among the early genes, the expression of the sigma factor ComX and ComW, induces the transcription of 80 "late genes" through the activation of a hilghly-regulated promoter, shown in purple in Figure 6.

iii. Fratricide and bacteriocin secretion

Two bacteriocins encoded among the late competence genes, CibA and CibB, and also two hydrolases, CbpD and LytA, cause lysis of non-competent S. pneumoniae populations, a process called fratricide (Claverys et al., 2007;[START_REF] Guiral | Competence-programmed predation of noncompetent cells in the human pathogen Streptococcus pneumoniae: genetic requirements[END_REF]. These competence-induced proteins are involved in specific lysis of S. pneumoniae combined with a constitutively expressed hydrolase LytC. Of these five proteins, four directly cause cell lysis by digesting the cell wall. The last protein, CbpB, stays attached to the attacker cell wall to enable the killing of non-competent cells in a contact-dependent manner [START_REF] Perez-Dorado | Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC[END_REF]. In order to be protected against its own lysis components, competent cells express two immunity proteins at the cell surface, ComM and CibC, which respectively preserve the cell from LytA/LytC/CbpB and CibA/CibB [START_REF] Johnsborg | A predatory mechanism dramatically increases the efficiency of lateral gene transfer in Streptococcus pneumoniae and related commensal species[END_REF]. Phosphorylated ComE also induces the expression of the pneumocins for which the maximum expression is observed 100 minutes after CSP induction [START_REF] Wholey | Coordinated Bacteriocin Expression and Competence in Streptococcus pneumoniae Contributes to Genetic Adaptation through Neighbor Predation[END_REF]. During this process, expression of the blp locus stimulates the secretion of BlpC, another peptidic pheromone targeting neighboring organisms [START_REF] Wholey | Coordinated Bacteriocin Expression and Competence in Streptococcus pneumoniae Contributes to Genetic Adaptation through Neighbor Predation[END_REF]. Pre-BlpC is cleaved and secreted through the BlpAB and ComAB transporter. This mature pheromone is detected by the membrane histidine kinase receptor, BlpH, which phosphorylates BlpR. This last protein upregulates the operons of the blp locus including the bir genes, which encode bacteriocins and immunity proteins [START_REF] Pinchas | An electrostatic interaction between BlpC and BlpH dictates pheromone specificity in the control of bacteriocin production and immunity in Streptococcus pneumoniae[END_REF].

While similar auto-induction processes are found in the competence and the bacteriocin expression, the blp locus composition depends on the pneumococcal strain. Four pheromone/receptor couples are found which provide a competitive advantage to one strain compared to its neighbors [START_REF] Bogaardt | Genomic analyses of pneumococci reveal a wide diversity of bacteriocins -including pneumocyclicin, a novel circular bacteriocin[END_REF]. However, in addition to the induction by CSP, an antibiotic-induction is observed for the pneumocin expression [START_REF] Kjos | Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System[END_REF]. The roles of this system could be to liberate nutriments and open the niche by lysing competing bacteria. Also, during antibiotic stress, it could be a way to acquire new antibiotic-resistance genes. Indeed, the coordination of the fratricide and bacteriocin systems may allow a selective advantage by increasing the pool of exogenous DNA available for internalization by natural transformation.

iv. DNA binding and uptake

The proteins involved in the binding of exogenous DNA and its internalization are encoded by three operons, comE, comF and comG locus [START_REF] Pestova | Isolation and characterization of three Streptococcus pneumoniae transformation-specific loci by use of a lacZ reporter insertion vector[END_REF].

Among these genes, all but one are exclusively expressed during compentence. The membrane-associated nuclease EndA is the only known component of the system to be constitutively expressed and recruited during competence [START_REF] Berge | Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation[END_REF]. In S.

pneumoniae, this apparatus is assembled within 15 minutes after CSP induction under laboratory conditions. The transformation pilus, a type-IV pilus, recognizes the doublestranded DNA [START_REF] Laurenceau | A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae[END_REF] and an unknown mechanism transfers the DNA to the cell surface. EndA digests one DNA strand while the other strand is translocated across the membrane through the pore ComEC. The potential ATP-binding protein ComFA could provide the energy required for this process and allow ssDNA incorporation at a speed of 90 nucleotides per second with a 3' to 5' end polarity [START_REF] Mejean | DNA processing during entry in transformation of Streptococcus pneumoniae[END_REF].

v. Homologous recombination

Specific DNA processing proteins such as DprA, SsbB, RadA and CoiA are expressed during competence (Figure 6Figure 6). These proteins are involved in the homologous recombination process after ssDNA incorporation. SsbB binds to incoming DNA to protect it against endonuclease degradation and to make a DNA reservoir before its recombination in the bacterial genome [START_REF] Attaiech | Role of the single-stranded DNA-binding protein SsbB in pneumococcal transformation: maintenance of a reservoir for genetic plasticity[END_REF]). Yet ssbB-null mutant only shows a transformation deficit of three-to five-fold (Bergé et al., 2003). In fact a paralogous Ssb protein, SsBA, involved in recombinational repair, is constitutively expressed and could replace SsbB [START_REF] Thanassi | Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae[END_REF]. The recombinase RecA is the universal recombinase involved in genome maintenance and is constitutively expressed.

It co-localizes with competence proteins during natural transformation [START_REF] Kramer | Multiple interactions among the competence proteins of Bacillus subtilis[END_REF]. RecA is recruited during competence by DprA (Figure 7.B), the competencespecific recombination mediator protein. The DprA dimer is described to displace SsbB [START_REF] Mortier-Barriere | A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA[END_REF] and to be a RecA loader on ssDNA [START_REF] Martin | Identification of the recA gene of Streptococcus pneumoniae[END_REF][START_REF] Mortier-Barriere | A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA[END_REF][START_REF] Quevillon-Cheruel | Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation[END_REF] in order to allow RecA polymerization on the ssDNA and enable its homologous recombination with the transformasome is composed of 20 to 50 proteins further described in the following sections.

C. Natural transformation in other bacterial species

Sequencing of bacterial genomes allowed the identification of many bacteria carrying the genes involved in natural transformation (Johnston et al., 2014b). So far more than 80 bacterial species such as Gram-positive bacteria like B. subtilis or Gramnegative bacteria like V. cholerae, N. gonorrhoeae, H. influenzae were found naturally transformable. The many components of the transformation machinery are conserved.

While the molecular basis of the DNA binding, entry and recombination is likely to be conserved, several important differences have been reported.

i. Regulation of competence

The transformasome expression is not induced by the same mechanisms in all competent bacteria (Figure 8).

In B. subtilis an autoinducer peptide analog to the CSP has been identified. This peptide, ComX, is cleaved during secretion by ComO and is bound by the response regulator ComP, thus inducing competence in non-competent cells [START_REF] Claverys | Induction of competence regulons as a general response to stress in gram-positive bacteria[END_REF].

The induction is also different in Gram-negative bacteria. For example in V. cholera, chitin is required to activate competence [START_REF] Meibom | Chitin induces natural competence in Vibrio cholerae[END_REF] but not the quorum sensor systel as in pneumonoccal competence. In H. pylori, competence is induced by DNA damage and induces RecA-dependent expression of both a lysozyme-like protein and the comT4SS involved in an increase of foreign DNA uptake [START_REF] Dorer | DNA damage triggers genetic exchange in Helicobacter pylori[END_REF].

DNA damage also induces competence in Legionella pneumophila due to genotoxic stress such as antibiotics and UV radiation [START_REF] Charpentier | Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila[END_REF]. In addition, in V. cholerae, instead of the CSP, Al-2 and CAl-2, two autoinducer peptides are observed. These peptides activate a cascade for the induction of the sigma factor qstR that is similar to the ComX sigma factor (Johnston et al., 2014b).

Finally, in some species such as N. gonorrhoeae, the transformation machinery is constitutively expressed [START_REF] Biswas | Factors affecting genetic transformation of Neisseria gonorrhoeae[END_REF]. In addition, the pathogen H. pylori is able sporulation [START_REF] Gonzalez-Pastor | Cannibalism by sporulating bacteria[END_REF]. Extracellular killing factors are secreted in order to lyse sibling cells which do not express the immunity receptor [START_REF] Ellermeier | A three-protein signaling pathway governing immunity to a bacterial cannibalism toxin[END_REF]. A recent review characterized cannibalism and competence as "chemical warfare" involved in the release of nutriments to feed competent bacteria (Claverys and Havarstein, 2007).

In addition, a link between the competence event and expression of the type VI secretion system (T6SS) in V. cholerae was recently reported [START_REF] Borgeaud | The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer[END_REF].

The cluster of genes encoding for the T6SS is positively regulated by the competence regulators TfoX and QstR. This co-regulation replaces the fratricide phenomenon of S.

pneumoniae for the release of DNA to increase the DNA pool available for uptake.

D. DNA uptake in Archaea

Archea are unicellular organisms which are able to survive and adapt in extreme environments such as extreme temperature, pH or salt concentration. Therefore, gene transfer systems are essential in order to cope with these ecological changes. Natural transformation has been observed in several species such as Methanobacterium thermoautotrophicum, Halorubrum or Pyrococcus furiosus. This phenomenon was first observed in Methanobacterium thermoautotrophicum by a similar study performed by

Griffith [START_REF] Griffith | The Significance of Pneumococcal Types[END_REF]. In fact a wild type strain was transformed by DNA from a strain resistant to 5-fluorouracil [START_REF] Worrell | Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg[END_REF]. Another archeae, Pyrococcus furiosus, is also naturally competent since a pyrF-null mutant was mutated and the gene was restored by adding to the culture media DNA encoding for the gene [START_REF] Lipscomb | Natural competence in the hyperthermophilic archaeon Pyrococcus furiosus facilitates genetic manipulation: construction of markerless deletions of genes encoding the two cytoplasmic hydrogenases[END_REF]. Pyrococcus furiosus was shown to be able to uptake both circular and linear DNA.

A recent study highlighted a new role for DNA uptake in the halophilic Archaea, Haloferax volcanii. This strain is able to use exogenous dsDNA as a source of phosphorus nutriment [START_REF] Chimileski | Extracellular DNA metabolism in Haloferax volcanii[END_REF].

IV. The DNA uptake apparatus in S. pneumoniae

A. The DNA uptake system

The transformasome regroups all proteins involved in DNA uptake and its recombination. My PhD thesis focuses on ComFA, a major component of the DNA uptake system involved in transformation. Below, I will give more details on this fascinating molecular system that translocates DNA across the bacterial membrane. Although the DNA uptake system and the recombination system are two different machineries, a recent study using FRET demonstrates their co-localization in B. subtilis [START_REF] Kramer | Multiple interactions among the competence proteins of Bacillus subtilis[END_REF]. The transformation complex is localized at midcell in S. pneumoniae since two proteins involved in the preparation of the incoming DNA, ComEA and EndA, colocalize with end-labeled dsDNA [START_REF] Berge | Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation[END_REF]. Moreover, ComGG pull down revealed a copurification of ComFA, ComEC, RecA, SsbB and DprA [START_REF] Mann | Complex formation and processing of the minor transformation pilins of Bacillus subtilis[END_REF]. This result indicates the existence of a stable complex, but direct protein interaction has not yet been identified. Despite the identification of all competence proteins, the role of ComFC and its position remain unknown in the current model described in the literature (Figure 11). This process relies on two different protein complexes, the transformation pilus and the DNA translocation apparatus. The first complex forming a type IV pilus is involved in DNA binding and is composed of proteins encoded by the comG operon. The second complex is involved in DNA preparation and DNA translocation and is formed by proteins encoded by the comE and comF operons.

assembly platform formed by ComGA and ComGB is mandatory for pilus assembly since ADN binding is completely abolished in the comGA knockout (Figure 12).

Another protein, ComEA, is involved in DNA attachment in order to stably bind the incoming dsDNA to the cell surface, probably close to the pore entrance of the DNA translocation apparatus. DNA uptake is abolished in the comEA knockout (Figure 15). These data suggest that the DNA binding occurs in two steps: first, the pilus captures the dsDNA and then transfers it to ComEA, which keeps it close to the membrane.

C. DNA degradation

The nuclease EndA, an extracellular membrane-associated protein, is constitutively localized over the entire surface of S. pneumoniae, unlike other proteins of the DNA uptake apparatus which are only expressed during competence. So far no homolog has been identified in other transformable bacteria. The EndA nuclease could be recruited by ComEA. It degrades one strand of exogenous dsDNA while the other strand is integrated into the bacterium. Nevertheless, when this digestion is abolished in an endA-null mutant, a significant reduction of the DNA entry is observed (Figure 12).

D. Translocation of the DNA

The helicase-like membrane-associated ATPase ComFA and the integral membrane ComEC are both required for DNA translocation across the bacterial membrane. In fact, this incorporation is abolished in both comEC and comFA knockout strains (Figure 12) while DNA attachment and degradation is preserved in these mutants. Indeed, a ΔcomFA mutation increased DNA binding by two-fold, indicating a possible interdependence of the transformation pilus and the DNA translocase apparatus since ComGA and ComFA are co-localized and show a stability dependency [START_REF] Hahn | Transformation proteins and DNA uptake localize to the cell poles in Bacillus subtilis[END_REF]. Furthermore, in the presence of ComEC channel and in absence of ComFA the DNA cannot enter the cell, indicating that ComFA is an essential source of energy for DNA uptake.

B. ComEA

ComEA is predicted to be an integral membrane protein, with the presence of a Nterminal transmembrane segment and to be involved in DNA binding. In addition, this protein is probably releated to the processing and the uptake of transforming DNA [START_REF] Hahn | Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA[END_REF]. Further investigations confirmed that ComEA is required for DNA binding and uptake in B. subtilis [START_REF] Inamine | ComEA, a Bacillus subtilis integral membrane protein required for genetic transformation, is needed for both DNA binding and transport[END_REF]. DNA binding is due to the helix-hairpin-helix domain present in the C-terminal domain of the protein that allows binding without any sequence specificity in B. subtilis [START_REF] Provvedi | ComEA is a DNA receptor for transformation of competent Bacillus subtilis[END_REF].

Although ComEA binds ssDNA and dsNDA, the protein interacts preferably with dsDNA.

In S. pneumoniae ComEA is also required, and the protein shares 40% identity with the B. subtilis homolog [START_REF] Pestova | Contribution of topoisomerase IV and DNA gyrase mutations in Streptococcus pneumoniae to resistance to novel fluoroquinolones[END_REF]. A recent study [START_REF] Takeno | Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis[END_REF] demonstrated that comEA-null mutant in B. subtilis shows a transformation efficiency decreased of 2.7*10 -6 compared to the wild type. In addition, the DNA uptake velocity was significantly reduced because the protein is involved in the transfer of the exogenous DNA into the DNA channel. ComE is the homolog of ComEA in Gram-negative bacteria, such as N.

gonorrhoeae or V. cholerae (Table 1). It does not contain a transmembrane domain but a N-terminal signal peptide for its secretion in the periplasm [START_REF] Chen | ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity[END_REF][START_REF] Seitz | DNA-uptake machinery of naturally competent Vibrio cholerae[END_REF]. Surprisingly, comE genes in Neisserial species are present in four copies in the genome, and their deletion decrease transformation efficiency of 4.10 4 fold. Only one copy, ComE1, is involved in DNA internalization whereas others copies are only responsible for DNA binding and accumulation in the periplasm. The latest study in the Gram-negative bacterium Thermus thermophilus highlighted the dsDNA binding activity of an atypical ComEA that is anchored in the inner membrane (Salzer et al., The N-terminal extracellular loop L19-Q176 contains two cysteines forming an intermolecular disulphide bond formed between C131 and C172, which is involved in the folding of the protein [START_REF] Draskovic | Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds[END_REF]. In addition of its role in the stabilization of ComEC, this N-terminal loop is involved in DNA uptake as transformation is abolished when this loop is deleted [START_REF] Draskovic | Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds[END_REF]. Several cysteines are present in the N-terminal domain of the pneumococcal ComEC, but their positions are not conserved. The transmembrane domain, L226-W443, is responsible for the oligomerization of the protein thanks to three cysteines C482, C483 and C494. At least two subunits are involved in an oligomeric assembly stabilized by this intermolecular disulphide bond and might contribute to the formation of an aqueous pore for the DNA transport. The N-terminal domain, S497-G700, is an extracellular loop with a metallo-betalactamase function. These domains are often involved in the breakdown of antibiotics due to its zinc peptidase activity [START_REF] Palzkill | Metallo-beta-lactamase structure and function[END_REF] and the 3D structure (PDB: 1BMC) reveals the presence of a Zn 2+ in the structure [START_REF] Carfi | The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold[END_REF]. Unfortunately, its role in the DNA uptake has not being explored.

In addition to its function in DNA uptake, a recent study in T. thermophilus indicates a role in the regulation of the competence and the pilitation genes [START_REF] Salzer | The Thermus thermophilus comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type IV pili[END_REF]. In this species, the comEC-null mutant impaired the piliation of T4P genes such as pilA4. In fact its transcription is decreased, as shown by the decrease of the mRNA level by 2.6-fold. The bacterium twitching motility is also impacted because the motility is abolished in this mutant. This result is in agreement with data obtained in A. baumanii, in which a ΔcomEC strain also shows an eradication of the twitching motility [START_REF] Wilharm | DNA uptake by the nosocomial pathogen Acinetobacter baumannii occurs during movement along wet surfaces[END_REF]. It might explain why ComEC homologs are found in non-competent bacteria. Moreover ComEC has been demonstrated to play a major role in the adaptive response to nutriment limitation and temperature stress since its transcription is triggered by nutriment limitation and low temperature [START_REF] Wilharm | DNA uptake by the nosocomial pathogen Acinetobacter baumannii occurs during movement along wet surfaces[END_REF].

VI. The comF operon

A. Architecture of the operon

The operon comF was first described in B. subtilis [START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF] in which three genes encoding three proteins have been identified named ComFA, Previous study determined that ComFA was a membrane-associated protein (Londono-Vallejo and Dubnau, 1994a) but only a short hydrophobic segment, between the amino acids 332 and 248, is identified. Lonodoño and colleagues compared ComFA to an E. coli PriA (Lee et al., 1990). In this protein, two steps were observed during its function: the DNA translocation and the DNA unwinding. Further studies indicated that this specific DNA helicase activity found in PriA was required to initiate the DNA replication by unwind dsDNA [START_REF] Gabbai | Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival[END_REF]. The motion observed of the translocation is from 3' to 5' end [START_REF] Lasken | The primosomal protein n' of Escherichia coli is a DNA helicase[END_REF]) at a rate of about 90 nucleotides/second [START_REF] Lee | Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein[END_REF]. These two data are compatible with the entry of the ssDNA at a rate of 90 nucleotides/second in the same orientation in the bacterium during transformation. The activity of PriA is helped by an interaction with ssDNA binding proteins (SSB), which stimulates the helicase activity [START_REF] Cadman | PriA helicase and SSB interact physically and functionally[END_REF]). In the transformasome, a single-strand DNA binding protein, called SsbB, is co-localizing with ComFA and might assist the protein for DNA uptake. The tridimensional structure of PriA from Klebisiella pneumoniae was recently solved [START_REF] Bhattacharyya | Structural mechanisms of PriA-mediated DNA replication restart[END_REF] and highlighted an atypical Zn 2+ binding domain between the DEAD box and the helicase C domains, which seems to be involved in the helicase activity but not the ATPase activity. A zinc finger domain (Figure 16) was also observed in PriA from E. coli and involved 8 cysteines instead of 4 in the pneumonoccal ComFA [START_REF] Zavitz | Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNAdependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities[END_REF]. This study highlighted a bidirectional helicase activity in presence or in absence of zinc but the metal binding increases the function of PriA of 6fold. The core structure of the zinc-cluster is comparable in ComFA and in PriA respectively and . ComFA could be part of a C2-C2 type of zincfinger [START_REF] Klug | Zinc fingers: a novel protein fold for nucleic acid recognition[END_REF] and should be able to bind several types of metal such as zinc and copper [START_REF] Hutchens | Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry[END_REF]. According to sequence homology ComFA could be part of the fold group 4 of zinc finger called zinc ribbon [START_REF] Krishna | Structural classification of zinc fingers: survey and summary[END_REF]. This family includes domains from proteins involved in translocation machineries. Zinc ribbon proteins are characterized by a long secondary hairpin and a three-stranded antiparallel β-sheet, that folding is involved in DNA-binding. The four cysteines residues are coordinating the metal binding that can be a zinc ion but also in some case cadmium, cobalt, copper or nickel. Both cobalt and copper have been shown to inhibit the DNA binding of the DNA-binding domain of the estrogen receptor in vitro [START_REF] Predki | Metal replacement in "zinc finger" and its effect on DNA binding[END_REF].

the self-assembly and the C-terminal domain involved for the motion amount the ssDNA incoming in the cell.

C. ComFC the unknown phosphorybosyl transferase

This soluble protein discovered in B. subtilis [START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF] has been poorly studied because the protein was described as not essential for transformation. Indeed, the deletion of comFC only leads to 5-fold decrease in transformation efficiency in these bacteria. Nonetheless, its homolog Com101A from H.

influenzae, that shares 21% identity with the B. subtilis protein, is required for transformation since null mutant cannot uptake DNA (Table 4). This protein, Com101A, has been demonstrated to be involved in the process of uptaking the incoming ssDNA because the amount of nucleotides release into the supernatant is reduced [START_REF] Larson | Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae[END_REF]. Nevertheless no direct nuclease activity has been identified. A ComFC homolog has been studied in Synechocystis sp. strain PCC 6803, which shares 21 % identity with S. pneumoniae ComFC [START_REF] Nakasugi | The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation[END_REF]. Unlike null mutant in B. subtilis, the knock out of comFC abolished transformation but also induced a modification of the piliation characteristics. A higher pilus bundling is observed probably due to an increase of the expression level of PilA, the major pilin, of 3 to 5-fold that might also be involved in the generation more rigid pili. Finally, the protein seems to be involved in the motility, in fact, null-mutant bacteria aggregate in culture. This

Multiple interactions among competence proteins of S. pneumoniae

Annick Dujeancourt, technician in the laboratory, has greatly contributed to the two-hybrid assay.

I. The translocation apparatus complex

In our working model, four proteins are known to be involved in the uptake:

ComEA the DNA receptor; EndA which converts dsDNA into ssDNA for internalization;

ComEC the DNA channel and ComFA the putative translocase [START_REF] Claverys | The genetic transformation machinery: composition, localization, and mechanism[END_REF].

Surprisingly, the second protein of the comF operon, ComFC, has never been tested before because its role during transformation in Bacillus subtillis is described to be minor [START_REF] Dubnau | Genetic competence in Bacillus subtilis[END_REF]. First, we focused on ComFA and ComFC to characterize the interaction between ComF proteins. Then, we tested other proteins of the transformasome together with ComFA or ComFC to understand their contacts with the rest of the transformation system.

All these interactions were determined using the bacterial two-hybrid (BacTH) assay [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF]. In this system, the adenylate cyclase gene from Bordetella pertussis is split into two coding sequences encoding two domains called T18 and T25

that cannot interact together unless they are fused to proteins that directly interact.

Therefore, proteins of interest are fused to these complementary domains, and if these proteins are associated, T25 and T18 fragments will reconstitute a functional adenylate cyclase. It will trigger the expression of the β-galactosidase in the bacterium and lead to a colony colored in blue after 48 hours on galactopyranoside-plates.

A. Interaction between the proteins of the comF operon

The PCR products obtained from the amplification of genomic DNA of S.

pneumoniae R800 were digested and cloned in all the plasmids used for the BacTH system (pUT18, pUT18C, pKNT25, pKT25). Constructs with C-and N-terminal T18 domain were easily obtained, whereas only two out of four constructs with the T25 domain where obtained (pAD041/42/43/44/45/46, material and method Table 7).

BTH101 reporter cells producing the indicated N-and C-terminal domain ComFA and ComFC fused to the T18 or T25 domain grown on plates supplemented with IPTG and X-Gal. Interaction between two proteins is resulting to a blue color colony as shown is the control.

Based on these results, we can hypothesize that the lack of the first 72 amino acids does not disturb ComFA interaction in vivo, which indicates that the zinc finger domain is not involved in the ComFA/ComFA interaction. The interaction domain would be the loop between the zinc finger domain and the DEAD box domain according to our data and previous study. In addition, ComFC full-length needs to be expressed to form homodimer, mabye because the domain of interaction required a specific tertiary structure around site where the cleavage was engineered. The interaction between

ComFA and ComFC also requires full-length ComFA to take place.

The other explaination of the lack of interaction could be because the moieties T25 and T18 are too far from each other to be able to react.

C. Adding ComEC to the system

In a previous model of the DNA translocation apparatus (Introduction, Figure 11), the protein ComFC was not represented. We demonstrated its interaction with ComFA but the question remains open regarding interactions with other proteins of the system.

As EndA and ComEA are extracellular, it is not plausible that they would interact with 7), noticed that one construct with the N-terminal soluble domain fused to the T25 on the C-terminus could not be obtained.

IPTG and X-Gal. Interaction between two proteins is resulting to a blue color colony as shown is the control.

Among the protein of the transformasome, no direct interaction with ComEC could be identified. Indirect data can only be considered with caution. The only information available is that ComFA is destabilized in comEC-null mutant indicating a functional interaction between proteins of the translocation apparatus. A tomography cryo-electron microscopy study of the DNA translocation apparatus would help to understand protein-protein interaction within the complex. However, S. pneumoniae constructs with a ComEC fused to GFP do not display fluorescence while being functional in transformation assays. This indicates a very low level of the expression and suggests that only few assembled DNA translocation apparatus are present in the bacterial membrane.

II. Extending the BacTH assay to the full DNA uptake apparatus

The DNA uptake apparatus is composed of two complexes: the DNA translocation apparatus and the transformation pilus. Localization experiments were performed on ComGA and ComFA [START_REF] Hahn | Transformation proteins and DNA uptake localize to the cell poles in Bacillus subtilis[END_REF], and both proteins were found to co-localize at the cell pole. A complementary FRET analysis in B. subtilis cells showed a close proximity between these proteins [START_REF] Kramer | Multiple interactions among the competence proteins of Bacillus subtilis[END_REF]. Co-transformations between my plasmids and Chiara Rapisarda's constructs of ComGA fused to T25 and T18 were performed to study the interaction between ComFA or ComFC with ComGA.

There is an interaction between ComFA and ComGA as shown by the pair ComGA-T25

and ComFA-T18 (Figure 27Figure 27). The constructs of ComFC and ComGA also show interaction between these two proteins.

and the last group of proteins spelled in red are involved in the homologous recombination. Pink arrows represent directs interactions determined by BacTH and black arrows represent protein co-localization and stabilization effect.

IV. Link between comF operon and the cell division system

Cell division is blocked during the expression of competence in S. pneumoniae [START_REF] Dagkessamanskaia | Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells[END_REF] but no link between proteins of the cell division system and those of the transformation system have being identified so far. Our collaborators from Toulouse University performed a pull-down purification with a SPA-tag [START_REF] Puig | The tandem affinity purification (TAP) method: a general procedure of protein complex purification[END_REF] on the C-terminus of ComFC (unpublished data). The mass spectrometry analysis carried out demonstrated the enrichment of several expected proteins such as ComFA or

ComEC but also two proteins involved in the cell division process: GpsB and spr1506.

GpsB is a key component during the localization of the divisome at the cell septum [START_REF] Pompeo | Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis[END_REF]. The interaction with GpsB could be the reason of the inhibition of the cell division but also an explanation of the septal localization of the DNA translocation apparatus. The second protein spr1506 is a hypothetical protein similar to DivIVA, also a key component of the divisome [START_REF] Fadda | Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context[END_REF].

Preliminary data obtained were very encouraging, however protein expression was very inconsistent due to media issue. The project was stopped after several unsuccessful attempts in various media (LB and TB) and in different bacterial strains (BL21-DE3 or Rosetta). Other constructs with the distinct cleavage site by the thrombine were also tried but once again the protein expression was a limitation for the study of the protein. In the future, protein expression platform like yeast or insect cells could be considered.

II. ComFA

ComFA has not been studied for more than 20 years, since David Dubnau and colleagues, showed that the protein was essential for transformation and was located in the bacterial membrane. The structural study of the protein is essential to understand the function of this unusual DEAD box protein.

A. Heterologous expression in E. coli of several construct from various species

The protein ComFA is essential for DNA uptake during natural transformation in B. subtilis (Londono-Vallejo and Dubnau, 1994b) but the structure of the protein has never been solved. Structural models based on homology could be obtained. However the closest templates (Recg from Thermanotoga maritima or the transcription-repair coupling factor from E.coli) share only 23 % and 32 % homology with the C-terminal domain of ComFA. In addition, the N-terminal domain does not share any homology with proteins known structure solved. As protein crystallization has a low probability of success in addition of ComFA S. pneumoniae R6 could not be obtained, I also cloned a homolog from Gram-positive bacteria: Geobacillus thermodenitrificans (Uniprot A4ISW3) that shares 22 % similarity with ComFA S. pneumoniae R6 (Figure 37).

SDS-Page gel of the test expression of pAD020, pAD021 and pAD022 respectively the construct ppSUMO_His-SUMO-ComFA/ComFC, pET21_ComFA/comFC-His and pProEX_His-ComFA/ComFC

This result is very interesting, in fact, for the first time both proteins were expressed and seen as physically interacting in vitro. Even if all constructs allow the obtention of the complex, the construct further used for the study was pAD021, since the interaction was the strongest. The second interesting part is the double band at about 50 kDa for pAD021 and pAD022, according the analysis of ComFC it could be due to a dimer of ComFC but a band at 50 kDa is not present on the last construct pAD020, which is the construct with His-SUMO-ComFA. In addition, a WesternBlot anti-His allows the identification of the tag protein, confirming that ComFC was not forming a dimer in presence of ComFA. This doublet seems to be due to a degradation of ComFA or posttranslational modifications and was already observed by a previous study (Londono-Vallejo and Dubnau, 1994a).

Other constructs based on pAD021 were engineered, in order to improve the stability of the complex. Finally, a similar construct to pAD020 was created with the genome of Geobacillus thermodenitrificans, in order to know is this construct could be more stable. But alike for the expression of ComFC G. thermodenitrificans the ComFC protein is precipitating in the membrane.

In order to study the structure of this newly discovered complex formed by ComFA and ComFC proteins, the main construct used is pAD021. Depending on the final protein concentration two structural approaches can be considered, X-ray crystallallography and electron microscopy. The main point to keep in mind is that large protein complexes are less likely to crystallize than smaller protein.

B. Purification optimization

Alike ComFA complex purification, first purifications attempt were performed using gel filtration before the implementation of the GraFix method in order to stabilize the complex.

i. Gel filtration purification

ComFA/FC S. pneumoniae with a C-terminal His6-tag on ComFC is expressed in high amount and mostly present in the soluble fraction. Identical purification protocol than for ComFA protein was used: a gentle lyse followed by an ultracentrifugation. The ComFA/ComFC complex would be 450kDa. This is compatible with the molecular weight observed of the complex purified.

A) Class average of ComFA/FC complex obtained from sample purified by gel filtration. B) Final volume of the complex obtained by projection matching and angle refinement done with Imagic. Description from the left to the right panel, top, side and bottom.

The formation of a complex between ComFA and ComFC, both two protein of the operon comF is a completely new discovery and is very encouraging for the future. A new grid made of ComFA/FC cross-linked by the GraFix approach will allows to improve the quality of sample. A new data colletion on this homogenous sample will allow a better 3D-structure resolution with automatic data collection.

ΔcomFA (Figure 59.C). Interestingly, the deletion of comFC also leads to an abolition of the transformation, as well as the deletion of both genes.

According to previous study [START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF], we were not expecting a such high impact of ComFC in the transformation. We demonstrated that this protein is essential for the transformation. The explanation is that ComFC might be a key partner between ComFA and ComEC. Those data are consistent with the previous chapter, suggesting that ComFC is a membrane-associated protein and important of the functional folding of ComFA.

B. Protein expression validation

Since comFA and comFC genes are in an operon, it is important to confirm that the deletion of one gene is not affecting the expression of the second protein.

The first technique proposed is to detect the protein by a Western blot. The bacteria strain used to knock out the genes did not contain any FLAG or His-tag on the proteins, so anti-His and anti-FLAG antibodies cannot be used. Unfortunately, no commercial antibodies against ComFA and ComFC were available. In some cases, it is possible to generate antibodies against proteins of interest, but a high amount of protein is required. As described in the previous chapter, ComFA purification could allow antibodies production but ComFC final concentration was too far from the required concentration of about 6 mg.mL -1 .

Proteins expression could be determined by localization using GFP fused to the protein. The protein ComFA was fused with the yellow fluorescent protein (YFP) in B.

subtilis [START_REF] Hahn | Transformation proteins and DNA uptake localize to the cell poles in Bacillus subtilis[END_REF], but the ComFA-YFP was not stable. In fact, the tag YFP was cleaved in vivo and the functional activity of the protein could not be guaranteed.

Regarding the second protein ComFC, no assay has been done, but due to the failure on ComFA, we decided not to use this approach.

The last approach was to complement mutants AD002, AD003, AD004 with the gene deleted in the genome in order to confirm that we recover the initial transformation value. If the initial value of about 10% of efficiency is not restored it would indicate that the knock out of the gene disturbs the expression of the second protein. Two plasmids could be used: the pCEP and the pCEP synthetic, containing respectively the kanamycin and the streptomycin resistance gene. The knock out of the gene of interest already introduced a kanamycin resistance so I had to use the pCEP synthetic. Unfortunately, this plasmid could not be manipulated efficiently. The plasmid stock digested with NcoI and BamHI was ligated with three different inserts ComFA;

ComFC; ComFA/FC and no clones were obtained. Several attempts of cloning in that plasmid failed no matter the ratio of inserted compared to the plasmid. Previously, other members of the team also tried to construct clones with this plasmid and did not succeed. We suspect that either the plasmid is super coiled so cannot be digested by the restriction enzymes, either the sequence synthetized might be wrong which prevents us to design proper primers for cloning.

We could not verify the expression of the second protein when one of the comF gene is knocked out, but it was confirmed by our collaborators in Harvard (Hannah Foster, PhD candidate) because they obtained the same result in B. subtilis.

We assumed that the expression was not disturbed based on the primer design.

According to data collected by two-hybrid and heterologous expression, the guess is that ComFA is the ATPase driving the DNA uptake and the passive transport alone cannot translocate the DNA. The second protein, ComFC, is the link between the membrane protein ComEC and ComFA, and in absence of ComFC the helicase ComFA cannot be localized near by the ComEC channel.

II. DNA binding assay

Helicases are capable of binding different types of nucleotides in order to unwind nucleotides duplexes. This activity is involved in the separation of two strands of DNA or separation of self-annealed RNA using the energy released by ATP hydrolysis.

Bioinformatics analysis using BLAST, predicted that 9 amino acids (Figure 60) are involved in that binding according to homology with the RNA helicase domain from the hepatitis C virus NS3 structure. The second protein of the operon comF, ComFC does not contain identified DNA binding site in its sequence. because the amount of free DNA is comparable with and without ComFC. The assay would have been better if it was performed on the complex co-purified, because according to the gel filtration of the in vitro reconstructed complex, the interaction cannot be recovered by mixing the proteins. The role of ComFC was impossible to identify due to the impossibility to recover the complex.

Raphael Laurenceau, previous PhD student in the laboratory, Petya Violinova Krasteva, postdoctoral fellow and Sahra Ouarti a bachelor student have greatly contributed in the identification of the protein.

Discussion and perspectives

The goal of my PhD project was to study the function and the structure of the DNA upkate machinery from S. pneumoniae. I focused on the comF operon and the expression of ComFA and ComFC, which are conserved among Gram-positive bacteria.

The main conclusions of this work are summarized and discussed in the perspective of future works.

I. The improved model of the DNA-uptake machinery of naturally competent S. pneumoniae

S pneumoniae competent cells require the expression of ComFA and ComFC to transport the DNA across the membrane. The impact of ComFC was underestimated in the previous study [START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF] due to an incomplete deletion in the bacterial genome of B. subtilis. The double hybrid approaches in this work allowed a better understanding of the interaction between the proteins involved in the uptake and the recombination of the DNA. Based on the literature and these results, we propose an improved model (Figure 73) that includes ComFC, a membrane associated protein that would be the intermediary between ComEC and ComFA. ComFA is clearly a soluble protein and interacts with more proteins downstream of the recombination process.

Moreover, ComF, which is a ComFC homolog, is expected to be interacting with ComEC in Gram-negative bacteria whereas ComFA is missing in those bacteria. This hypothesis could not be confirmed due to the toxicity of ComEC fused with T25/T18 and because the core domain or both N-terminal and C-terminal soluble domains of the protein are not sufficient to interact with ComFA or ComFC.

II. Structural model of ComFA and ComFC

Two proteins complexes structures, ComFA hexamer and ComFA/ComFC complex have been solved using electron microscopy. Also ComFA monomer and dimer structure were revealed by cross-linking mass spectrometry.

The purification of protein ComFA was performed as a soluble protein, because the protein neither has a signal peptide nor several transmembrane domains. Previous study (Londono-Vallejo and Dubnau, 1994a) of ComFA B. subtilis has shown than only a minor part of the protein is found in the soluble fraction, because the protein is highly unstable expressed alone. We found a way to prevent the precipitation during heterologous expression, with the SUMO tag that helps the solubilization of the protein.

However, the protein was expressed without tag when we co-expressed it with ComFC.

Somehow both proteins were stabilizing each other, leading us to think that inclusions bodies previously obtained were due to this missing protein and that ComFA required

ComFC to form a tight controlled complex. I was not possible to determine an atomic model of ComFA by X-ray crystallography because the protein did not show any sign of crystallization even in presence of DNA and/or ATPase inhibitor. The model proposed in this work is based on residue proximity determined by cross-linking mass spectrometry.

The determined model is atypical compared to other DEAD box helicase, but the linker of ComFA is longer than other and can allow this inversion. However the technic used may create uncertainty because the constraints proximity is around 20 Å and is expected size of the protein is 30 Å. A similar analysis of the N-terminal domain of the protein was done but due to the presence of few lysines, the specific structure could not be determined, but it would help us to understand protein-protein interaction. However the BacTH study did not highlight the formation of oligomer within N-terminal domain mentioned by previous studie (Londono-Vallejo and Dubnau, 1994b). In fact it seems that the dimeric of ComFA interacts orthogonally around the C-terminal domain based on the dimer modeling. A bigger structure is assembled, a hexamer that forms a ring like some helicase according to electron microscopy analysis. It was assumed that ComFA was a SF2 based on its putative activity but its structure tends to indicate that it is a hexameric helicase of the SF5 family. In order to solve the structure at a higher resolution, direct cross-linking has to be improved in order to obtain a higher protein concentration of ComFA. In fact with the GraFix technic the final concentration is around 0.001 mg.mL -1 , whereas a concentration of at least 0.1 mg.mL -1 is required for cryoelectron microscopy. Both techniques, GraFix and cross-linking, are introducing biais in the structure because of the cross-linker so an improvement of the complex stability without linker will allow the resolution of the native structure.

The structure determination of ComFC could not be determined neither by X-ray nor cross-linking mass spectrometry because of its extreme instability. The protein was equality distributed in the membrane and the soluble fraction, and none of these forms were purified in sufficient amount for further analysis. However we shown that ComFC was forming an oligomer according to the BacTH study, and the purification tends to demonstrate the formation of a dimer. A recommendation for future work would be to express the protein in a new system of expression such as S. pneumoniae in order to obtain the right posttranslational modification for a better behavior. Since the number of copy per cell is not known, an over-expression system might be mandatory.

Finally, the structure of the complex formed between ComFA and ComFC was obtained at a low resolution by negative stain electron microscopy. We propose a molecular model of a completely understudied complex with two hexamer assembled on the top of each other. The BacTH study allowed identifying that both C-terminal domains are involved in subdomain interaction, but the complex did not behave correctly during mass-spectrometry so the exact residues involved in the interaction could not be identified. As well as for the ComFA complex, the improvement of a direct cross-linking followed by a gel filtration is required to obtain a high-resolution structure by cryoelectron microscopy. At a resolution of 4 to 6 Å it will be possible to identity amino acid involved in the interaction. For both structure solved, we need to keep in mind that in vivo studies are essential because the oligomer conformation would be an artifact due to the protein concentration.

Further studies are currently performed on ComFA and ComFC. The NMR analysis of the N-terminal domain of ComFA, is planned to find out if the protein is properly folded and to solve the zinc-finger structure. Preliminary results show that only one third of the protein is folded. The purification protocol is now optimized for both complexes ComFA and ComFA/ComFC, but the optimization of the cryo-electron microscopy needs to be operated to finish the structural study of comF operon. As mentioned before, the pore channel was badly behaving when the protein is expressed to the Walker A, but no data were obtained by gel shift assay on the Walker B mutant because this construction was not stable enough to be tested. However the first mutant seems to bind the DNA more strongly than the wild type, which could be related to the ATPase or the helicase activity. In fact the ATP hydrolysis is suggested to be only involved in DNA release but not its binding. Though the protein can bind both type of DNA, the ATPase activity is only revealed with ssDNA its native substrate which disabled the last study on ComFA performed in vivo in Bacillus subtillus [START_REF] Takeno | Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis[END_REF]. The same study claimed that ComFA was only involved in the uptake velocity but was not paying the role of a translocase and/or helicase. We tested both hypotheses in vitro, first the translocase activity was confirmed and tends to prove that ComFA allows the DNA to be drawn is the cell. The protein has a key role in the mechanism of the DNA uptake as demonstrated by the fact that the comFA-null mutants are not able to incorporate the DNA anymore. ComFC is also mandatory for the uptake of the DNA, but its function remained unclear. A hypothesis was that the protein allows the midcell localization of the complex but no interaction with cell division system was found by BatCH. Now, we suspect that ComFC could be involved in the stabilization of other proteins such as

ComFA and ComEC. Also ComFC might be involved in the hypothetical pilus retraction, which would explain the absence of transformation in comFC-null mutant. Negative stain analysis of AD003 strain during competence will allow identifying if the pilus formation is modified or if the numbers of pilus increase.

Further studies are currently performed concerning the helicase activity, but preliminary data seems to show that the protein is not able to unwind dsDNA. It is consistent with the fact that ComFA is residing in the cytoplasm and only ssDNA is translocated though ComEC. The ATPase activity ssDNA-dependent shows that this energy drives the DNA pulled inside the bacteria and the Brownian motion can not integrate 90 nucleotide per second on his own [START_REF] Lee | Differential ATP requirements distinguish the DNA translocation and DNA unwinding activities of the Escherichia coli PRI A protein[END_REF] as proposed previously [START_REF] Takeno | Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis[END_REF].

Materials and methods

I

E. Strains and plasmids

All

II. Protein biochemistry

A. Protein expression in E. coli

Colonies from freshly transformed BL21 (DE3) cells were grown at 37°C at 180 rotation per minute (rpm) in 100 mL of Terrific broth (TB) with the appropriate selective antibiotic, either with ampicillin (Sigma-Aldrich) at a concentration of 100 μg.mL -1 or kanamycin (Sigma-Aldrich) at a concentration of 50 μg.mL -1 . After 3 hours, 4

L of TB were inoculated with the preculture and cultivated under the same condition. At OD600 = 0.8, protein cultures were placed at 16°C, 180 rpm for 30 min before protein expression induction overnight. Most of the plasmid are under T7 promoters (pET21, ppSUMO, pProEX) and were induced with Isopropyl β-D-1-thiogalactopyranoside (Sigma-Aldrich) at 1 μg.mL -1 . IBA plasmid with tet promoters were induced by andydrotetracycline (Sigma-Aldrich) at 200 μg.mL -1 . Finally pBAB plasmid were induced with 0.1% arabinose (Sigma-Aldrich).

B. Protein purification i. Soluble protein purification

Cells were harvested and resuspended in lysis buffer at the ratio 5 mL per grammes of cells. The basic composition of this buffer is 25 mM TRIS pH=8; 5 mM MgCl2; 0.130 mM CaCl2; lysozyme (Anatrace); 1.25 μg.mL -1 DNAse, protease inhibitor cocktail (Roche) and NaCl at 70 mM for complex purification or 700 mM for single protein purification. Other components were added depending on the purification such as reducing agents like TCEP or DTT, but also vanadate. After 30 min incubation cells were lysed using a cell disruptor at 15,000 PSI, 4°C. Lysate was cleared by two centrifugation 20,000 g for 15 min then 100,000g for 30min and passed on an affinity chromatography resin. Two types of resin were used TALON Metal Affinity Resin (Clontech) for Histagged protein of 5 mL Strep-Tactin Superflow (IBA) for Strep-tagged protein. Elution fractions were concentrated on a Millipore concentrator of different cut off, 100 kDa for the complex, 30 kDa for ComFA, 10 kDa for ComFC, and 3kDa for subdomains. The concentrated proteins were then purified by size exclusion chromatography on Superdex 75/200 (GE Healthcare) for single protein and Superose 12 (GE Healthcare) for the complex. Eluted fractions were ran on SDS-PAGE and used directly for microscopy study and activity assay or concentrated again for protein crystallization.

ii. Membrane protein purification

For membrane protein such as ComEC, membranes were pelleted by ultracentrifugation at 100,000 g for 45 min at 4°C. Membranes were resuspended in solubilization buffer (300 mM NaCl; 20 mM HEPES pH=8; 35 mM imidazole; 1% (w/v) CYMAL-6 (Anatrace)) using a potter. After 20 min incubation at 22°C the sample was centrifuged one last time at 100,000 g for 20 min at 4°C to remove unsolubilized material. The sample was then purified over affinity resin and size exclusion column with 0.04% w/v CYMAL-6 in the buffer to avoid precipitation of the protein.

iii. Cleavage of the tag

Protein expressed with the C-terminal His6-SUMO-tag were cleaved by adding 20 μg of 5 mg.mL -1 homemade SUMO protease after the affinity purification. The sample was incubated overnight à 4°C during dialyse in a 3 kDa pore membrane (Spectrum lab) in 400 mL of buffer (20 mM Tris pH=8; 500 mM NaCl; 1 mM TCEP) to dilute the imidazole. The cleaved sample was run against a NiNTA resin column and the cleaved protein in flow through was kept. The sample is further purified using the Superdex 200, Superdex 75 or the Superose 6 (GE Healthcare).

Membrane proteins tagged with the C-terminal His6-Mystic were cleaved on bed overnight with the homemade thrombin. The flow through containing the protein of interest was collected the next day and further purified by size exclusion column using the Superdex 200 (GE Healthcare).

iv. Membrane floating assay

During the purification of ComFC, half of the protein was found in the membrane fraction. In order to know if the protein was a membrane protein or only precipitated in the membrane a liposome floating assay was done. After cells lysing, the membranes were pelleted at 100,000g for 45 min. The membrane pellet was resuspended in 8 mL of solubilization buffer (20 mM TRIS pH=8, 20% (w/v) sucrose, 1% (w/v) LDAO) and passed 21 times through the extruder to form liposomes. A linear gradient from 20% to 60% (w/v) sucrose was made using the gradient maker, and 200 μL of the sample was

C. Structural analysis

Three different techniques were used in order to do solve the structure of ComFA, ComFC and the complex.

i. Protein crystallization

The screening of crystallization conditions and crystal optimization were Several compounds were added to the concentrated ComFA protein before crystallization screeing in order to prevent the ATPase activity of the protein or to fill the ATP binding pocket. Two ATP analogs (Jena Bioscience) were used ATPγS (Adenosine-5'-γ-thio-triphosphate, lithium salt) and AppCp (Adenosine-5'-[β,γmethyleno]-triphosphate, sodium salt) but also vanadate.

During the X-ray exposure, crystal would be degraded by these radiations because free radicals and ionization phenomena. Crystals need to be protected against this radiation damage; they are frozen at a temperature of -196°C in liquid nitrogen.

Cryoprotectant is also required to prevent water from freezing because it forms ice rings on diffraction patterns at multiple resolutions. Various cryoprotectants can be used as glycerol, methylpentanediol (MPD) or polyethylene glycol (PEG).

Data were collected with the help of Dr Chiara Rapisarda using the X-ray source at ESRF (European Synchroton Radiation Facility) the synchroton of Grenoble.

ii. Negative stain electro-microscopy

Homemade 400 mest copper grids coated with a thin carbon film were activated by glow-discharging. Due to the low protein concentration, 5 μL of the sample after GraFix cross-linking was incubated for 4 minutes on the grid, and washed with 0.1% (w/v) uranyl acetate solution (Merck) and dried on a filter paper. Transmission Electron Microscopy (TEM) analysis were carried out on a FEI Tecnai T12 for grids screening and on a FEI Tecnai F20 the data collection with a direct electron detector the Falcon II and the software EPU for automatic data collection.

Particles were picked semi automaticly using EMAN2 soflware and class averaging and structure resolution were solved using Relion

iii. Cross-linking mass spectrometry

The cross-linking study was performed by Martial Rey from the Mass Spectrometry facility in Institut Pasteur. Fresh proteins were cross-linked in solution with NNP9 [START_REF] Nury | A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis[END_REF] at room temperature or at 4°C, then digested by the trypsin. Peptides given were analyzed by mass spectrometry, and results were studied to identify cross-linked residues. Further bioinformatics study allowed modeling single protein folding and multi-protein complexes using the proximity constraints of 20 Å.

iv. Bacterial two-hybrid

The bacterial two-hybrid (BACTH) system is used to determine protein interaction in an E. coli strain. In this system the adenylate cyclase gene from Bordetella pertussis, is split into two domains T18 and T25 that cannot interact together. Proteins expected to be interacting are fused to these complementary domains, when the proteins are associed T25 and T18 fragments will be functionally active and leads to the synthesis of cyclic AMP. That signal will activate the transcription of the catabolic operon lacZ gene encoding for the β-galactosidase leading to a colony colored on MacConkey plates after 48 hours.

III. Activity assay

A. Transformation efficiency assay in Streptococcus pneumoniae

For transformation efficiency assay, competence in S. pneumoniae was induced at OD600 = 0.15 for 10 min at 37°C using 50 μg.mL -1 of Competence-Stimulating Peptide (CSP). Bacteria were incubated for 20 min at 30°C with 100 ng of DNA amplified from S.

pneumoniae R304 genomic DNA containing the streptomycin resistance gene str41.

Bacteria were plated in presence and absence of 100 μg.mL -1 streptomycin on Columbia Agar supplemented with 5% horse blood. Colonies were counted after an overnight growth at 37°C.

B. ATPase activity assay

An ATPase assay (Innova Bioscience) was performed to test ComFA activity in presence or absence of nucleic acids oligomer, following the protocole provided by the company. The hydrolysis of ATP by the protein released free phosphate, which was further titrated with colorimetric assay readout on a 96-wells plated reader at 600nm.

C. DNA binding assay

Random sequence of 20; 30 and 40 mer oligonucleotide single and double strained labeled with DY682 were used. Forward single strained oligomer labeled and unlabeled reverse sequence were order from Eurofins MWG operon. The labeled double stranded was generated by mixing 500 mM of each oligonucleotide in 10 mM Tris pH=8, 1 mM EDTA and annealed using a thermocycler (Biorad). The DNA was boiled for 3 min at 95°C then the temperature was slowly decrease from 1°C per minute until 25°C.

Several concentration of protein was incubated at room temperature for 20 min in the dark with 0.02 μM of oligonucleotide. The sample was then mixed with loading buffer (20 % w/v sucrose, 0.12% bromophenol blue) to be load on an EMSA gel at 8% acrylamide in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA). The gel was run for 1h50 at 80 V at 4°C and revealed by a Licor Odyssey scanner.

Annexes

Annexe 1: Scientific publication published

As mentioned in the chapter "Confirmation of the shape of transformation pilus in S.

pneumoniae" I participated to the following work that as being published. A mutation of S. pneumoniae was performed to delete the gene adhE, in order to study the impact on the transformation efficiency and to investigate the presence the pilus.

L'appareil de translocation de l'ADN impliqué chez

The DNA translocation apparatus involved in Streptoccoccus pneumoniae

Summary:

Bacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S.

pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.

In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome.

In addition, heterologous expression of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA.

  expression in E. coli of several construct from various species ... B. Purification optimization ........................................................................................................... i. Gel filtration purification ........................................................................................................ II. Protein biochemistry ................................................................................. A. Protein expression in E. coli .................................................................................................. B. Protein purification ................................................................................................................... i. Soluble protein purification ............................................................................................... ii. Membrane protein purification ....................................................................................... iii. Cleavage of the tag ............................................................................................................... iv. Membrane floating assay .................................................................................................. v. Limited proteolysis ............................................................................................................... vi. Oligomerisation state .......................................................................................................... vii. GraFix cross-linking ........................................................................................................... C. Structural analysis ..................................................................................................................... i. Protein crystallization ........................................................................................................... ii. Negative stain electro-microscopy ................................................................................ iii. Cross-linking mass spectrometry .................................................................................. iv. Double hybrid ........................................................................................................................ III. Activity assay .............................................................................................. A. Transformation efficiency assay in Streptococcus pneumoniae ............................. B. ATPase activity assay ............................................................................................................... C. DNA binding assay ..................................................................................................................... References ............................................................................................ Annexes .................................................................................................

Figure 1 :

 1 Figure 1: Transduction process .................................................................................................................

Figure 2 :

 2 Figure 2: Conjugation process ....................................................................................................................

Figure 3 :

 3 Figure 3: Natural transformation process ............................................................................................

Figure 4 :

 4 Figure 4: Pneumococcal virulence factors ............................................................................................

Figure 5 :

 5 Figure 5: Natural transformation peak in S. pneumoniae culture ...............................................

Figure 6 :

 6 Figure 6: Competence state in S. pneumoniae .....................................................................................

Figure 7 :

 7 Figure 7: Protein recruitment for D-loop formation involved the homologous recombination ...................................................................................................................................................

Figure 8 :

 8 Figure 8: Comparison of natural transformation in different bacterial species ...................

Figure 9 :

 9 Figure 9: Transformasome model in V. cholerae ................................................................................

Figure 10 :

 10 Figure 10: Comparison with T4P system involved in twitching motility ................................

Figure 11 :

 11 Figure 11: Transformasome model in S. pneumoniae ......................................................................

Figure 12 :

 12 Figure 12: Identification of binding, degradation and internalization of DNA in wild type and null mutant strains .................................................................................................................................

Figure 13 :

 13 Figure 13: Comparison of comE operon .................................................................................................

Figure 14 :

 14 Figure 14: Prediction domain of the pore channel ComEC ............................................................

Figure 15 :

 15 Figure 15: Comparison of comF operon .................................................................................................

Figure 16 :

 16 Figure 16: Prediction domain of the DEAD like helicase ComFA ................................................

Figure 17 :

 17 Figure 17: Sequence and structural organization of the helicase SF2 proteins ...................

Figure 18 :

 18 Figure 18: Sequence alignment between ComFA and DnaT .........................................................

Figure 19 :

 19 Figure 19: Prediction domain of ComFC ................................................................................................

Figure 20 :

 20 Figure 20: Bacterial two-hybrid assay on ComFA and ComFC ....................................................

Figure 21 :

 21 Figure 21: Bacterial two-hybrid assay on ComFA and ComFC full length and their subdomains ................................................................................................................................................................

Figure 22 :

 22 Figure 22: Bacterial two-hybrid assay on ComFA and ComFC sub-domains ........................

Figure 23 :

 23 Figure 23: Bacterial two-hybrid assay on ComFA and ComFC with ComEC ..........................

Figure 24 :

 24 Figure 24: Bacterial two-hybrid assay comF locus with ComEC domain ................................

Figure 25 :

 25 Figure 25: Bacterial two-hybrid assay ComEC domain ...................................................................

Figure 26 :

 26 Figure 26: Bacterial two-hybrid assay of ComEA and ComEC domain ....................................

Figure 27 :

 27 Figure 27: Bacterial two-hybrid assay on ComFA and ComFC with ComGA .........................

Figure 28 :

 28 Figure 28: Model of the DNA uptake apparatus in S. pneumoniae .............................................

Figure 29 :

 29 Figure 29: Bacterial two-hybrid assay on ComFA and ComFC with the protein of the homologous recombination ........................................................................................................................

Figure 30 :

 30 Figure 30: Model of the transformasome in S. pneumoniae ..........................................................

Figure 31 :

 31 Figure 31: Bacterial two-hybrid assay on ComFA and ComFC with GpsB and spr1506 ..

Figure 32 :

 32 Figure 32: Model of transformasome and cell division in S. pneumoniae ...............................

Figure 33 :

 33 Figure 33: Transmembrane domain of ComEC ...................................................................................

Figure 34 :

 34 Figure 34: Sequence alignment of ComEC proteins ..........................................................................

Figure 35 :

 35 Figure 35: Text expression of ComEC from Geobacillus species .................................................

Figure 36 :

 36 Figure 36: Purification of ComEC-Core ..................................................................................................

Figure 37 :

 37 Figure 37: Sequence alignment of ComFA proteins ..........................................................................

Figure 38 :

 38 Figure 38: Text expression of ComFA G. thermodenitrificans .......................................................

Figure 39 :

 39 Figure 39: Purification of ComFA ..............................................................................................................

Figure 40 :

 40 Figure 40: Trypsin digestion assay ..........................................................................................................

Figure 41 :

 41 Figure 41: ComFA complex analysis by cross-linking .....................................................................

Figure 42 :

 42 Figure 42: ComFA complex analysis by GraFix ...................................................................................

Figure 43 :

 43 Figure 43: Purification of ComFA Walker A mutant .........................................................................

Figure 44 :

 44 Figure 44: Purification of ComFA N-terminal domain .....................................................................

Figure 45 :

 45 Figure 45: Oligomeric state of ComFA N-terminal domain ...........................................................

Figure 46 :

 46 Figure 46: Spectrum of ComFA N-terminal domain .........................................................................

Figure 47 :

 47 Figure 47: Molecular weight determination of ComFA complexes ............................................

Figure 48 :

 48 Figure 48: Spectrum of the trimer sample of ComFA full-length ................................................

Figure 49 :

 49 Figure 49: ComFA structure determination by cross-linking mass spectrometry analysis ..................................................................................................................................................................................

Figure 50 :

 50 Figure 50: ComFA dimer structure determined by cross-linking mass spectrometry analysis .................................................................................................................................................................

Figure 51 :

 51 Figure 51: Sequence alignment of ComFC proteins ..........................................................................

Figure 52 :

 52 Figure 52: Purification of ComFC ..............................................................................................................

Figure 53 :

 53 Figure 53: ComFC crystals ...........................................................................................................................

Figure 54 :

 54 Figure 54: Test expression of comF operon .........................................................................................

Figure 55 :

 55 Figure 55: Complex ComFA/ComFC purification and negative stain observation .............

Figure 56 :

 56 Figure 56: ComFA and ComFC complex analysis by GraFix ..........................................................

Figure 57 :

 57 Figure 57: In vitro reconstruction of complex .....................................................................................

Figure 58 :

 58 Figure 58: ComFA and ComFC complex structure resolution ......................................................

Figure 59 :

 59 Figure 59: comF-null mutants study .....................................................................................................

Figure 60 :

 60 Figure 60: Prediction of nucleotide binding site .............................................................................

Figure 61 :

 61 Figure 61: Gel shift assay on dsDNA and ssDNA .............................................................................

Figure 62 :

 62 Figure 62: Gel shift assay on different mer ........................................................................................

Figure 63 :

 63 Figure 63: ATPase activity assay with several substrates ..........................................................

Figure 64 :

 64 Figure 64: ATPase activity assay with ssDNA of different length ............................................

Figure 65 :

 65 Figure 65: Translocation activity assay ..............................................................................................

Figure 66 :

 66 Figure 66: Structure of a type IV pilus versus a type II secretion pseudopilus-like ........

Figure 67 :

 67 Figure 67: Plated filament next to a type IV pilus ...........................................................................

Figure 68 :

 68 Figure 68:Plaited filament in pilus-deficient pneumococcal strains ......................................

Figure 69 :

 69 Figure 69: Plated filament in non-induced R1501 cells ...............................................................

Figure 70 :

 70 Figure 70: Identification of AdhE spirosomes ..................................................................................

Figure 71 :

 71 Figure 71: Protein expression during induction .............................................................................

Figure 72 :

 72 Figure 72: AdhE-null mutant study .......................................................................................................

Figure 73 :

 73 Figure 73: Improved model of the DNA-uptake machinery of naturally competent S. pneumoniae .....................................................................................................................................................

Figure 74 :

 74 Figure 74: Cyro-fixation of S. pneumoniae cell .................................................................................

  ComFA or ComFC. Therefore, we focused on the channel ComEC. The topology of the Nterminal transmembrane domain of the protein is unknown, so I decided to remove the seventy-four first amino acids of ComEC encoding a potential transmembrane segment to insure that the moieties T18 and T25 fused to this domain are expressed inside of the bacterium. The partial full-length protein was amplified but could not be cloned due to its toxicity (Figure23.B), so we only cloned its core domain (222-481) and its two soluble domains, N-terminal domain (residues 75-220), and the C-terminal domain (480-746) (Figure 23.A). PCR screening and sequencing confirmed the sequence of the clone (pAD048/49/50/51/52/53/62/63/64, material et method Table

  For example the Walker A motif of ComFA was mutated. A Strep-tag was inserted at the C-terminus of ComFA (pAD026) or a His-tag at the C-terminus of ComFC but unfortunately the expressed proteins were precipitating. The last construct designed contained the N-terminal domain of ComFA and ComFC full-length. The goal was to investigate the domain of interaction between both proteins, but E. coli cells were dying right after IPTG induction, indicating the toxicity of the construct.

  sample was then passed throught a TALON-resin and ran on size-exclusion chromatography (Figure 55.A). The purity of the sample reached more than 95 %, because only ComFA at 50 kDa and ComFC at 25 kDa are visible on the SDS-Page gel after Comassie staining (Figure 55.B). The sample molecular mass is about 550 kDa according to the gel filtration calibration, and was big enough to be studied by negative stain electron microscopy. Large complexes were observed, and two mains shapes are observed: some ring-shaped and rectangle-shaped objects (Figure 55.C). According to the observation of the complex we assumed that a hexamer is formed, alike ComFA hexamer. A SYPRO stain allows the determination the protein stoichiometry indicating a ratio of 1:1 between ComFA and ComFC. With this stoichiometry an hexamer of the

Figure 60 :

 60 Figure 60: Prediction of nucleotide binding site Alignment of the Helic C domain of ComFA and cd00079 sequence cluster of Helic C family. The recognition motif of binding is shown in highlighted letters.

  in E. coli DNA sequences of ComFA, ComFC or ComEC were amplified from genomic DNA of Streptoccoccus penumoniae strain R800, Geobacillus thermodenitrificans or Geobacillus thermoleovorans and inserted in commercial plasmids using restriction enzymes sites.Plasmids were transformed in E. coli Top10 and plaited on selective antibiotic Luria Broth (LB) agar plate with 100 μg.mL -1 ampicillin (Sigma-Aldrich) or 50 μg.mL -1 kanamycin (Sigma-Aldrich). After an overnight growth, clones were screened by PCR of the insert, and only the positive clones were cultivated during 6 hours at 37°C in 4 mL of selective LB in order to extract the plasmid using Qiagen kit. The sequence of the plasmid was confirmed by sequencing (Eurofins MWG) using primers covering the inserted sequence. Once the sequence was validated, the plasmid was transformed in BL21 DE3 cells for test expression.B. Cloning in S. pneumoniaeNull-mutants strains were made by transformation of R1501 strain with a DNA cassette of kanamycin resistance gene from the plasmid pR411 instead of the gene. Different cassettes were designed using PCR overlapping of three fragments with the amplification of the kanamycin resistance gene, and the two other fragments corresponding to 1000 base pairs fragment of the genomic region upstream and downstream of the genre of interested. Thanks to the complementarity of the design primers, the three PCR products were assembled by overlap extension PCR and the purified DNA was used for transformation of pre-competent S. pneumoniae R1501 cells.Null-mutants were positively selected by overnight growth on Columbia agar with 5% horse blood and 250 μg.mL -1 kanamycin. Clones were PCR screened then plated again for sub-cloning on two plates with and without antibiotic before validation by DNA sequencing.C. Single point mutationPoint mutation of the protein were made in order to determine the function of predicted active sites like Walker A and B motif of ComFA, by inserting different mutations and selecting the most stable mutant. Primers complementary with 18 base pairs upstream and downstream of the mutation site were used to amplify the selected plasmid by PCR. The product was purified and digested by the enzyme DpnI (Thermo Scientific) to eliminate the template plasmid. The linearized plasmid was then transformed in E. coli Top10 cells, recircularized in the bacteria and selected with the associated antibiotic and positive clones were confirmed by sequencing.D. Sub-domain cloningProteins sub-domains were analyzed with bioinformatical tools and cloned independently for crystallization assay. Templates used were the full plasmid, and amplified by PCR except the domain to remove. The extremities 5' end were phosphorilyzed with the T4 polynucleotide kinase (New England Biolabs) to allow the recircularization of the plasmid by the T4 DNA ligase (Thermo Scientific). The plasmid was then transformed in E. coli Top10 cells and selected with the associated antibiotic and positive clones were confirmed by sequencing.

  performed with the help of Ahmed Haouz, Patrick Weber and Cedric Pissis from the Crystallography facility at Institut Pasteur. The technique used was the vapor diffusion by hanging drop. It is based on the thermodynamic properties which generate a balance by diffusion solvent vapor. At the beginning, a drop of protein was diluted with different ratios with a solution which contains precipitant(s). This drop is placed in a well hermetically closed with a large volume of the precipitant solution. The plates were incubated at 18°C or 4°C for a slow return to equilibrium by evaporation of the water and other volatile components. The decrease of the volume of the drop at the same time leads to increase the concentration of protein and precipitant(s) and reach the super saturation. Commercial kits such as Jena Bioscience or Hampton research were used with the Mosquito robot for large screening, then reproduced and optimized in bigger volum to obtain crystals of sufficient size and quality of diffraction.

Table 1 : Sequence identities of ComEA proteins Proteins

 1

	S. pneumoniae ComEA	42 %	49 %	53 %	40 %
	B. subtilis ComEA	-	30 %	33 %	28 %
	N. gonorrhoeae ComE1		-	53 %	71 %
	V. cholerae ComEA			-	46 %

B. subtilis N. gonorrhoeae V. cholerae T. thermophilus identities of ComEA proteins from the naturally transformable S. pneumoniae, B. subtilis, N. gonorrhoeae, V. cholerae and T. thermophilus.

Table 4 : Sequence identitites of ComFC proteins Proteins

 4

identities of proteins from the naturally transformable S. pneumoniae, B subtilis, H. influenza and Synechocystis sp.

Strain PCC 6803.

Table 6 : Bacterial Strains

 6 S. pneumoniae strains and E. coli plasmids used during this thesis are listed in the following tables. Most of the plasmids are made with S. pneumoniae R800 genomic DNA.

	Organism	Strain number	Genotype	Reference
	S. pneumoniae	R800	R6 derivative	(Lefevre et al., 1979)
		R1501	R800, but ΔcomC	(Dagkessamanskaia et al., 2004)
		AD001	R1501, but ΔadhE;	(Laurenceau et al., 2015)
			KanR

Table 7 :

 7 Plasmids

Streptoccoccus pneumoniae

  RésuméLa transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques.Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèses et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFA.

Acknowledgments

First among all the scientists I met during my PhD, I want to give warm thank you to Rémi Fronzes for allowing me to perform my PhD research under his direction. He taught me all the different techniques related to structural biology and kindly enabled me to learn electron microscopy single particle analysis at international workshops.

I want to thank Annick Dujeancourt who is a tremendous laboratory manager that helped me a lot all over my three years of work.

Many thanks to Francesca Gubellini, Chiara Rapisarda, Sahra Ouarti, Raphaël Laurenceau, Thomas Perry, Cédric Pissis, Quentin Merel, Esther Marza, Matteo Tassinari, Eric Durand and Petya Violinova Krasteva, all brilliant researchers that have been very generous in sharing their experience with me. I thank my friend and collaborator Gerard Pehau-Arnaudet, for guiding my first steps and mentoring me in the art of electron microscopy. I thank Hannah Foster and Briana Burton. They have been invaluable collaborators for the activity assay performed to complement my work. I thank all the other collaborators to this work: Martial Rey, Christian Malosse and Julia Chamot-Rooke from the mass spectrometry facility; and Calum Johnston and Patrice Polard from Toulouse University. They have been researchers that I enjoyed to work with, whether the outcome of several projects over my work.

I thank Olivera Francetic, my tutor, for his kind and insightful guidance. I thank also Frederic Saul for his for corrections of languages he has brought to my manuscript.

Finally I want to thank my family and friends who pushed me forward all along these 3 years.

pAD045 comFC in pUT18C ColE1, AmpR This study pAD046 comFC in pKNT25 ColE1, KanaR This study pAD047 comEC (75-222) in pUT18C ColE1, AmpR This study pAD048 comEC (75-222) in pUT18 ColE1, AmpR This study pAD049 comEC (75-222) in pKNT25 ColE1, KanaR This study pAD050 comEC (480-746) in pUT18C ColE1, AmpR This study pAD051 comEC (480-746) in pUT18 ColE1, AmpR This study pAD052 comEC (480-746) in pKNT25 ColE1, KanaR This study pAD053 comEC (480-746) in pKT25 ColE1, KanaR This study pAD054 comFA (2-72) in pUT18C ColE1, AmpR This study pAD055 comFA (72-432) in pUT18 ColE1, AmpR This study pAD056 comFA (2-72) in pKT25 ColE1, KanaR This study pAD057 comFA(72-432) in pKT25 ColE1, KanaR This study pAD058 comFC (2-93) in pUT18 ColE1, AmpR This study pAD059 comFC (93-220) in pUT18 ColE1, AmpR This study pAD060 comFC (2-93) in pKNT25 ColE1, KanaR This study pAD061 comFC (93-220) in pKNT25 ColE1, KanaR This study pAD062 comEC (222-481) inpUT18C ColE1, AmpR This study pAD063 comEC (222-481) inpKT25 ColE1, KanaR This study pAD064 comEC (222-481) in pKNT25 ColE1, KanaR This study pAD065 comEA in pUT18C ColE1, AmpR This study pAD066 comEA in pKT25 ColE1, KanaR This study pAD067 comEA in pKNT25 ColE1, KanaR This study pAD068 spr1506 in pUT18 ColE1, AmpR This study pAD069 spr1506 in pUT18C ColE1, AmpR This study pAD070 spr1506 in pKT25 ColE1, KanaR This study pAD071 spr1506 in pKNT25 ColE1, KanaR This study pAD072

before. Unlike ComFA, the position of the T18 moiety does not disturb the interaction between ComFC subunits with the construct ComFC-T25. Therefore, the plasmids used for ComFC expression are designed to introduce a tag at the N-terminus or C-terminus of the protein.

The most interesting result of this set of experiment is the detection of interactions between ComFA and ComFC. Various combinations between ComFA and ComFC were tested and all showed interactions between both proteins, as shown by the pairs ComFA-T18/ComFC-T25 or T25-ComFA/T18-ComFC. Again, the intensity depends on the position of the T25 and T18 moieties. Again, this point was crucial to define coexpression and co-purification strategies. The discovery of a potential ComFA/ComFC interaction is very interesting because it is assumed in the field that ComFA does not require other proteins of the competence system for its stability or its activity, nevertheless ComFC seems to be an important partner.

B. Identification of interaction between sub-domains of comF genes i. Domain predictions for ComFA and ComFC

Several fold recognition servers such as HHpred and Phyre2 identified several domains in ComFA. At its N-terminus, ComFA would contain a zinc finger domain, which could either be a protein-protein interaction region (Londono-Vallejo and Dubnau, 1994b)

ii. Bacterial Two-Hybrid assay

First, full-length proteins were tested with each sub-domains (Figure 21.C). As we noticed that some interactions seem to be prevented by the fused T25 and T18. We only tested the positive constructs in order to determine if the region of interaction was the N-or the C-terminal domain.

added to the top of the gradient. After an ultracentrigation of 60,000 g for 15 hours, the sample was collected and run on a gel. If the protein is present around 30% (w/v) sucrose it means that the protein in the liposome and so it is a membrane protein, otherwise the protein is only precipitated in the membrane.

v. Limited proteolysis

Limited proteosysis with the tryspin protease (Sigma-Aldrich) was used to identify sub-domains of ComFA that could be more likely to crystallize. The protein was incubated for 30 min at 4°C with the protease at a ratio 1:1500. The reaction was stopped by adding a 1 μg.mL -1 protease inhibitor cocktail (Roche) and the sample was purified on a Superdex 75 (GE Healthcare).

vi. Oligomerisation state

The oligomerisation state of each purified proteins, ComFA and ComFC, were tested by multi-angle static light scattering (MALS). The sample was run over on a Superdex 200 Increase (GE) to separate different conformation based on the size and shape. The molar mass of each peak is determined and the oligomerique state is then calculated based on the molecular weight of a single protein.

vii. GraFix cross-linking

Complex of interest were not stable enough for structural analysis, the GraFix (Gradient Fixation) method was used. After a gel filtration the fraction containing the hexamer was put on a top 4.4 mL tube containing a gradient of glycerol from 10% to 30% (w/v) glycerol and 0 to 0.1% glutaraldhehyde. The sample was gently cross-linked and purified over density gradients for 14 hours by ultracentrifugation at 150,000 g. The complex of interest contained at 0.05% glutaraldehyde fraction was collected and directly fixed on grids for negative stain electro-microscopy analysis.