Keywords: Spirosomes, Cryo-electron microscopy, Alcoholic fermentation, Alcoholacetaldehyde dehydrogenase, AdhE, Filamentation Spirosomes, Cryo-electron microscopy, Alcoholic fermentation, Alcoholacetaldehyde dehydrogenase, AdhE, Filamentation

Acetaldehyde-alcohol dehydrogenase (AdhE) enzymes is a key metabolic enzyme in bacterial physiology and pathogenicity. They are responsible for the conversion of acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in absence of oxygen. This two-step reaction is associated to NAD + regeneration, essential for the glycolysis. This protein is composed of two domains: the N-terminal acetaldehyde dehydrogenase (ALDH) domain linked to the C-terminal alcohol dehydrogenase (ADH) domain. This bifunctional AdhE enzyme is well-conserved in all bacterial kingdoms and also in more phylogenetically distant microorganisms such as green microalgae. AdhE is well-studied for its involvement in biofuel production, but as well for its role in bacterial pathogenicity. This enzyme is commonly found as an oligomeric form called spirosomes. Despite the great interest in the AdhE enzyme, the structure and function of these filaments remain unknown since their discovery in the 1970's. Depending on ligands incubated with, Escherichia coli spirosome are found as two different conformational states. During this thesis, the structure of the extended and the compact conformation have been solved at high-resolution using cryo-electron microscopy. Based on these structures, AdhE mutants have been designed in order to disrupt the filamentation and study its role. We have shown that spirosomes contain active AdhE monomers and the AdhE filamentation is essential for its activity in vitro and in vivo. Again based on the obtained structure of the extended conformation, we identified that a channel within the filament which could be responsible for the channeling of the toxic acetaldehyde intermediate. Like other examples of metabolic enzyme filamentation or micro-compartments encapsulating dehydrogenases, spirosomes are a form of bacterial compartmentalization. In order to look deeper in this compartmentalization, immunolabelling experiments have been performed in order to localize AdhE filaments inside the bacteria. Observations revealed a peripheral localization for E. coli AdhE wild-type and a diffuse localization for the nonfilamentous AdhE. These differences are not understood but might suggest a difference of function between oligomeric and monomeric form.

The structure of the Streptococcus pneumoniae spirosome has also been solved using cryo-EM. Compared to E. coli spirosomes, AdhE filaments from S. pneumoniae have always been purified as extended conformation. S. pneumoniae spirosomes seem to be naturally found in extended conformation and so active form. This could be due to differences present in the two homologous AdhE primary sequences.

The determination of the E. coli and S. pneumoniae structures at high-resolution and the analysis of the function reveal that these filaments are required for bacterial physiology in absence of oxygen. The study of the regulation of spirosome activities might lead to the discovery of molecules stimulating or inhibiting conformation change in E. coli AdhE filaments. These molecules would present a great interest in biotechnology or to prevent pathogenic anaerobes.

Titre : Structure et fonction des spirosomes bactériens

Résumé : L'enzyme acétaldéhyde-alcool déshydrogénase (AdhE) est une enzyme clé dans la physiologie et pathogénicité bactérienne. Cette enzyme est responsable de la conversion de l'acétyl-CoA en acétaldéhyde puis en éthanol au cours de la fermentation alcoolique en absence d'oxygène. Ces deux réactions sont associées à la régénération du NAD + qui est essentiel pour la glycolyse. AdhE est composée de 2 domaines : le domaine Nterminal acétaldéhyde déshydrogénase (ALDH) et le domaine C-terminal alcool déshydrogénase (ADH). Cette enzyme bi-fonctionnelle est très conservée dans le règne bactérien et également chez d'autres micro-organismes tels que des algues vertes. L'enzyme AdhE est étudiée pour son implication dans la production de biocarburants mais également pour son rôle dans la pathogénicité bactérienne. Cette enzyme est communément retrouvée sous sa forme oligomérique appelée spirosome. Malgré l'intérêt porté au monomère AdhE, la structure et la fonction des filaments restent inconnues depuis leur découverte dans les années 1970. En fonction des ligands présents, les spirosomes d'Escherichia coli peuvent être retrouvés dans 2 états distincts. Durant cette thèse, les structures de la conformation étendue et compacte ont été résolues à haute résolution par cryo-microscopie électronique. A partir des structures obtenues, des mutants d'AdhE ont pu être générés afin d'empêcher la filamentation et ainsi étudier son rôle. Nous avons montré que les spirosomes contiennent des monomères d'AdhE actifs et que la filamentation est essentielle à son activité in vitro et in vivo. Lors de l'analyse de la structure étendue, nous avons identifié un canal dans le spirosome qui serait responsable de la canalisation de l'intermédiaire toxique qu'est l'acétaldéhyde. Comme d'autres exemples de filamentation d'enzymes ou de micro-compartiments enfermant des déshydrogénases, les spirosomes sont une forme de compartimentation bactérienne. Dans le but de l'étudier plus en détails, des expériences d'immuno-marquages ont été réalisées afin de localiser les filaments d'AdhE à l'intérieur de la bactérie. Ces observations ont révélé une localisation périphérique de la protéine sauvage d'AdhE alors que la protéine mutante non-filamentaire présente une localisation diffuse. Même si cette observation ne peut encore être expliquée, elle pourrait suggérer une différence de fonction entre la forme oligomérique et monomérique.

La structure des spirosomes de Streptococcus pneumoniae a aussi été résolue par cryo-ME. Ces filaments sont toujours purifiés sous leur forme étendue. Cette structure est hautement similaire à celle obtenue pour E. coli dans leur conformation étendue. Les spirosomes d'S. pneumoniae semblent être naturellement stabilisés en conformation étendue et donc active. Ceci pourrait être expliqué par les différences des séquences primaires des deux protéines homologues.

La détermination des structures des spirosomes d'E. coli et S. pneumoniae à haute résolution puis l'étude de la fonction révèlent que ces filaments sont indispensables à physiologie bactérienne en absence d'oxygène. L'étude de la régulation de l'activité des spirosomes pourrait mener à la découverte de molécules stimulant ou inhibant le changement de conformation des filaments d'AdhE de E. coli. Ces molécules pourraient présenter un intérêt en biotechnologie ou dans le but d'inhiber la croissance de micro-organismes anaérobies pathogènes.

Mots clés : Spirosomes, Cryo-microscopie électronique, Fermentation alcoolique, Alcoolacétaldéhyde déshydrogénase, AdhE, Filamentation

Unité de recherche

Microbiologie Fondamentale et Pathogénicité, UMR 5234, 2 rue robert escarpit 33600 Pessac

Acknowledgments

Before anything else, I would like to thank all the members of the jury Dr. Axel Magalon, Dr. Andrea Dessen, Dr. Celia Plisson-Chastang and Dr. Julien Bergeron for making this defense exciting and stimulating.

I would like to express my sincere consideration for my supervisor Dr. Remi Fronzes for giving me the opportunity to start this project.

Through your passion for science, you inspired me in the way of deeply understanding bacterial nanomachines. Thank you for sharing your expertise with high generosity.

You introduced me to the art of solving atomic structures with real enthusiasm and pushed me give to the best of me. You guided me into how to develop my analytical and critical mind. I am very grateful for having you as a mentor.

I could not have hoped for more kindness from Esther Marza. With your pedagogy and your patience, you knew how to ensure that I would be autonomous quickly. Even with your teaching, you were always available and took time for teaching and mentoring me. I am sure that all these qualities will be highly appreciated by the future members of this lab.

I want to acknowledge Chiara Rapisarda and Thomas Perry. You were the best colleagues for starting this new adventure. I really enjoyed to be part of the beginning of the IECB lab with you. Chiara, nobody helped me as you did and gave me faith in myself. I am very proud of worked with a such talented scientist. I admire the way you live the science and the invaluable energy you put into testing so many hypotheses.

With eternal good humor, Thomas you have made these years very easy to live. I really enjoyed sharing science with you two talented scientists.

I thank Marion Decossas for introducing me to electron microscopy and Armel Bezault for helping during spirosome collects. And both of you for your support.

I also want to thank people who helped me during this project, Laurent Terradot for the collaboration in its project but also for your interesting advices and your support and Claire Stines-Chaumeil for your suggestions.

I also want to thank all the members of the lab, Jean-Paul Bourdineaud whose conversations were so interesting. Robin Anger, Esther Gavello-Fernandez, Nina

Lopez-Lozano, Pierre Nottelet for your help and for the good mood that reigned in this lab. Leonardo Talachia Rosa for your trust in myself and your big support. Thibaud

Renault for your support. And I would like to express my sincere thank you for Laure Bataille, so motivated.

Last but not the least, my special thanks to Nadia El Mammeri for your unconditional and sincerely consideration.

Thank you.

List of figures

INTRODUCTION

Chapter I: Anaerobic life

I. Anaerobes

Anaerobic micro-organisms are among the most ancient form of life capable to survive under molecular oxygen-free atmosphere. Anaerobic life is at the origin of the development of micro-organisms which were able to evolve in absence of oxygen, acquiring novel capabilities allowing the emergence of divergent microbial lineages [START_REF] Martin | Early Microbial Evolution : The Age of Anaerobes[END_REF]. They were able to colonize many numerous environments from soils or air to human body.

Anaerobic micro-organisms can be classified in different categories based on their oxygen tolerance. Strict anaerobes cannot tolerate molecular oxygen while moderate anaerobes are able to grow in presence of low concentration of oxygen. Aerotolerant anaerobes do not use oxygen even when it is present in the environment. Facultative anaerobic micro-organisms are highly versatile, they are able to switch their metabolism between anaerobic and aerobic allowing to survive under anoxic condition.

This diversity of anaerobic organisms is dependent on the expression of enzymes such as superoxide dismutase, catalase and peroxidase [START_REF] Hentges | Medical Microbiology[END_REF]. These proteins are responsible of the detoxification of oxygen radicals generated during aerobic metabolism [START_REF] Ighodaro | First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid[END_REF].

II. Metabolism without oxygen

A. Energy sources

All the functions performed in a biological cell require energy. This energy can be provided by the photosynthesis, the fermentations and the respiration depending on cells, substrates and environmental conditions. During aerobic respiration, the final electron acceptor of the electron transport chain is molecular oxygen. These oxidationreduction reactions are coupled with the generation of an electrochemical gradient across the inner membrane. This proton motive force is used to ATP synthases for converting ADP into ATP.

But in absence of molecular oxygen, other compounds such as nitrate, sulfate or carbon compound like fumarate can also be used as final electron acceptor, it is called anaerobic respiration [START_REF] Kroger | Fumarate respiration of Wolinella succinogenes : enzymology, energetics and coupling mechanism[END_REF]. Usually compared to O2, aforesaid molecules imply that the final ATP yield is lower from 36 to 2 ATP.

With the exception of acetogenic and methanogens organims which can conserve energy and produce acetyl-coenzyme A associated with the CO2 fixation (Wood-Ljungdahl pathway) [START_REF] Ragsdale | Acetogenesis and the Wood -Ljungdahl pathway of CO2 fixation[END_REF]. In absence of oxygen or other final electron acceptors or when organisms are not able to use respiration pathways, fermentation is the only pathway to produce energy in order to survive which was described as "C'est la vie sans air, c'est la vie sans gaz oxygène libre " by Pasteur in 1876 [START_REF] Pasteur | Etudes sur la bière, ses maladies, causes qui les provoquent, procédé pour la rendre inaltérable, avec un théorie nouvelle de la fermentation[END_REF].

a. Fermentations

The first step called glycolysis, is the conversion of carbohydrates into pyruvic acid associated to the generation of adenosine triphosphate (ATP) and the reduction of nicotinamide adenine dinucleotide (NAD) cofactor. After uptake, the glucose is converted into pyruvate during the glycolysis. This metabolic pathway generates the production of 2 ATP and 2 NADH. The second step is the re-oxidation of NADH into NAD + required for glycolysis. The recycling of NAD + can be done by several metabolic pathways ending up to unique or many end-products. In the middle of the XIXth century, Louis Pasteur investigated fermentation processes and understood that different ferments were responsible for different types of fermentation. Anaerobes are present in all three domains of life: bacteria, archaea, eukaryotes.

Eukaryote fermentative micro-organisms such as Saccharomyces cerevisiae are wellknown to switch their metabolism to react to changing environmental conditions. Yeast fermentation metabolism is highly studied for its contribution to the production of beverages or bakery products like wine making or bread production.

Other micro-organisms like anaerobic or facultative bacteria are able to ferment in order to keep growing under anoxic condition. In this thesis, I will focus on bacterial fermentation that lead to ethanol production.

b. Acid mixed fermentation (Escherichia coli -Streptococcus pneumoniae)

Escherichia coli is a Gram-negative facultative anaerobic bacterium which is able to survive in absence of oxygen by converting sugars to a mixture of products. This process is called mixed fermentation. E. coli bacteria accept many carbohydrate sources such as glucose, galactose, mannose, arabinose ... and produce also a mixture of compounds under anoxic conditions as presented in the Figure 2. E. coli bacteria mainly employ the Embden-Meyerhof-Parnas (EMP) glycolytic pathway to convert glucose into pyruvate [START_REF] Hollinshead | Biotechnology for Biofuels Examining Escherichia coli glycolytic pathways , catabolite repression , and metabolite channeling using Δ pfk mutants[END_REF]. Pyruvate can be metabolized to acetyl-CoA and formate by the pyruvate-formate lyase (PFL) enzyme. Then, lactate dehydrogenase, acetaldehyde-alcohol dehydrogenase, malate dehydrogenase, fumarate reductase enzymes regenerate the NAD + necessary for maintaining the NAD + /NADH ratio required for the glycolysis. This type of fermentation leads to the production of different end-products. From [START_REF] Jiang | Regulation of the ldhA gene , encoding the fermentative lactate dehydrogenase of Escherichia coli[END_REF] Streptococcus pneumoniae is a Gram-positive facultative anaerobic bacterium, which is able to metabolize 32 different sugars [START_REF] Bidossi | A Functional Genomics Approach to Establish the Complement of Carbohydrate Transporters in Streptococcus pneumoniae[END_REF]. In the presence of glucose, the pneumococcal metabolism is mainly homofermentative with the formation of lactic acid by the lactate dehydrogenase whereas when galactose is present the bacterial metabolism switch to a mixed-acid metabolism with the production of ethanol, acetate and formate by the alcohol dehydrogenase, acetate kinase and pyruvate formate lyase, respectively. Metabolism switch is due to transcriptional regulation of gene expression induced by end products. [START_REF] Al-Bayati | Pneumococcal galactose catabolism is controlled by multiple regulators acting on pyruvate formate lyase[END_REF] (Figure 3). Anaerobic bacteria are found in many types of environmental conditions. They also represent the largest part of the composition of the human microbiota. Anaerobes are essential for the development and the function of biological systems like immune [START_REF] Maier | Understanding How Commensal Obligate Anaerobic Bacteria Regulate Immune Functions in the Large Intestine[END_REF] or digestive [START_REF] Flint | Plant Cell Wall Breakdown by Anaerobic Microorganisms from the Mammalian Digestive Tract[END_REF] systems . They are involved during the digestion process by metabolizing different compounds, influencing lipid metabolism or during the development of the immune system.

Anaerobes that colonize human body can be classified in two categories: exogenous and endogenous anaerobes. Despite their fundamental physiological role, they can be responsible for anaerobic infections. These infections can be caused by both Grampositive and Gram-negative anaerobic bacteria. They are mainly due to a trauma to mucosal membranes or skin letting endogenous anaerobes to extend its colonization area. But some exogeneous anaerobic infections can be mentioned. For example, Clostridium infection such as botulism or tetanus can be developed by the inoculation of Clostridium botulinum and tetani bacteria which secrete toxin that affect the function of nervous system. Studying and understanding these highly versatile micro-organisms is crucial to establish appropriate treatments in case of infection. Bacterial anaerobes have been neglected for a long time due to the difficulty to identify, isolate from infectious sites and cultivate them. They usually have a slow growth and can be found as polymicrobial culture with aerobic bacteria.

b. Food preservation and beverages

Since the Neolithic, fermentation is used to preserve food and make beverages like beer and wine. Many civilizations used micro-organisms to transform natural products into fermented food. Many examples could be mentioned, but the most common ones are Lactobacilli that are used to convert milk into yoghurt or cheese and the yeast Saccharomyces cerevisiae which are responsible for the alcoholic fermentation in beer.

c. Industrial applications

The study of the fermentative metabolism of micro-organisms revealed biological pathways and interesting capabilities which are useful in biotechnological applications.

A wide variety of end-products is generated during fermentative metabolism such as (amino) acids, sugars, vitamins, antibiotics … Many factors such as temperature, pH, and composition of the medium influence the type and the proportion of these final products. Nowadays, the easier manipulation of bacteria genome has simplified the engineering of novel strains able to produce these chemicals of interest in large quantities. This allows the large-scale production of highly demanded chemicals such as biofuels or solvents.

Chapter II: Dehydrogenase enzymes

I. Classification

Enzymes are proteins with a unique tri-dimensional structure that gives them a specific biological activity. Enzymes are capable of speeding up biochemical reactions that could not happen or will take longer period of times without their action. These special proteins have a dedicated active site which is composed of a binding site for one or more substrates and of catalytic sites. By specifically binding the substrate into the active site, enzymes lower the activation energy of the reaction, stabilize the transition state and allow the conversion of substrates into products. These proteins are required in all biological metabolic processes in order to catalyze enzymatic reactions. The enzymatic activity can be modulated by activator or inhibitory molecules.

Depending on the type of mechanism, these biocatalysts can be classified into 6

categories.

E.C. Class name

II. Structural details of dehydrogenase enzymes

Usually, dehydrogenase enzymes are proteins able to oligomerize into larger structures [START_REF] Minárik | Malate Dehydrogenases -Structure and Function[END_REF]. They are composed of different domains. Many are comprising one of the most famous tertiary structural motifs: the Rossman fold.

After obtaining and/or analyzing different dehydrogenase structures such as lactate or malate dehydrogenases [START_REF] Adams | Structure and Mechanism of Lactate Dehydrogenase. In Pyridine Nucleotide-dependent dehydrogenases[END_REF][START_REF] Hill | Polypeptide Conformation of Cytoplasmic Malate Dehydrogenase from an Electron Density Map at 3.0 A Resolution[END_REF][START_REF] Rossmann | Chemical and biological evolution of a nucleotide-binding protein[END_REF], Michael Rossman and colleagues highlighted common structural domain which binds dinucleotide coenzyme such as NAD, FAD. This Rossman domain also called bab is formed by a series of beta strands and alpha helices (Figure 6). This nucleotide binding domain is a highly conserved structural motif but it can be formed by a different number of series of bab and the amino-acid length between secondary structures can vary.

Figure 6: Rossman motif representation [START_REF] Rossmann | Chemical and biological evolution of a nucleotide-binding protein[END_REF] Separately from the nucleotide binding domain, the catalytic domain is responsible for the binding of the substrate involved in the enzymatic reaction.

Another domain is also usually found in dehydrogenase enzymes such as aldehyde dehydrogenase which is the oligomerization domain composed of a b hairpin which also forms a b strand responsible for the association of monomers with each other.

This domain seems to be important for the function of aldehyde dehydrogenases [START_REF] Rodriguez-Zavala | Structural Aspects of Aldehyde Dehydrogenase that Influence Dimer -Tetramer Formation[END_REF].

III. Dehydrogenases involved in ethanol fermentation pathways

During the reaction, hydrogen is transferred to an electron acceptor. This acceptor can be nicotinamide coenzyme NAD + /NADP + or flavin coenzyme FAD/FMN (Figure 7). The nicotinamide adenine dinucleotide coenzyme is required for glycolytic pathways and will be regenerated during ethanol fermentation in bacteria. In the alcohol metabolism as ethanol fermentation, pyruvate can be oxidized using two pathways (Figure 8).

The first one using the pyruvate decarboxylase (PDC) enzyme to converts pyruvate to acetaldehyde which is metabolized by an alcohol dehydrogenase (ADH) generating ethanol. The other one is more common in bacteria, which used either pyruvate From [START_REF] Eram | Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles[END_REF] A. Aldehyde dehydrogenases (PF00171)

Aldehyde dehydrogenase enzymes are essential in all living micro-organisms. These reactions are involved in many catabolic pathways such as sugar, lipid, amino-acid metabolisms and are also responsible for maintaining cellular redox balance. These enzymes are also involved in cytoprotective reactions under oxidative stress conditions.

ALDHs convert aldehydes into carboxylic acids. This reaction is always coupled with the reduction of NAD(P) + . Aldehydes are highly electrophilic reactive compounds. They can interact with nucleic acids and proteins leading to cellular damage. A high concentration of this toxic molecule can alter the cellular physiology, so it must to be regulated.

These enzymes can be classified depending on their substrate-specificity and by comparing their sequences. A sequence usually contains about 450 to 520 aminoacids. In 2010, 16765 aldh genes entries in the Pfam database have been listed in all kingdoms of life. It was found that several types of ALDHs could be expressed in an organism. For example, in 2011 19 aldh genes have been detected in Escherichia coli [START_REF] Jackson | Update on the aldehyde dehydrogenase gene ( ALDH ) superfamily[END_REF].

Furthermore, it has been determined that some ALDHs are fused with other proteins such as in the multicomplex proline utilization A (PutA) or acetaldehyde-alcohol dehydrogenase (AdhE) enzyme.

B. Alcohol dehydrogenases (PF00465)

Alcohol dehydrogenase enzymes can be classified in different categories depending on the cofactor used NAD(P)-dependent, NAD(P)-independent which use pyrroloquinoline quinone (PQQ) cofactor, heme group or F420 cofactor and flavin dependent enzymes.

NAD-dependent alcohol dehydrogenases catalyzes the reduction of carbonyl group to alcohol compound associated to the oxidation of the NADH to yield NAD + .

Among the NAD-dependent ADHs, several type can also be distinguished:

• Type I It comprises the first alcohol dehydrogenase discovered, they are found in yeast, bacteria, mammals and plants. They require Zn 2+ ions for their activity. Their sequence contains about 350 amino-acids, qualified as long/medium chain.

They are dimeric enzymes, with each subunit binding Zn 2+ ion but only one domain is active. The other domain is a structural domain.

• Type II They are found in drosophila. They do not require any metal for their activity. Their sequence contains about 250 amino-acids.

• Type III This family comprises iron-containing/activated enzymes. It was first discovered in Zymomonas mobilis. The sequence usually contains about 385 amino-acids but it could be more such as the multi-enzymatic bifunctional acetaldehyde-alcohol dehydrogenase (AdhE) which contains about 900 amino-acids for the two domains [START_REF] Fontaine | Molecular Characterization and Transcriptional Analysis of adhE2 , the Gene Encoding the NADH-Dependent Aldehyde / Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824[END_REF]. The Fe 2+ -dependent alcohol dehydrogenase domain is about 450 amino-acids.

Different types of ADHs are often found in an organism, reflecting the high adaptation of these organisms regarding the diversity of substrate.

Chapter III: Filamentation of enzymes

I. Introduction

In eukaryotic cells, the compartmentalization of the functions necessary for the cell survival can be done through internal membranes from endosymbiotic origin or not, based on protein-protein interactions, using gradients … [START_REF] Diekmann | Evolution of intracellular compartmentalization[END_REF]. As in eukaryotes, in prokaryote organisms, the spatial organization is also fundamental for biological processes. Bacterial cytoplasm contains all the metabolic pathways necessary for cell metabolism and cell division. Different strategies are used to organize a single environment which regroup many different functions. One way to isolate metabolic processes within bacteria is enzyme filamentation. This nonmembranous compartmentalization was observed in both Gram-positive and Gramnegative bacteria. They can be tubes, helices or linear polymers. Two types of protein oligomers can be mentioned: cytoskeletal-like structures such as MreB, FtsZ and noncytoskeletal filaments composed of metabolic enzymes.

Metabolic enzymes are also found to be involved in filament formation. These selfassemblies are not only found in bacteria but also other kingdoms of life like yeast, plants, mammals. Many roles for enzyme filamentation have been speculated like storage of enzymes [START_REF] Petrovska | Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation[END_REF], enzyme protection from oxidation or degradation (Park & Horton, 2019), regulation of active enzyme pool [START_REF] Aughey | Metabolic regulation via enzyme filamentation[END_REF].

Non-cytoskeletal enzyme polymerization can be found under physiological condition like the acetyl-CoA carboxylase [START_REF] Kleinschmidt | Acetyl Coenzyme A Carboxylase: Filamentous Nature of the Animal Enzymes[END_REF]) (Figure 9A) or CTP synthase filaments (Figure 9B). CTP filaments from bacteria present different architectures and regulation modes than the ones from humans. Interestingly, conserved metabolic enzyme filamentation can evolved differently depending on organisms (Park & Horton, 2019).

The filamentation can also be triggered after a stress like hypoxia or nutrient starvation associated with pH variation which trigger glutamine synthestase filamentation for example [START_REF] Petrovska | Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation[END_REF], by substrate/product concentration like for glucokinase filaments [START_REF] Stoddard | Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity[END_REF]. It has been reported that the enzyme filamentation gives additional levels either in compartmentalization by protecting the cell from the toxicity of metabolite intermediates (as observed in carboxysome an organelle which encapsulate such volatile compound [START_REF] Penrod | Conserving a Volatile Metabolite : a Role for Carboxysome-Like Organelles in Salmonella enterica[END_REF]) or in regulation by allosteric and/or cooperativity modes like for the inosine monophosphate dehydrogenase (IMPDH) assemblies (Figure 9D). (Anthony, Burrell, Johnson, Duongly, & Barber, 2017). But all the details related to enzyme filamentation is not fully understood. Adapted from (Park & Horton, 2019).

II. Spirosome filaments

Spirosomes are filaments observed for the first time in Gram-positive bacteria by Kawata in the 1970's (Kawata, Masuda, & Yoshino, 1975) (Figure 10A). Later these fine spirals were also found in Gram-negative bacteria as Escherichia coli [START_REF] Kawata | Presence of Spirosomes in Escherichia coli[END_REF]) (Figure 10B). Spirosome filaments are conserved across all bacterial kingdoms and they have also been reported in more phylogenetically distant organisms such as photosynthetic unicellular eukaryotic organisms [START_REF] Laurenceau | Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence[END_REF][START_REF] Van Lis | Concerted up-regulation of aldehyde/alcohol dehydrogenase (ADHE) and starch in Chlamydomonas reinhardtii increases survival under dark anoxia[END_REF]. These filaments were localized inside the cell where they are formed but they are also present in the extracellular environment which is probably due to cell lysis [START_REF] Laurenceau | Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence[END_REF]. Lactobacillus casei (A) from (Kawata et al., 1975) and Escherichia coli (B) from [START_REF] Kawata | Presence of Spirosomes in Escherichia coli[END_REF].

Scale bar (A) 100 nm and (B) 10 nm.

In the early 1990's, Matayoshi identified the spirosomal subunit called so far spirosin as being the bifunctional acetaldehyde -alcohol dehydrogenase enzyme [START_REF] Matayoshi | Relationship between the Production of Spirosomes and Anaerobic Glycolysis Activity in Escherichia coli B[END_REF].

III. Acetaldehyde-alcohol dehydrogenase enzyme (AdhE)

The bifunctional acetaldehyde -alcohol dehydrogenase (AdhE) is composed of two domains a N-terminal Coenzyme A-dependent acetaldehyde dehydrogenase (ALDH) and a C-terminal iron-dependent alcohol dehydrogenase (ADH) domain (Figure 11).

This 96 kDa protein is encoded by the adhE gene which originated from the fusion an acetaldehyde oxidoreductase and an ethanol oxidoreductase [START_REF] Leonardo | Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli[END_REF]. The ADH domain classify as type III is activated by metal ion such as iron [START_REF] Espinosa | The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact Cterminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity[END_REF]. These two reactions are coupled with the regeneration of two NAD + necessary during the glycolysis. This bifunctional enzyme participates in alcohol fermentation catalyzing the two final reactions (Figure 12). Depending on the conditions such as substrate availability or/and pH, these two reactions are reversible in vitro [START_REF] Van Lis | Concerted up-regulation of aldehyde/alcohol dehydrogenase (ADHE) and starch in Chlamydomonas reinhardtii increases survival under dark anoxia[END_REF].

Figure 12: Involvement of the bifunctional AdhE enzyme during the regeneration of NAD+ by producing ethanol end-product (A). Metabolic reactions of the reversible catalytic activities containing into the AdhE enzyme (B).

In specific conditions, it has been shown that bacteria could grow on ethanol using an AdhE containing a single mutation. The substitution of glutamate 568 in lysine in the ADH domain allows to activate the AdhE activity under aerobic conditions (Hollandstaley, [START_REF] Holland-Staley | Aerobic Activity of Escherichia coli Alcohol Dehydrogenase Is Determined by a Single Amino Acid[END_REF].

In Entamoeba histolytica, the two homologous dehydrogenase domains were expressed separately in order to test ALDH and ADH activities. The two expressed ALDH (1-446) and ADH (417-870) domain showed in vitro dehydrogenase activity.

But ALDH domain present lower activity than ALDH activity in AdhE enzyme. The truncated ALDH domain is purified as dimer or oligomer (>1000kDa) suggesting that the dimer is the minimum active unit. The expression of this truncated protein is not able to restore the growth of E. coli strain under anaerobic condition [START_REF] Chen | Structural analysis of the acetaldehyde dehydrogenase activity of Entamoeba histolytica alcohol dehydrogenase 2 ( EhADH2 ), a member of the ADHE enzyme family[END_REF]. The truncated ADH domain is also found as tetramers and present the same activity as ADH activity in AdhE. But the expression of this truncated ADH domain is not able to restore the growth of E. coli strain under anaerobic condition [START_REF] Espinosa | The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact Cterminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity[END_REF].

These results also confirms that the adhe gene is generated after a gene fusion of two distinct ALDH and ADH enzymes.

IV. AdhE expression

It has been reported that the adhe gene is highly expressed, about eight-fold larger under anaerobic conditions than under an aerobic environment which correspond to approximately 30 000 copies per cell [START_REF] Dalle-Donne | Redox Proteomics: From protein modifications to cellular dysfunction and diseases[END_REF]. This expression is regulated at transcriptional and translational levels [START_REF] Leonardo | Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli[END_REF]. The AdhE enzyme is only active under anaerobic condition meaning that it is also regulated at post-translational level. It has been suggested that the exposure of oxygen trigger spirosome disassembly associated to the loss of enzymatic activity. The glutamate 568 could be critical for the inactivation of the enzyme in aerobic condition. [START_REF] Holland-Staley | Aerobic Activity of Escherichia coli Alcohol Dehydrogenase Is Determined by a Single Amino Acid[END_REF]. This enzyme seems to also have a role in aerobic condition and could have a protective role against oxidative stress acting as H2O2 scavenger causing AdhE inactivation (Echave, Tamarit, Cabiscol, & Ros, 2003b).

Studies have shown that the deletion of adhe gene nearly abolish the ethanol production and the flux is redirected towards lactate and acetate production [START_REF] Zhu | Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D -lactate production under microaerobic condition[END_REF]. Further, in Escherichia coli the expression of adhe gene is essential for bacterial growth in minimal media in absence of oxygen [START_REF] Yong | Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs[END_REF].

AdhE is also expressed in biofilm condition and involved in 1-propanol synthesis. It has been suggested than AdhE could convert propionyl-coenzyme A into proionaldehyde and then into 1-propanol. So, AdhE enzyme is also required for threonine fermentation pathway [START_REF] Létoffé | Biofilm microenvironment induces a widespread adaptive aminoacid fermentation pathway conferring strong fitness advantage in Escherichia coli[END_REF].

AdhE generates a lot of interest for biofuel production. But also, in fundamental research, studies have reported that this bifunctional enzyme could be involved in bacterial pathogenicity.

V. Biotechnology

Biofuel production became a great alternative to replace fossil fuel energies. Among all microbial biofuels, bioethanol happens to be an interesting substitute for renewable energy. A lot of efforts are made to understand the metabolic pathways ending up to biofuel in order to engineer novel high-yield associated species and also to optimize the micro-organism cultures. In this way, investigations have been undertaken on the fundamental AdhE enzyme for its ability to produce ethanol. Different studies have been performed on the enzymology or the regulation of this bifunctional enzyme (Tian, Cervenka, Low, Olson, & Lynd, 2019). Mutations in adhE gene lead to higher ethanol yield and tolerance. It has been reported that metabolic engineered T. saccharolyticum strain produce ethanol with a yield of 90% of the theoretical maximum [START_REF] Zheng | Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum[END_REF].

VI. Bacterial pathogenicity

In addition to its interest in biotechnology application, more recent studies mentioned the role of the bifunctional AdhE in bacterial pathogenicity. In several bacterial species, AdhE seems to be involved in bacterial virulence.

For example, in Listeria monocytogenes, the Listeria adhesion protein LAP (lmo1634)

was identified as an alcohol-acetaldehyde dehydrogenase by searching genetic sequence homology (K. [START_REF] Kim | Adhesion characteristics of Listeria adhesion protein ( LAP ) -expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor[END_REF]. LAP is secreted by SecA2 system into its environment and promotes bacterial paracellular translocation through epithelial cell junction [START_REF] Burkholder | LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species[END_REF]H. Kim & Bhunia, 2013). In Streptococcus pneumoniae the deletion of adhe gene alters cell functions such as hemolytic activity and bacterial colonization under ethanol condition. In addition, host cells of the streptococcus strain show less inflammatory activity [START_REF] Luong | Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae[END_REF]. To this extent, bifunctional AdhE enzyme might be considered as a S. pneumoniae virulence factor. Also, Beckham et al, reported that the deletion of adhE gene in pathogenic enterohaemorrhagic bacteria Escherichia coli (EHEC) cause a bacterial virulence decrease associated to a high decrease of the Type 3 secretion system (T3SS)

expression. The deletion of adhE gene leads to an acetate accumulation which could be at the origin of transcriptional regulation of flagellar components. [START_REF] Beckham | The metabolic enzyme AdhE controls the virulence of E scherichia coli O157:H7[END_REF]. This T3SS is used for mediating Gram-negative bacteria attachment to the cell host and for delivering bacterial virulence factors during infection [START_REF] Wagner | Bacterial type III secretion systems : a complex device for the delivery of bacterial effector proteins into eukaryotic host cells[END_REF].

This deletion leads to the overexpression of non-functional flagella. Also, correlation of the pyruvate metabolism and the virulence of Salmonella typhimurium has been investigated. S. typhimurium adhE and pflB, which codes for the pyruvate formate lyase I enzyme are up-regulated during bacterial infection. These genes could alter the expression of genes involved in the S. typhimurium T3SS coded by the Salmonella pathogenicity island (SPI) 1. [START_REF] Abernathy | Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPI1-mediated gene expression and infectivity[END_REF].

All these examples suggest that the bifunctional AdhE enzyme is not only involved during ethanol fermentation but also in other crucial bacterial processes such as bacterial virulence or colonization.

VII. Non-bacterial AdhE

The presence of the bifunctional acetaldehyde -alcohol dehydrogenase AdhE enzymes is reported in more distant phylogenetical organisms such as photosynthetic unicellular eukaryotic green algae Chlamydomonas reinhardtii. AdhE is located in the chloroplast where it was found as dimers or spirosomes (Van Lis et al., 2017) (Figure 13A). Studies reported that cells with higher AdhE abundance correlate with better adaptation to stresses [START_REF] Van Lis | Concerted up-regulation of aldehyde/alcohol dehydrogenase (ADHE) and starch in Chlamydomonas reinhardtii increases survival under dark anoxia[END_REF]. AdhE homologous enzyme called EhADH2 is also found in an ancient parasitic protozoan Entamoeba histolytica as spirosome oligomers. (Bruchhaus & Tannich, 1994) (Figure 13B).

VIII. AdhE oligomerization

The exploration of the oligomerization of this enzyme is only at the beginning.

Nevertheless, an interesting paper published by Kessler et al, showed that spirosomes from Escherichia coli could been found in two different conformations depending on the ligands incubated with (Kessler, Herth, & Knappe, 1992) (Figure 14 A). In absence of ligands called apo-form, spirosomes present a compact state (close) but after incubation with coenzymes such as NAD + , Coenzyme-A and Fe 2+ which is necessary for the activation of the alcohol dehydrogenase domain, spirosomes are in an extended conformation (open). They also have shown that the extended spirosomes are less susceptible to the chymotrypsin proteolysis suggesting that the incubation with ligands and so the extended conformation has fewer accessible sites for its action. ( Kessler et al., 1992) The structure of the alcohol dehydrogenase domain of the bifunctional AdhE enzyme from Geobacillus thermoglucosidasius has been determined using X-ray crystallography at 2.5Å resolution. Using homology with the homologous acetaldehyde dehydrogenase domain of AdhE from V. parahaemolyticus and a putative ALDH from Listeria monocytogenes, the acetaldehyde dehydrogenase domain of the AdhE enzyme was modelling and associated with the ADH domain calculating interaction prediction [START_REF] Extance | Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius[END_REF].

In the course of this project, the structure of the compact conformation of the spirosome was published (G. [START_REF] Kim | Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity[END_REF]. This structure is presented in absence of any ligands and showed the architectural organization between each ALDH and ADH domains and between AdhE monomers. They also could observed ligands-induced conformational change, but the structure of the extended conformation is absent. In addition, the structure does not present the helical parameters.

They were able to disrupt the filamentation by mutating a single amino acid (670) located at the ADH-ADH interface. This mutation altered also the acetaldehyde dehydrogenase activity of the enzyme. It suggests that the filament could regulate the enzymatic activity.

I. Introduction

In 60 years, the determination of tridimensional macromolecular structure has been revolutionized. Since the first 3D structure solved by John Kendrew and Max Perutz in 1958, structural biology has emerged as a method to understand the function of biological systems through their 3D structure. Indeed, the unique 3D configuration of each protein is crucial for their function and so for the action of these proteins in important biological processes. Nobel awarded in 1962 John Kendrew and Max Perutz established X-ray crystallography in order to solve the position of each atom in a globular protein structure. Since the development of the X-ray crystallography, over than 160 000 structures have been deposited in the Protein Data Bank (PDB) (Figure 15). Other powerful techniques for structure determination have been established. Among these techniques, nuclear magnetic resonance (NMR) spectroscopy is widely used to determine 3D structure of small proteins in solution, to study interactions between biological molecules and proteins or also to look into protein dynamics associated to conformational changes. A variant, solid state NMR (ssNMR) is also able to provide structural information about large assemblies displaying a level rigidity and order at the molecular level (e. g., viral capsids, fibrils). ssNMR us a useful biophysical method in order to study protein interaction with lipid bilayers or about the dynamic of noncrystalline samples. [START_REF] Wel | New applications of solid-state NMR in structural biology[END_REF] Cryo-electron microscopy (cryo-EM) is now used to determine high-resolution structure of proteins.

II. Cryo-electron microscopy

A. Resolution revolution

Ernst Ruska (Nobel Prize for physics in 1986) and Max Knoll designed the first electron microscope in 1933 initiating the future revolution in structural biology. Indeed, the development of electron microscope allows to overpass the theoretical resolution limit (200nm) of the optic microscope, which uses light with a longer wavelength. Since this breakthrough, electron microscopes have never stopped to be optimized. Originally used for material science, transmission electron microscopy (TEM) allows to get internal structural information of non-biological samples whereas scanning electron transmission (SEM) developed by M. Knoll in 1935 and later by Zworykin is useful in order to get sample surface information by capturing backscattered electrons [START_REF] Bogner | A history of scanning electron microscopy developments : Towards '"wet-STEM[END_REF]. The main limitation in using biological specimens in EM is in the preservation of sample integrity. Highly energetic electrons cause radiation damage when they interact with biological samples. Moreover, the necessity of being in high vacuum environment in order to avoid to scatter electrons is not compatible with maintaining sample integrity. Since the development of techniques allowing to preserve sample integrity (sample inclusion or fixation) and the improvement of image contrast (staining with heavy atoms) [START_REF] Bai | How cryo-EM is revolutionizing structural biology[END_REF], TEM could be used to analyze biological specimens. But all these methods can induce artifacts into the final image. Technique that maintain the integrity of the specimen in its native environment without interfering with its quality was needed.

One of the major advances has been the vitrification process. In the 1980's Jacques

Dubochet and colleagues developed a new method of sample freezing in an amorphous ice instead of crystalline or cubic ice. Indeed, aqueous sample must be rapidly frozen in liquid ethane or propane cooled by nitrogen liquid leading to a thin layer of vitreous ice. This development of vitrification resulted in preserving the sample integrity and avoiding radiation damage. Improvements of the method allowed to get the first high-resolution structure of membrane protein, bacteriorhodopsin. (R. [START_REF] Henderson | Model for the Structure of Bacteriorhodopsin Based on High-resolution Electron Cryo-microscopy[END_REF][START_REF] Henderson | Three-dimensional model of purple membrane obtained by electron microscopy[END_REF]) (Figure 16).

D. Image formation

A biological sample is mainly composed of light atoms (C, H, O, N, P, S) which are highly subjected to radiation damage. Indeed, electrons from the electron beam interacting with the biological sample can ionize and break bonds within the sample. It can also create free radicals which can induce damage. So in order to minimize irradiation damage, it is necessary to reduce the electron dose to preserve sample integrity. In consequence, the contrast in the image is very low. In contrast to heavy atoms such as metals, electrons weakly interact with light atoms, the contrast must be increased to distinguish signal over noise. Two types of contrast can be differentiated, amplitude contrast and phase contrast. Electrons behave differently when they interact with sample (Figure 20). Backscattered electrons are not captured by camera device which are placed under the specimen in TEM. When electrons interact with the specimen, they can transfer a part of their energy that is responsible for radiation damage, they are called inelastically scattered electrons. In contrast, the others are called elastically scattered electrons and do not transfer energy during the interaction.

The amplitude contrast is produced when an electron path is highly affected by the sample (heavy metal, thickness, …) which is poor in cryo-EM.

The phase contrast occurs when there is a shift between scattered and non-scattered electrons interacting with the sample. Henderson and colleagues also contributed to the development of new devices able to detect single electron events in order to limit noise in background without decreasing the DQE.

Since the 2010's, the emergence of new direct detection devices (DDD) capable of single electron counting upgraded cryo-EM. DDDs are based on metal oxide semiconductor devices that are capable of directly counting a single electron event.

Direct detection system presents much higher DQE at high frequencies than previous systems that in application improve signal-to-noise ratio (Figure 21). The higher DQE allows also automated data acquisition thanks to a faster readout and gives the opportunity to record movies instead of unique images. The main benefit is that all the subframes composing the movie can be realign with each other in order to compensate the drift occurring during the acquisition. After alignment, all the subframes are averaged to generate a single image. The final electron final dose of one image is the sum of each subframe dose. Now, DDD associated to an automated data acquisition microscope are the state-of-art of the cryo-EM. The 2D electron crystallography is based on the principle of the X-ray crystallography.

In crystals, proteins form a 2D lattice of the repeated unique information. Electrons are diffracted by the interaction with the sample resulting in a diffraction pattern composed of diffraction spots. These spots can be correlated to the amplitude of the protein structure. In 1968, De Rosier and Klug introduced the acquisition of titled micrographs in order to reconstructed 3D protein structure based on the amplitudes and the phases (DeRosier & Klug, 1968). This technique was used by Henderson and his colleagues to solve the structure of the bacteriorhodopsin at 7Å in 1975.

Another technique taking advantage of protein crystal sample is the micro-electron diffraction (micro-ED) which allows the electron dose to be decreased in order to minimize damage of biological specimens (3D protein crystals). [START_REF] Nannenga | The cryo-EM method microcrystal electron diffraction (MicroED)[END_REF] Cryo-electron tomography (Cryo-ET) is a remarkable technique allowing the 3D reconstruction of an object in its native environment such as a cell. Multiple images of the same sample have to be collected with different angles. The sample is tilted relative to the electron beam using incremented angle. By realigning and combining all the 2D different projections, the object of interest can be reconstructed in 3D using image processing softwares (e. g. IMOD). Different tomograms can be averaged with each other in order to improve the signal over noise ratio and reach sub-nanometer final resolution. [START_REF] Zhang | Advances in cryo-electron tomography and subtomogram averaging and classification[END_REF] In this study, I used both the single particle analysis and helical reconstruction techniques.

a. Helical reconstruction

The sample can be an object with a helical symmetry. In this case, the refinement can get advantage of this geometric repetition. Indeed, multiple (hundreds or thousands) views of the same unit are present in one unique helical object.

Two types of refinements can be described: the Fourier-Bessel and the real-space reconstruction methods.

Fourier-Bessel reconstruction is used to determine parameters (helical rise associated to helical twist or rotation or the pitch associated with the number of units per turn) and the three-dimensional structure of helical symmetry. Because it can be difficult to index all the layer lines in the Fourier transform especially for non-straight and long repeated helical assembly, other methods have been developed.

Another techniques can also be described in order to determine helical symmetry and solve the 3D structure of helical assemblies, the Iterative Helical Real-Space Reconstruction (IHRSR) was developed by Egelman and colleagues [START_REF] Egelman | The iterative helical real space reconstruction method: Surmounting the problems posed by real polymers[END_REF] and is also implemented in image processing softwares. In this method, the helical object is segmented into smaller segments which overlap with each other. Then, these extracted segments containing asymmetric unit of the helical assembly are compared with different projections generated from a 3D reference. The 3D reference can be a cylinder. All the different views can be extracted due to the helical symmetry of the object. This strategy doesn't need long and straight filaments as the previous one. After comparison, extracted segments are back projected in order to build the 3D reconstruction without imposing any symmetry. Helical parameters are determined in real space from the 3D reconstruction. Then, these parameters can be applied to improve the quality of the refinement during the next iterations. [START_REF] He | Helical reconstruction in RELION[END_REF] b. Single particle analysis

In 1995, Frank and his colleagues presented the first structure of E. coli ribosome solved using 4300 single particles (Franck et al, 1995). This technique is now widely used in order to reconstruct the tridimensional structure of isolated asymmetric biological objects such as purified proteins.

The method takes advantage of random orientation of biological particles. During the vitrification process, purified particles are spread into the grid and are frozen into multiple different orientations. Hundreds of thousands of 2D projections of single particles in different orientations are recorded in micrographs. After sorting in different 2D classes, identical projections are averaged to increase the signal to noise ratio.

One challenge is to build the 3D reconstruction from all views of the object. Among these methods, random conical tilt (RCT) [START_REF] Radermacher | Threedimensional structure of the large ribosomal subunit from Escherichia coli[END_REF]) can be described. Two images have to be recorded, one with a high tilt angle and the other without it. It allows to have access to different orientations of the particle but not all of them which is the main limitation of this method. Moreover, the sample is submitted to higher electron dose because two images of the same area is recorded. 3D reconstruction can also be performed from an initial reference from a lower resolution structure, from a based on already known homologous structure or using electron tomography.

Otherwise, initial density map can be generated using ab initio methods. Powerful algorithms were developed and implemented in image processing softwares. In cryoSPARC [START_REF] Punjani | cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination[END_REF] or RELION softwares [START_REF] Scheres | RELION : Implementation of a Bayesian approach to cryo-EM structure determination[END_REF], initial density can be produced without external reference using stochastic gradient descent (SGD) algorithm [START_REF] Zivanov | New tools for automated high-resolution cryo-EM structure determination in RELION-3[END_REF].

Back-projections of the reference is generated in order to be compared with all the 2D classes. Maximum likelihood optimization target methods have been developed in order to align 2D classes and the back-projections (Scheres, 2011). After several rounds of refinement based on Bayesian approach, the higher resolution map is generated.

The development of robust algorithms allowing the 3D reconstruction is one of the factors leading to the cryo-EM revolution (Kuhlbrandt, 2014).

During the vitrification, the orientation of purified proteins is theoretically random. But in practice, specific orientations can be adopted on the carbon surface during the freezing. The preferential orientation of the protein into the ice can lead to aberrations during the reconstruction and finally to anisotropic resolution. In order to recover all the information of the 3D structure, the sample can be tilted during the acquisition. But this method can present some disadvantages like the need to estimate a gradient of defocus into the tilted micrograph or increase the beam-induced motion. [START_REF] Lyumkis | Challenges and opportunities in cryo-EM single-particle[END_REF]

c. Post-processing and validation

It is now common to obtain maps displaying resolution better than 4 Å. In order to limit the noise presents in the resulted map and improve the contrast at high-resolution, Rosenthal and Henderson presented a technique to maximize the quality of the map (Rosenthal and Henderson, 2003). Sharpening is a method to adjust the quality of the map depending on the resolution. This can highly improve the readability of the electron map. However, aberrations can be introduced due to over-sharpened map. Bfactors are calculated depending on the resolution and applied on the map in order to increase the amplitude of the structure factors.

The comparation between the two half maps obtained using independent data set is used to determine the quality of the processing and then to validate the final map. This is done by calculating the Fourier shell correlation (FSC) between the two half maps.

So, the final resolution of the map can be estimated using a threshold (Saxton &Baumeister, 1982, Harauz andvan Heel, 1986). This threshold is commonly 0.143.

The resolution is not uniform across all the Cryo-EM map and could not represent the quality of the map. Advanced methodologies are now used to estimate local resolutions in the map.

In order to avoid the noise surrounding the biological object of interest, a mask is applied. The resolution is calculated for the unmasked and the masked map.

The validation of the reconstructed model based on the map will also be appreciated by calculated the FSC between the map and the model.

F. Limitations and perspectives

Different limitations can be mentioned about the cryo-EM technique. At the microscope level, the wavelength of electrons at 300kV is 2 pm meaning that it is not the limiting factor for the final resolution. But one of the limitations is the radiation damage caused by inelastic scattering of the electrons. Sample is altered by the energy transmitted during the exposure which degrade the structure of the object.

Also, the sample itself and its preparation seem to be the main limitation to get a highresolution. Depending on the specimen, pure samples can be difficult to get. Small and flexible protein structures can be difficult to solve at high-resolution and are still very challenging. Furthermore, in order to obtain high resolution structures, a large dataset is required that involve large memory storage and powerful computational devices.

In order to get in situ structural information, cryo-electron microscopy can be combined with fluorescence microscopy. Proteins or macromolecular assemblies can be localized using optic microscope. Saved coordinates can be implemented into electron microscope to acquire the targeted object at high-resolution. The correlative light electron microscopy (CLEM) can even rely on super-resolution optical microscopy in order to localize with higher precision object of interest [START_REF] Tuijtel | Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins[END_REF].

Chapter V: Aims of the project

The aim of this project is to study the role of spirosomes in bacteria. The observation of native spirosomes from different species showed a difference of phenotype.

Spirosomes from Gram-positive anaerobic bacteria such as Clostridium difficile, Streptococcus sanguinis or pneumoniae present an extended conformation whereas spirosomes from Gram-negative facultative bacteria such as Escherichia coli are found in a compact conformation which can be extended using ligands. We decided to solve the structure of spirosomes from Escherichia coli and Streptococcus pneumoniae in order to compare these two structures. Based on these structures, it is possible to consider the function, the role and the regulation of these filaments. This thesis is divided into two parts.

At the beginning of this project, none study was able to reveal the spatial arrangement of the bifunctional acetaldehyde -alcohol dehydrogenase (AdhE) within the spirosome. The atomic structure of the AdhE enzyme is also unknown. Previously in the lab, the observation of E. coli and S. pneumoniae spirosomes uncovered significant structural differences. So, our structural analyses were focused on these two specific species. The first part was dedicated to the determination of the atomic structure of different conformations of E. coli spirosomes and S. pneumoniae spirosomes using cryo-electron microscopy.

The second part is focused on the investigation of the role of the filamentation based on the obtained structures. It could be achieved by molecular biology techniques such as directed mutagenesis in order to understand the role of major structural features.

Enzymatic assays were performed to understand how catalytic domains are affected by these mutations/deletions. Then, these modifications were studied in vivo through complementation assays. Finally, the spirosome sub-cellular localization was also investigated by immunofluorescence analysis and super-resolution microscopy.

RESULTS

STRUCTURAL STUDY OF BACTERIAL

SPIROSOMES

I. Compact conformation of Escherichia coli spirosomes

A. Structure determination of compact Escherichia coli spirosomes

The AdhE E. coli gene was cloned into a pET15 vector as detailed in Materials & Methods. Constructed plasmid was incorporated into BL12(DE3) bacteria by chemical transformation. The cells were cultivated in Luria-Bertani media at 37°C, 180r.p.m. to exponential phase. Overexpression was induced using IPTG during 16h at 16°C. After purification using affinity chromatography, purity of purified fractions was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). AdhE was directly purified in its oligomeric state. Then, pure AdhE fraction was observed using negative stain electron microscopy. We reproducibly observed polydisperse filaments in the same sample preparation. We clearly visualized E. coli spirosomes in their compact conformation after overexpression in aerobic environment (Figure 22).

Then, these filaments were vitrified on Cryo-EM grids into liquid ethane cooled by liquid nitrogen using Vitrobot as detailed in Material & Methods.

After obtaining grids with a good filament dispersion and ice thickness, thousands of movies were recorded using Talos Arctica cryo-electron microscope equipped with K2 direct electron detector. Two different strategies were used to solve the structure of compact and extended spirosomes. Purified filaments were rather short and flexible, so single particle analysis was used to solve the structure of the asymmetric unit and helical reconstruction analysis was performed to get helical parameters in order to reconstruct the filament.

B. Structure analysis of compact Escherichia coli spirosomes

Compact structure of E. coli spirosomes could be solved using spirosomes in absence of ligand (the apo-form), which could be due to the expression in aerobic condition or to their dissociation due to a weak affinity to the compact filament.

b. Comparison with monofunctional dehydrogenases

The structure of AdhE was compared with the structure of monofunctional ADH or ALDH enzymes. We selected homologous monofunctional enzymes with active sites of similar topology than AdhE using the COACH server (Yang, Roy, & Zhang, 2013a[START_REF] Yang | Protein -ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment[END_REF]. For AdhE ALDH domain, the crystal structure of Rhodopseudomonas palustris propionaldehyde dehydrogenase (PDB 5JFL) was identified (Zarzycki, Sutter, Cortina, [START_REF] Zarzycki | In Vitro Characterization and Concerted Function of Three Core Enzymes of a Glycyl Radical Enzyme -Associated Bacterial Microcompartment[END_REF]. For AdhE ADH domain, the crystal structure of the lactaldehyde:1,2-propanediol oxidoreductase of E. coli (PDB 2BL4) was identified [START_REF] Montella | Crystal Structure of an Iron-Dependent Group III Dehydrogenase That Interconverts L -Lactaldehyde and L -1 , 2-Propanediol in Escherichia coli[END_REF]. This homologous structure of the ADH domain is also in presence of ligands: NAD + and Fe 2+ in contrast to the structure of the ADH domain of the bifunctional AdhE enzyme from Geobacillus thermoglucosidasius (PDB 3ZDR) which is in its apo-form. These monofunctional enzymes were compared with the corresponding ALDH and ADH domains in AdhE. As shown in Figure 27, the overall canonical fold found in ALDH and ADH enzymes is conserved in AdhE. Interestingly, the monofunctional enzymes are all active as dimers or tetramers. By superimposing these structures with AdhE, we could observe that the interfaces present in dimers of monofunctional enzymes correspond to the ADH -ADH and ALDH -ALDH interfaces of the extended spirosomes (Figure 27). Of note, in the AdhE filament in its compact state, the conformation of the ALDH domain and the ALDH -ALDH interface are not superimposable with their counterpart in the monofunctional enzymes.

n β-ALDH n ⍺-ALDH n β-ADH n+1 ⍺ -ADH n-1 β-ADH n ⍺-ADH (α-ALDH)n/(β-ALDH)n (α-ADH)n/(β-ADH)n-1 (β-ADH)n/(α-ADH)n+1 (α-ALDH)n/(β-ADH)n (α-ADH)n/(β-ALDH)n Erb, &

c. Two distinct active sites linked by a channel

In the compact spirosome structure, the NADH density was only present in the ADH domain. In the extended spirosome structure, NAD + and Fe 2+ densities can be clearly identified in the cryo-EM map in the ALDH and ADH domains of AdhE but the densities are not well resolved (Figure 28A). Again, highly conserved amino-acids composing both active sites have been identified using the superimposition of the AdhE structure with monofunctional homologous enzymes. Both active sites are located in a Rossman fold motif which is typical of nicotinamide binding site which is highly conserved (Figure 28B). Amino-acids involved in the interaction with the ligands are strictly conserved. The ALDH domain of the AdhE was superimposed with the monofunctional propionaldehyde dehydrogenase from R. palustris. We could observe that important catalyzing-reaction amino-acids are also conserved. We identified the Cys246, as the nucleophile residue important for the proton transfer during the reaction, helped by Thr368 and His 367 as well as Glu335 (Cys330, Thr450, His449 and Glu419 in the homologous aldehyde dehydrogenase of R. palustris respectively).

Thanks to the following multiple sequence alignment analysis with monofunctional aldehyde and alcohol dehydrogenase enzyme sequences, we could identify conserved channels in the ALDH and ADH dehydrogenase domains of AdhE. So, a global channel from the ALDH domain to the ADH domain was identified (Figure 29). After analyzing the structure of the compact conformation, we could not observe such a channel. This channel was only found in the extended conformation. In the compact conformation, we observe that the loop 1 of the ALDH domain is placed between the loops 2 and 3 of the ADH domain and obstruct the channel (Figure 31).

III. Filamentation study

A. Strategy

Structural analysis of a protein allows the identification of important features which will further be targeted in order to understand their role. For example, these specific features can be interfaces between domains, charged-pockets, loops, active sites …

In order to understand the filamentation of spirosomes, we decided to try to disrupt the filamentation of AdhE enzyme. To do so, different zones and amino-acids were targeted to alter this polymerization. The ADH -ADH was also targeted in order to disrupt the filamentation. Indeed, as

shown before, the spirosome corresponds to the stacking of AdhE dimers through this interface. So, we identified the glutamate 669 as a putative important amino-acid for stabilizing this interface. It could make a hydrogen bond with the nearby nitrogen of the arginine 702 (Figure 36A). To do so, we constructed two different mutant Glu669Lys

and Glu669Leu in order to invert the amino-acid charge or neutralize the charge and disrupt the interaction with the arginine. After expression and purification, both mutants were still present in the void volume of the gel filtration profile suggesting this mutant is still able to form filaments (Figure 36B). The D376A/N377K and the D376A/N377A mutants were constructed in order to confirm this hypothesis, at this moment the purification of these mutants has not yet been done and we could not present results about these mutants.

D. Positively-charged pocket

Interestingly, a density located at the external surface of the ALDH -ADH interface could not be assigned to AdhE or known ligands. The presence of the density was difficult to identify due to a poor repetition along the filament. Moreover, the density is not clearly defined and we were not able to fit any compound with certainty (Figure 38A). Nevertheless, this density is present at the edge of the ALDH-ADH which is the interface responsible for the channeling of intermediate of reaction. It could be a site for a regulatory molecule interfering with the enzyme activity or for stabilizing the filament in extended conformation. The characterization of this density could therefore be crucial in understanding the function of the spirosome. Even if we are not able to identify the molecule of interest for the moment, we could localize many positivelycharged residues surrounding this density such as arginine (265, 772-774, 768, 790), lysine (228) and histidine (269) (Figure 38B). In order to study the function of this pocket and try to identify the chemical compound responsible for this intriguing density, we decided to mutate some of these amino-acids R772L, R773L, R774L, R790I as described in Materials & Methods.

After expression and purification, we could observe that filaments were still formed, and they were purified in their compact conformation (Figure 39). Part II: Structural study of Streptococcus pneumoniae spirosomes

I. S. pneumoniae spirosomes preparation

The AdhE S. pneumoniae gene was cloned into pET21 vector in the lab before the beginning of my thesis. As for AdhE E. coli expression, the constructed plasmid was incorporated into BL12(DE3) bacteria by chemical transformation. The cells were cultivated in Luria-Bertani media at 37°C, 180r.p.m. to exponential phase. Overexpression was induced using IPTG during 16h at 16°C. After purification using affinity chromatography, purity of purified fractions was analyzed with SDS-polyacrylamide gel electrophoresis and Western-blot. We observed that the integrity of the sample is affected, a fraction of the sample is degraded. So, sample was applied into size exclusion chromatography in order to keep the longest spirosomes. Then, spirosome fraction was observed using negative stain electron microscopy.

We observed filaments in an extended conformation in contrast to E. coli spirosome which were purified in a compact conformation in the same conditions. We obviously presumed that a structural difference induces a permanent extended state in the S. pneumoniae spirosome.

Cryo-EM grids were prepared as explained in Materials & Methods. After collecting thousands of images and reconstructed S. pneumoniae filaments, the resolution was limited around 5Å.

II. Structure analysis of S. pneumoniae spirosomes

The structure of purified S. pneumoniae spirosomes reveals a filament with a width of 165.6 Å and a rise of 59.5 Å (Figure 40). The filament is intrinsically present in an extended conformation, meaning that the incubation with ligands is not necessary to trigger the conformational change. ALDH and ADH domains are organized as in the Escherichia coli spirosome structure.

III. Sequence analysis between Gram-positive and Gram-negative bacteria

The comparison between Gram-positive and Gram-negative bacteria using the multiple sequence alignment of the bifunctional AdhE highlights substantial differences (Annexes 1-4). The arginin amino-acids delimiting the positive-pocket present in the AdhE of Gram-negative bacteria is absent in the AdhE of Gram-positive bacteria. It could be a large discrepancy explaining the difference of conformational state. Indeed, one major difference is that Gram-negative spirosomes are found in compact conformational state whereas Gram-positive spirosomes are purified in an extended conformation even in aerobic condition. This density could be responsible for the regulation of the AdhE enzyme within the filament in Gram-negative bacteria. Further analyses have to be carried out in order to elucidate these first observations.

In order to analyze the structure of S. pneumoniae spirosomes in greater details, we will consider to purified native S. pneumoniae spirosomes or to incubate filaments with ligands as it has been done for E. coli spirosomes. Even if the S. pneumoniae spirosomes are present in an extended conformation, it could stabilize flexible filaments and improve the final resolution.

FUNCTIONAL STUDY OF BACTERIAL

SPIROSOMES

I. Disruption of the filamentation

Based on the structure, we could identify that the filamentation correspond to the dimerization of AdhE dimers. As presented before, we identified that the linker zone between the ALDH and ADH domains was stabilizing the AdhE dimer through the ALDH-ALDH interface. We noticed that the deletion of the 446-449 aborted the spirosome formation. In order to study the impact of this deletion, we studied the enzymatic activities of native AdhE forming spirosomes and Δ446-449AdhE which cannot form filaments.

A. Enzymatic assays of the dehydrogenase activities

The bifunctional acetaldehyde -alcohol dehydrogenase is a reversible enzyme, responsible for the following reaction: Our results confirm that the spirosome fraction exhibit both ALDH and ADH activities of AdhE. However, the AdhE Δ 446-449 mutant, which does not assemble as spirosomes, does not present ALDH activity but the ADH domain is still active. The reverse reaction is highly affected by the deletion whereas the physiological reaction remains intact (Figure 44). Using the correlation between the NADH absorbance and the concentration, the specific activities have been calculated. In the physiological reaction, 1 mg of AdhE WT consumed 0.112 mmol of NADH per minute whereas 1 mg of AdhE D446-449 consumed only 0.035 mmol of NADH per minute. In the reverse condition, the specific AdhE D446- 449 activities are not affected compared with AdhE WT (0.351 mmol min -1 mg -1 and 0.336 mmol min -1 mg -1 respectively). These results show that the ALDH activity is strongly WT ( 7) Mutant ( 7)

WT ( 8 affected by the AdhE filamentation disruption while the ADH activity seems to be still functionally active.

In their study, Kim et al, have also disrupted the AdhE filamentation (G. [START_REF] Kim | Aldehyde-alcohol dehydrogenase undergoes structural transition to form extended spirosomes for substrate channeling[END_REF]. They mutated the 670 phenylalanine which stabilizes the ADH-ADH interaction and allow to obtain dimer of AdhE (about 200kDa). They showed that the ALDH activity was affected only for the physiological reaction and is undisturbed by the mutation in the reverse reaction.

Overall, the filamentation seems to be required for the ALDH activity of the AdhE enzyme. However, neither AdhE D446-449 nor Adhe F670E structures was solved. We hypothesized that the deletion of the 446-449 amino-acids generated a dimer through the ADH-ADH interface, but we are not able to confirm it. Indeed, homologous monofunctional alcohol dehydrogenases and the ADH domain of the AdhE expressed separately seems to be active only as dimer or tetramer.

Regarding to the Adhe F670E , it would be more likely to think that it corresponds to the minimal unit of the AdhE enzyme, namely a dimer of AdhE generated through the ALDH-ALDH interface.

B. Study of anaerobic growth

Thanks to the Keio collection [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection[END_REF], we could obtain the DAdhE K12 Escherichia coli strain. This strain is unable to grow under anaerobic condition in minimal media. We decided to carry out complementation experiments in order to try to restore the anaerobic growth in minimal media. To do so, we decided to clone wild-type adhE and mutant adhE genes into the pKG116 plasmid available in the lab. This is a low-copy chloramphenicol resistant and sodium salicylate-inducible expression plasmid. Cloning of wild-type adhE and D446-449 adhE genes were performed as described in Materials & Methods.

Our results show that the expression of the AdhE WT in the DAdhE E. coli strain allows to restore the anaerobic growth as observed for the wild-type E. coli strain. However, the expression of the AdhE Δ 446-449 cannot rescue the phenotype and present a growth similar to the DAdhE strain (Figure 45) . This complementation analysis confirms that the filamentation of the AdhE associated to the functionally active ALDH enzymatic activity of the AdhE enzyme is required for the anaerobic bacterial growth in minimal media. The presence of the full-length wild-type AdhE and the Δ 446-449 AdhE was confirmed by Western -blot using anti-AdhE antibody (Supplementary Fig. 8).

The structural analysis of the extended conformation of spirosomes revealed a specific loop which could stabilize the ALDHn-ADHn+1 interface. Complementation assay (n=1)

has been performed with two different mutants (D376A-N377A and D376A-N377K) in order to check the capacity of these to restore the anaerobic growth of K12DAdhE strain. The early experiments suggest that D376A-N377A mutant could grow in fermentative condition in minimal media whereas D376A-N377K could not restore this phenotype (Figure 46). This preliminary data could be important to target specifically the ALDHn-ADHn+1 interface. Further experiments are needed in order to confirm the capacity of these two mutants to form filaments.

II. Study of the extension phenomenon

A. Conformational change

As shown by Kessler and colleagues, we incubated spirosomes with Fe 2+ , NAD + and Coenzyme A in order to trigger the conformational change along the filament.

Coenzyme A is not required for the conformational change which occurs also after incubation with only NAD + and iron. We could not be able to induce the filament extension using NADH and iron.

Because of the difficulty of reproducing the extension of spirosomes, we hypothesized that the extension phenomenon is also dependent on other parameters. Time incubation, pH, purification conditions or effectors may play a role for trigger the extension.

The identified positively-charged pocket may also play a role during this extension.

We compared the physiological in vitro activity of WT AdhE and R772L-R773L-R774L-R790I AdhE mutant. We observed that the physiological fermentative activity (Acetyl CoA consumption towards ethanol formation) is largely decreased compared to the WT AdhE activity (Figure 47). Experiment was performed at 37°C at pH 7.

Now, we have to check the activity in the reverse condition (Ethanol consumption) in order to confirm if both ALDH and ADH dehydrogenases activities are affected or only ALDH activity.

Two different mutants have been used in order to restore the anaerobic growth of K12DAdhE strain: R772l-R773L-R774L-R790I and R772E, R773E, R774E, R790E AdhE mutant. We observed that mutants are not able to restore the anaerobic growth in minimal media (Figure 48). These results are preliminary assays (n=2) and have to be reproduce in order to confirm this phenotype. But these first results allow to consider this pocket to be a regulation site which a charged molecule (probably negatively charged) required during the fermentation. As described before, the mutations R772L, R773L, R774L, R790I do not alter the filamentation of AdhE. After purification, AdhE is still purified as filament. We decided to compare the extension between WT AdhE filaments and R772L, R773L, R774L, R790I AdhE filaments. After incubated with NAD + , only WT AdhE filaments were found in an extended conformation whereas R772L, R773L, R774L, R790I AdhE filaments were still in a compact conformation (Figure 49). Even if this initial result has to be confirmed, we could suggest that this positively-charged pocket may be responsible for the binding of an effector which is important for triggering the extension or stabilizing the filament in an extended conformation. The presence of a small molecule linked to the positively-charged pocket in extended E. coli filament structure led us to think that we could facilitate and/or stabilize the conformational change. In order to identify this molecule, we would like to test different chemicals like (p)pp(G)pp, an alarmone present in E. coli which is known to regulate cellular processes such as transcription, protein biosynthesis, biosynthesis and uptake of nucleotides or stringent response [START_REF] Hauryliuk | Recent functional insights into the role of (p)ppGpp in bacterial physiology[END_REF]. We also would like to use nucleotides such as ADP or GDP and cyclic di-nucleotide such as di-AMPc or di-GMPc because they present many negatively charged phosphate which could bind many positively-charged amino-acids present in the pocket. In order to test these molecules, we will have several approaches. In the first instance, we would like to identify whether one of these molecules might affect the enzymatic reaction. In a second step, we will observe if one of these molecules limit the proteolysis of spirosomes using chymotrypsin. Indeed, it has been shown that the proteolysis is lower in presence of ligands such as NAD + , CoA and Fe 2+ (Kessler et al., 1992).

Another interesting observation was done after the enzymatic assay performed in the reverse condition. Indeed, after incubation with ethanol, CoA and NAD + we let the enzymatic reaction at pH 9 at 37°C for 1 hour. Then, we observed filaments in compact conformation. We concluded that in the presence of a large quantity of Acetyl-CoA and NADH, spirosomes are in compact conformation. However, we did not observe extended filaments after letting the physiological reaction occur. Nevertheless, these observations have to be done again in order to confirm these results. Another interesting observation would be to observe whether extended filaments are able to return to a compact state (Figure 50). Electron micrograph of negative stained spirosomes.

B. Study of the localization of AdhE

Interestingly, in some of our first micrographs, we observed filaments merged with membrane stacks. Some spirosomes were stuck to the border of the holes of the cryo-EM grids, this could indicate a hydrophobic affinity. We decided to take a deeper look into the intracellular localization of AdhE.

a. Confocal microscopy

The intracellular localization of AdhE enzyme was investigated using immunolabelling and fluorescence microscopy after bacteria grew in microanaerobic environment.

Micrographs were acquired using confocal microscope.

K12 E. coli strain cultivated under microanaerobic environment was used as a reference in order to compare the localization of expressed AdhE in DAdhE K12 strain. Further experiments need to be performed in order to visualize tagged WT and mutant AdhE in vivo in E. coli bacteria. Bacterial cultures and labelling are still in progress.

The objective of this thesis is to study the structure of bacterial spirosomes and based on these structures investigate the function of spirosomes in bacteria. Indeed, it has been proposed that filament formation could be a general mechanism in order to store inactivated metabolic enzymes during starvation [START_REF] Petrovska | Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation[END_REF]. However, it would appear that spirosomes contain activated AdhE with regard to their ability to II. Spirosomes are dynamic filaments

I. Conformational switch and filament regulation

After purification of E. coli spirosomes, we could trigger the extension of spirosomes using either NAD + and iron or NAD + , iron and coenzyme A. We were not able to extend them using NADH and iron as Kim et al did. Kessler et al., have shown that the extension of spirosome is related to a lower proteolytic sensitivity to chymotrypsin.

They also have reported than E. coli incubated in presence of NADH and iron were more resistant to this proteolysis, so they could be in their extended conformational state. Further investigations have to be carried out in order to elucidate if NADH and iron could trigger its conformational change and how. A rapid analysis of spirosomes incubated with NADH showed a compact conformation with NADH associated only in the ADH domain. Based on this result, it seems that NADH could not bind into the ALDH and trigger the conformation change.

We showed that two NAD + cofactors have to be bound to both the ADH and ALDH domains of AdhE in order to trigger the filament extension. Iron is also necessary to be Indeed, this rigid body motion could allow to turn the full filament into an active conformation. Moreover, the adjacent ALDHn+1 domain could then be activated without the need of cofactor binding. Cooperativity behavior is found into oligomers in order to regulate their activity (Park & Horton, 2019).

Our analyses of the 446-449 mutant reveal no ALDH activity in the dimer. Kim et al. have also abolished the ALDH activity by disrupting the ADH -ADH interface. The fact that the filamentation is required for the ALDH activity is also an argument for the regulation. But studies on the structure of the 446-449 mutant could not allow to reveal the organization of these dimer which impede the ALDH activity.

A study reported that Escherichia coli AdhE enzyme was found inactive under aerobic condition and active under anaerobic condition meaning that it exists also a posttranslation regulation [START_REF] Holland-Staley | Aerobic Activity of Escherichia coli Alcohol Dehydrogenase Is Determined by a Single Amino Acid[END_REF]. The conformational switch is triggered with ligands as NAD + and iron but it could also be summitted to other partners. The non-identified small molecule could be involved for the pre-activation of the spirosome. Indeed, a study presents a time-dependent activation of the multienzyme coenzyme A linked aldehyde dehydrogenase [START_REF] Shone | Steady-State and Pre-Steady-State Kinetics of Coenzyme A Linked Aldehyde Dehydrogenase from Escherichia coli[END_REF].

This enzyme includes an NAD-dependant alcohol dehydrogenase which is the now known AdhE enzyme. In their experiment, a lag phase was observed after incubating inactive ALDH enzyme with NAD + or analogues. The time of the activation of this enzyme is dependent on the NAD + concentration. Another study reported that this enzyme behaves as a hysteretic enzyme due to this lag phase after incubation with NAD + and a thiol group [START_REF] Rudolph | Initial Rate Studies of Adenylosuccinate Synthetase with Product and Competitive Inhibitors[END_REF]. A hysteretic behavior corresponds to a slow response to a rapid change in ligands concentration which is often related to a conformational change [START_REF] Frieden | Slow transitions and hysteretic behavior in enzymes[END_REF]. This regulatory molecule could be produced in large quantity under anaerobic condition.

Other enzymes are found as filament and present active and inactive conformations depending on the ligands/products concentrations. Such filamentation arrangement might establish different levels of regulation as cooperative regulation in CTPS2 filaments. [START_REF] Lynch | Filament formation by metabolic enzymes -A new twist on regulation[END_REF] The study of the enzymatic regulation must be done on spirosomes in order to elucidate the advantage of AdhE polymerization.

Bacteria used different strategies to respond to stresses like presence of antibiotics, starvation, or oxidative stress... It has been shown that Gram-negative and Grampositive use different pathways in order to regulate their metabolism [START_REF] Boor | Bacterial Stress Responses : What Doesn ' t Kill Them Can Make Them Stronger[END_REF].

Based on our results, we could assume that E. coli spirosome are found in inactive compact state and active extended state whereas S. pneumoniae, Clostridium [START_REF] Laurenceau | Conserved Streptococcus pneumoniae Spirosomes Suggest a Single Type of Transformation Pilus in Competence[END_REF], Lactobacillus spirosomes which were always purified and observed in an extended state. The spirosomes from the latter does not seems to be regulated as well as E. coli one. It could be interesting to study whether Gram-positive AdhE enzymes behave also as a hysteretic enzyme.

II. Structure-function relationship

Holland-Staley and colleagues have isolated a strain able to express active AdhE protein in aerobic conditions. They have shown that a mutation was present in the promoter and two mutations Ala267Thr and Glu568Lys were present in the structural gene adhE. The Glu568Lys mutation is responsible for the expression in aerobic conditions. The purification of this mutant did not allow to determine a single oligomerization state.

Another interesting question about the filament formation is how AdhE subunits associate into spirosomes. We still have no clue if other factors are recruited or if AdhE self-associate into filaments. We hypothesized that AdhE dimer is a minimal subunit found inside the cell. However, the factor which triggers the polymerization of AdhE remains unknown. It could be interesting to look into the intracellular NAD + /NADH ratio which is a play a central role in energy metabolism.

III. Compartmentalization

It is now well-know that the bacterial cytoplasm is not disordered but well-organized.

Different types of compartmentalization can be mentioned like spore formation, bacterial micro-compartment (Kerfeld, Aussignargues, Zarzycki, Cai, & Sutter, 2018), enzyme filamentation … [START_REF] Cornejo | Compartmentalization and Organelle Formation in Bacteria[END_REF] Dehydrogenases which generate toxic acetaldehyde are also found in bacterial microcompartments (BMCs) called metabolosomes (Figure 54). These assemblies encapsulate an alcohol dehydrogenase and an aldehyde dehydrogenase associated to phosphotransacylase which converts aldehyde into alcohol and carboxylic acid, especially at metabolic branch points. [START_REF] Castellana | Enzyme clustering accelerates processing of intermediates through metabolic channeling[END_REF] As a reminder, acetyl-CoA is one of the major branch points with pyruvate molecule during energy metabolism.

IV. Localization

The filamentation might be also involved in other AdhE role. Our preliminary results about the immunolabelling of the AdhE enzyme as spirosomes or as dimers seem to show different localization. Spirosomes, which are active form AdhE might be localized near the membrane whereas the localization of the mutant is diffused into the cell.

It might be rational to think that functional spirosomes which generate a large quantity of ethanol are present near the membrane in order to minimize the diffusion of the ethanol within the cell in order to maximize its diffusion out of the cell through the plasma membrane. Moreover, it has been reported that many enzymes involved in glycolytic pathway metabolism are localized on the bacterial surfaces (B. [START_REF] Henderson | Bacterial Virulence in the Moonlight : Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease[END_REF][START_REF] Kopeckova | Diverse Localization and Protein Binding Abilities of Dehydrogenase in Pathogenic Bacteria : The Key to its Multifunctionality ?[END_REF]. Further experiments need to be performed in order to confirm these first results about the spirosome localization.

One of the unfinished experiments of this PhD is to observe in vivo bacterial spirosomes using super-resolution optical microscopy. Using this technique, it will be possible to appreciate the cellular localization of the filament and the dynamic of the formation of the spirosome thanks to live cell methodology.

metabolic enzyme have additional biological actions such as a role in bacterial virulence (B. [START_REF] Henderson | Bacterial Virulence in the Moonlight : Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease[END_REF].

The understanding of the role of the AdhE filamentation and its regulation is central in order to correlate its involvement during bacterial pathogenicity. The fundamental research done on spirosomes could serve as basis for discovering molecules able to prevent the extension of AdhE filaments. These molecules would be useful for impairing fermentation in pathogens or optimize the alcoholic fermentation process in biotechnology.

I. Molecular biology

I. Cloning and Mutagenesis

All vectors used in this study are listed in the following table. All these clonings were performed using AQUAcloning technique. Primers were carefully designed using Snapgene software. The adhE E. coli gene was amplified from E. coli K12 genomic DNA using specific primers for each vector. Vectors were also amplified using adequate primers and then the template plasmid was digested using DpnI enzyme. After PCR purification of amplified vectors and inserts, DNA fragments were mixed with molar ratios 3:1 (insert:vector). Then, chemically competent Stellar or DH5a bacteria were transformed and plated on selective antibiotic media. Clones were tested by PCR colony. Positive clones were cultivated in LB media overnight at 37°C under agitation. After plasmid preparation, the adhE gene sequence was checked by sequencing and the reliable plasmid was transformed in chemically competent E. coli BL21(DE3).

All the point mutations were introduced in primer sequences used for amplifying the adhE gene. After digestion by DpnI and purification, the amplified fragment was phosphorylated by T4 polynucleotide kinase and circularized by T4 DNA ligase. Stellar or DH5a bacteria were transformed with the circularized plasmid. Some clones were cultivated overnight in LB media at 37°C under agitation. After plasmid purification, the inserted mutation was validated by sequencing.

All the pKG116 plasmids were transformed in the DadhE K12 strain using electroporation technique. At OD600 = 0.5, DadhE K12 bacteria were pelleted and washed twice with sterile cold water. The plasmids and bacteria were incubated during 5 minutes at 4°C. Then, bacteria were electroporated using Gen Pulser Xcell Electroporation system (Biorad). Electroporated bacteria were incubated with 1 mL of LB media during 45 minutes at 37°C. Finally, about 50 uL were plated onto LB agar plate with the appropriate antibiotic.

II. Protein purification

Bacteria were pelleted at 6000g for 30 min and resuspended in lysis buffer Tris-HCl pH 7.5, 200mM NaCl, cOmplete Protease Inhibitor Cocktail, Dnase (1.25 ug/ml), lysozyme (100 ug/ml). Then, cells were sonicated and debris were harvested at 11000g for 1h at 4°C. The supernatant was loaded on nickel-NTA affinity column and His6-AdhE was eluted with 300mM imidazole. The protein concentration was estimated using Bradford protein assay. The purity of fractions was checked by (12%) SDSpolyacrylamid gel electrophoresis. The expression of AdhE was checked by western blot analysis using anti-AdhE antibody diluted 1/1000 coupled with a secondary antirabbit-HRP antibody diluted 1/5000. Eluted fraction containing the protein of interest was either used for grid preparation or loaded in size exclusion chromatography column.

The presence of spirosomes was validated by negative stain electron microscopy. Five microliters of sample diluted at 0.05 mg/ml was applied on a glow-discharged carboncoated grids (300 mesh Cupper, EMS). After two washes with water and one wash with a drop of 0.75% uranyl formate, the sample was stained for 1 min with one drop of uranyl formate and blotted with Whatman paper. Micrographs were collected using CM120 microscope (FEI) operated at 120kK equipped with charge-coupled device camera.

III. In vitro enzymatic assays

Enzymatic assays were performed at 37 °C by following the absorbance of NADH at 340 nm with infinite M1000 plate-reader (TECAN). Reactions were started by adding of 0.04-4 μM of purified AdhE enzyme. Reductase activities of ALDH-ADH were monitored using 0.2 mM acetyl-CoA and 0.4 mM NADH in Tris-NaCl buffer pH 7.5.

Dehydrogenase activities of ALDH-ADH were monitored using 170 mM ethanol, 1.5 mM NAD + , and 0.2 mM CoA in Tris-NaCl buffer pH 8.8. Three independent experiments were performed each in triplicate.

IV. Multi-angle laser light scattering (MALS)

This part was performed by Laurent Terradot, our collaborator based in IBCP (Institut de biologie et chimie des protéines), Lyon. Size exclusion chromatography (SEC) experiments coupled to multi-angle laser light scattering (MALS) and refractometry (RI)

were performed on a Superdex S200 5/150 GL increase column (GE Healthcare). 25 μl of AdhE mutant protein were injected at a concentration of 10 mg/ml in buffer 0.05M

Tris HCl pH 7.5, 0.2M NaCl. On-line MALS detection was performed with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses (Mw) were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).

V. In vivo complementation

The DadhE K12 strain was kindly provided by the Keio collection [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection[END_REF].

For the complementation experiments, K12 E. coli and DadhE K12 complemented or not preculture were performed in LB containing kanamycin or kanamycin and chloramphenicol antibiotics. The induction of AdhE protein and mutant AdhE protein was induced by adding 1 uM of sodium salicylate. Then, the culture was performed in minimal media (6 g/L Na2HPO4; 3 g/L KH2PO4; 0.5 g/L NaCl; 1g/L NH4Cl; 4 g/L glucose; 0.2 mM MgSO4; 0.1 mM CaCl2; 10 mg/L Thiamine) in a tube filled to the top without agitation at 37°C.

Anaerobic growth curves were monitored every hour with optical density (O.D. 600nm) using spectrophotometer. Three independent experiments were performed each in triplicate using K12 WT as a positive control.

VI. Immunofluorescence and confocal microscopy

After culture, cells were diluted to OD600 = 1 and washed in water. A drop of cells was deposited in a coverslip coated with poly-L-lysin. Then, bacteria were fixed in 4% paraformaldehyde and permeabilized using 100ug ml -1 lysozyme. Non-specific sites were blocked using 2% BSA. AdhE proteins were immunolabelled using an Anti-AdhE polyclonal antibody diluted 1/100 (Agrisera) for 45 minutes followed by Alexa 488 conjugated secondary anti-rabbit antibody (Invitrogen) for 45 minutes. Negative control was performed by omitting the incubation with primary antibody (data not shown).

Observations were performed with a TCS SP8 confocal microscope (Leica) using 63x/1.40 oil objective.

III. Cryo-electron microscopy

VII. Grid preparation

Freshly purified protein was incubated with ligands (5 mM NAD + , 5 μM CoA, 0.3 mM Fe(NH4)(SO4)2 and 3 mM MgSO4) or not. Four microliters of sample diluted at 0.5mg/ml was applied onto glow-discharged (Elmo Cordouan) grids. Then, the excess of sample was immediately blotted away (4 s) with Whatman paper and samples were plunged into liquid ethane cooled by liquid nitrogen using a Vitrobot device (ThermoFisher) at 4°C at 100% humidity. Cryo-grids were clipped into autogrids in order to be transferred in the cassette and finally in the microscope.

VIII. Data acquisition

Movies were recorded on Talos Arctica (ThermoFischer) operated at 200 kV equipped with K2 direct electron detectors at ×36,000 of magnification and a pixel size of 1.13 Å per pixel using SerialEM. Micrographs were collected in a defocus range of -0.4 μm to -2 μm and with a dose of 0.77 electrons per Å 2 per frame.

IX. Image processing and 3D reconstruction

Movies were aligned for beam-induced motion using MotionCor2 and CTF parameters

were assessed using GCTF. The following steps were performed using RELION 3.0 software. Details and statistics about each dataset are provided in the Table 1. Two approaches were used to solve the 3D structure of spirosomes. For single particle analysis (SPA) approach, non-overlapping segments of the AdhE filaments were manually picked and particles were extracted using a box size of 260 pixels. These particles were 2D classified in ten classes. The 2D classes corresponding to distinct orientations of the filaments were selected and used as references to automatically pick segments in all the micrographs. After extraction, several rounds of 2D classification were performed. From a subset containing 10,000 particles, an initial 3D map was reconstructed without imposing symmetry. The whole dataset was then used to refine this 3D map. A homology model of E. coli AdhE was generated with SWISS-MODEL software using the crystal structure of Vibrio parahaemolyticus acetaldehyde dehydrogenase (PDB code 3MY7) of the V. parahaemolyticus AdhE and the structure of Geobacillus thermoglucosidasius ADH domain (PDB code 3ZDR) of G.

thermoglucosidasius AdhE. The generated AdhE model was fitted in the cryo-EM map using UCSF Chimera software. A 3D mask was generated in Chimera to contain only one AdhE dimer and the adjacent ADH domains. This 3D mask was then used for focused refinement in RELION. After CTF refinement and Bayesian polishing, the 3D map was further refined by focused refinement. The densities outside of the 3D mask used above for focused refinement were subtracted and a final refinement was performed. For the map of the extended AdhE filament, C2 local symmetry was applied during this refinement to obtain the final map. The final resolution was calculated with two masked half-maps, using 0.143 Fourier shell correlation (FSC) cut-off criterion.

Local resolution was estimated using RELION. Map visualizations were prepared using UCSF Chimera.

For helical reconstructions (HR), the procedure was similar with the following differences. During manual and automatic picking, overlapping segments of the filaments were used. No initial model was generated. Instead, a cylinder of 150 Å in diameter was used. A first refinement without any symmetry imposed was performed.

The helical twist and rise were determined in real space on this initial model using relion_helix_toolbox (Rotational search and correlation along Z) Then, these helical parameters were used and refined in further refinements.

X. Model building and refinement of atomic models

For the model of AdhE in its extended state, a homology model of AdhE E. coli was fitted in the refined SPA cryo-EM map with C2 local symmetry. Using these models as starting points, an initial 3D model of the AdhE dimer and adjacent ADH domains was manually built in Coot software. The map was sharpened in PHENIX (phenix.

autosharpen). The final model was refined by several rounds of manual refinement in Coot and real-space refinement using phenix.real_space_refine with noncrystallographic symmetry (NCS) restrains. The model was validated using MolProbity and phenix.validation_cryoem implemented in PHENIX software.

For the model of AdhE in its compact state, the models of the ALDH and ADH domains obtained for the extended state were docked in the cryo-EM map obtained by SPA.

ARTICLE

Filamentation of the bacterial bi-functional alcohol/ aldehyde dehydrogenase AdhE is essential for substrate channeling and enzymatic regulation Pauline Pony 1,2 , Chiara Rapisarda 1,2,4 , Laurent Terradot 3 , Esther Marza 1,2 ✉ & Rémi Fronzes 1,2 ✉ Acetaldehyde-alcohol dehydrogenase (AdhE) enzymes are a key metabolic enzyme in bacterial physiology and pathogenicity. They convert acetyl-CoA to ethanol via an acetaldehyde intermediate during ethanol fermentation in an anaerobic environment. This two-step reaction is associated to NAD + regeneration, essential for glycolysis. The bifunctional AdhE enzyme is conserved in all bacterial kingdoms but also in more phylogenetically distant microorganisms such as green microalgae. It is found as an oligomeric form called spirosomes, for which the function remains elusive. Here, we use cryo-electron microscopy to obtain structures of Escherichia coli spirosomes in different conformational states. We show that spirosomes contain active AdhE monomers, and that AdhE filamentation is essential for its activity in vitro and function in vivo. The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling within the filament and for the regulation of enzyme activity. F acultative anaerobe bacteria, such as Escherichia coli, are metabolically versatile and are able to grow under a wide range of oxygen concentrations, from anaerobic conditions in the gut to aerobic environments. This metabolic adaptability is vital for the fitness and survival of commensal and pathogenic bacteria. In the absence or low concentrations of oxygen, bacteria use fermentation to survive. The bifunctional alcohol-acetaldehyde dehydrogenase enzyme (AdhE) is a key metabolic enzyme of the alcoholic fermentation pathway. This 96 kDa enzyme is highly expressed in the absence of oxygen and is essential for ethanol production in E. coli 1 . It is composed of a N-terminal aldehyde dehydrogenase (AlDH) domain and a C-terminal iron-dependent alcohol dehydrogenase (ADH) domain. AdhE converts acetyl-coenzyme A to acetaldehyde and then to ethanol, in a two-step reaction that is coupled with the oxidation of two NADH molecules into NAD + . The biochemistry of AdhE enzymatic activities is well characterized 2,3 . In particular, a lot of investigations looked into this key fermentative enzyme to engineer novel ethanolproducing bacteria 4,5 or identify bacterial strains capable of growing under ethanol 6 .

Interestingly, the biological role of AdhE seems to go beyond alcoholic fermentation. This protein could also be directly or indirectly involved in bacterial pathogenicity. In Listeria monocytogenes, the Listeria adhesion protein LAP (homologous to AdhE) promotes bacterial adhesion by interacting with a receptor of intestine cells 7,8 . It has also been reported in Streptococcus pneumoniae that AdhE also promotes bacterial adherence 9 . In E. coli, this enzyme would play a role during colonization by regulating the expression of key virulence genes 10 . Finally, AdhE could also act as a peroxide scavenger under aerobic conditions 11 .

Although alcohol and aldehyde dehydrogenases are found as monofunctional enzymes in all kingdom of life, the bifunctional enzyme AdhE is found mostly in bacteria and in some unicellular eukaryotes such as microalgae. Intriguingly, AdhE is capable of oligomerization and forms a filament (identified as spirosome in early studies). These helical macromolecular assemblies were observed for the first time in the 1970s in bacteria 12 . These filaments of AdhE are widely conserved in bacteria 13 and also in phylogenetically distant organisms such as Chlamydomonas reinhardtii 2 and Entamoeba histolytica 14 . Although their wide conservation probably highlights an essential function, the exact role of AdhE filaments remains unknown. Recently, the cryoelectron microscopy (cryoEM) structure of the spirosome from E. coli was reported 15 . This study confirmed that AdhE is composed of canonical aldehyde dehydrogenase (AlDH) and ADH domains, interconnected by a short linker. As proposed in earlier studies 15,16 , the AdhE oligomerization interfaces are mediated by canonical ADH-ADH and AlDH-AlDH dimerization interfaces. In the spirosomes, AdhE monomers are interlocked in a head-tohead manner through the AlDH-AlDH dimerization interface. These dimers are further assembled into a helical filament through ADH domain dimerization. Finally, it was shown that spirosomes display AlDH and ADH activities, and that the integrity of the ADH-ADH interface, which is essential for the spirosome assembly, is also essential for AdhE activity in vitro 15 .

Here we used cryoEM to obtain structures of E. coli spirosomes in different conformational states using helical reconstruction (HR) and single-particle analysis (SPA). The detailed analysis of these structures provides insight showing that AdhE filamentation is essential for substrate channeling between the AlDH and ADH domains, and for the regulation of enzyme activity. Finally, we confirm that spirosomes contain active AdhE monomers and show that AdhE filamentation is essential for AlDH activity in vitro and AdhE function in vivo.

Results

CryoEM structure of E. coli spirosomes in different states. In early studies, Kessler et al. 17 reported that the E. coli spirosomes were found in closed (compact) or open (extended) conformations and would change their conformation upon the addition of ligands. Strikingly, we and others reported the observation of native spirosomes isolated from various bacterial and eukaryotic species, all found exclusively in the open/extended conformation. The conformational change observed, which seems to depend on the binding of the cofactors, could be an important feature for the regulation of its activity.

The adhE E. coli gene was cloned in a high copy number vector and expressed in BL21 E. coli cells. Recombinant N-terminally His 6 -tagged AdhE was purified by Nickel-nitrilotriacetic acid affinity chromatography followed by gel filtration. The fraction containing filaments was collected in the void volume of the gel filtration column (Supplementary Fig. 1b) (see Methods section for details). Purified spirosomes were incubated with various combinations of ligands to determine the conditions, which trigger this conformational change (Fig. 1a). Samples were deposited on cryoEM grids and vitrified in liquid ethane. Micrographs of frozen-hydrated spirosomes were collected using a Talos Arctica cryo-electron micoscope equipped with K2 summit direct electron detector. Contrast transfer function (CTF)-corrected and re-aligned movies were analyzed using RELION 3 software 18 . Non-overlapping helical segments were sorted with several rounds of two-dimensional (2D) classification. In the classes obtained, we could clearly distinguish the compact and extended conformations of the spirosomes, and we confirmed that in the absence of ligand (apo), the spirosomes are compact (Fig. 1a). Incubation with NAD + and Fe 2+ is sufficient to extend the filaments. The addition of Coenzyme A does not impair the conformational change triggered by NAD + and Fe 2+ (Fig. 1a). Interestingly, we show that in the same conditions, NADH and Fe 2+ are not able to trigger a conformational change from the compact to the extended form (Fig. 1a). However, it was reported that NADH could trigger the spirosomes extension 15 . Although similar conditions were used for protein and ligand concentrations, we could not reproduce this result.

The recently reported cryoEM structure of the E. coli spirosome was obtained in the absence of ligand (apo-form). Therefore, the spirosomes were observed in the closed/compact conformation. To obtain the structure of the E. coli spirosomes in their extended and compact form, both in the presence of ligands, we collected larger datasets of the spirosomes incubated with NAD + , Fe 2+ , and CoA or with NADH and Fe 2+ . As in both cases the filaments were rather short and flexible, we chose to use SPA to solve their structure. However, we also used HR in parallel to uncover the helical symmetry parameters of these filaments and obtain a cryoEM map of the complete assemblies. For SPA, after 2D classification, an initial model was generated and refined in RELION. The structure corresponding to the AlDH and ADH domains were identified in the map. The part of the map corresponding to one AdhE dimer in interaction with two adjacent ADH domains (colored in Fig. 1b,c) were further refined by focused refinement. The final map was obtained after Bayesian polishing, subtraction, and focused refinement. A resolution of 3.2 Å and 3.8 Å was obtained for the extended and compact spirosome, respectively (Supplementary Fig. 2). For HR, after 2D classification, an initial map was reconstructed without any symmetry imposed using a feature-less cylinder as a three-dimensional (3D) reference. The helical symmetry of the AdhE filaments was determined in real space and was imposed during further refinements. The final 3D reconstruction was obtained after Bayesian polishing. The overall resolution of the ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15214-y final masked map was estimated to be 4.4 Å and 5 Å for the extended and compact spirosomes, respectively (Supplementary Fig. 2). The refined helical symmetry was 56.5 Å rise and 164.4°t wist of the extended spirosome, and 34.7 Å and 154.5°twist for the compact spirosome. The maps used to reconstitute the complete spirosome assembly were obtained after imposing these symmetry parameters to the central 30% of the maps obtained (Fig. 1d, e and Supplementary Fig. 3).

Structure of the AdhE filament in its extended state. As this work was started before the publication of the work by Kim et al. 15 , homology models of AdhE AlDH and ADH domains obtained using Swissmodel 19 were docked in the map of the extended spirosome obtained by SPA. A complete structural model was rebuilt inside the densities using Coot and then refined and validated using PHENIX 20,21 (see Methods section for details). As described previously 15 , the AdhE monomer is composed of an N-terminal AlDH domain connected by a linker region to the C-terminal ADH domain (Fig. 2a). Each domain resembles the canonical AlDH and ADH domains found in the corresponding monofunctional enzymes. From N to C terminus of each domain, they are composed of a NAD-binding domain and catalytic domain. Two AdhE protomers are interlocked with apparent C2 symmetry to form the AdhE dimer (Fig. 2b). These protomers will be named (α or β) and their respective domains α-AlDH, α-ADH, β-AlDH, or β-ADH. Clear density for NAD + and Fe 2+ could be observed in the density map in the AlDH and ADH domains in both subunits of the AdhE dimer (Fig. 2a). The AdhE helical assembly can be described as one AdhE dimer repeated along a right-handed helix with a helical twist of 164.5 Å and a rise of 56.4 Å (Fig. 1c). To obtain a model of the AdhE filament, the AdhE dimer was docked in the map obtained by HR and duplicated using the helical parameters. Based on these helical parameters and polarity, the AdhE dimers will be numbered n, n + 1, n + … Structure of the AdhE filament in its compact state. The structure of the AdhE spirosome in its compact state was obtained using the same strategy. The overall structure of the AdhE monomer is unchanged (Fig. 2d). Clear density for Fe 2+ and NADH could be found in the ADH domain (Fig. 2d). No density is visible for NADH within the AlDH domain. Within the AdhE dimer, both subunits are related by apparent C2 symmetry (Fig. 2e). Using the helical parameters and map obtained by HR, we could build a structural model for the spirosome in its compact state (Fig. 2f). We will use the same nomenclature for AdhE domains and protomers than described above. We notice that this structure is highly similar to the structure recently published in its apo-form 15 (Supplementary Fig. 4).

Inter-domain interactions within the extended AdhE filament. In the extended AdhE filament, three different inter-domain interfaces are formed. Within each AdhE dimer, two interfaces are found. The dimer is maintained through an AlDH-AlDH interface (named (α-AlDH)n/(β-AlDH)n interface) and two symmetric AlDH-ADH interfaces (named (α-AlDH)n/(β-ADH) n and (α-ADH)n/(β-AlDH)n interfaces) (Fig. 2b, e and Supplementary Fig. 5). Along the helical assembly, on both sides of the dimer n, are found two equivalent interfaces between ADH domains (named (α-ADH)n -1/(β-ADH)n) and (α-ADH)n/(β-ADH)n + 1). The (α-AlDH)n/(β-AlDH)n interface is characterized by hydrogen bonds (Supplementary Figs. 5a and6b). The (α-AlDH)n/(β-ADH)n and (α-ADH)n/(β-AlDH)n interfaces are maintained by five salt bridges and a vast network hydrogen bonds scattered along the interfaces (Supplementary Figs. 5b and6a). Moreover, the dimer is stabilized by the interaction Comparison with monofunctional enzymes. The structure of AdhE was compared with the structure of monofunctional ADH or AlDH enzymes. We selected homologous monofunctional enzymes with active sites of similar topology than AdhE using the COACH server 22,23 . For AdhE AlDH domain, the crystal structure of Rhodopseudomonas palustris propionaldehyde dehydrogenase (PDB 5JFL) was identified. For AdhE ADH domain, the crystal structure of the lactaldehyde : 1,2-propanediol oxidoreductase of E. coli (PDB 2BL4) was identified. These monofunctional enzymes were compared with the corresponding AlDH and ADH domains in AdhE.

As shown in Fig. 3a, b, the overall canonical fold found in AlDH and ADH enzymes is conserved in AdhE. Interestingly, the monofunctional enzymes are all active as dimers or tetramers. By superimposing these structures with AdhE, we could observe that the interfaces present in dimers of monofunctional enzymes correspond to the ADH-ADH and AlDH-AlDH interfaces of the extended spirosomes (Fig. 3a,b). Of note, in the AdhE filament in its compact state, the conformation of the AlDH domain and the AlDH-AlDH interface are not superimposable with their counterpart in the monofunctional enzymes. Sequence alignments between E. coli AdhE and monofunctional ADH and AlDH enzymes reveal the presence of three loops that are specific to AdhE (Fig. 3c and Supplementary Data 1 and 2). Importantly, these loops are located close to the AlDH active site (loop1, residues 413-433) or at the AlDH-ADH interface in the AdhE dimer (loops 2 and 3, residues 561-589 and 758-772). Even if their sequence can vary, these loops are always present in the AdhE homologs (Fig. 2c and Supplementary Data 3).

The interface between the AlDH and ADH domains. In the monofunctional AlDH and ADH enzymes, access to the active site is possible by a continuous channel embedded between the catalytic and NAD-binding domains (Fig. 4a). On one side, one channel allows the binding of the NAD + /NADH (and of Acetyl-CoA for the AlDH) in the catalytic pockets. On the other side of the domains, a channel allows the entrance/exit of the substrates/products of the enzymatic reactions (ethanol and acetaldehyde). As shown in Fig. 4a, these channels are conserved in the AlDH and ADH domains of AdhE. Remarkably, the substrate/product channels of both the AlDH and ADH domains lead to the two cavities located at the AlDH-ADH interfaces within the AdhE dimer. The loops 2 and 3 seal this cavity by mediating the interactions between the AlDH and ADH domains (Fig. 4b). This allows a direct channeling between the AlDH and ADH domain active sites (Fig. 4c).

Extended spirosomes contain catalytically active AdhE monomers. The comparison of AdhE structures in the absence of cofactors 15 , in the presence of cofactor bound only to the ADH domain or to both ADH and AlDH domains provides valuable insight about the regulation of AdhE enzymatic activities. Although very little differences are observed between the structures of the Apo-ADH and NADH-bound ADH domains (Supplementary Fig. 4), the binding of NAD + in the AlDH domain induces a significant conformational change in this domain. Cofactor binding induces a closure of the AlDH catalytic site onto the NAD/NADH-binding domain (Fig. 5a). This induced-fit conformational change orients the key catalytic residues (Cys 246, His 367, and Glu 335) in the optimal orientation for catalysis (Fig. 5b). At the level of the whole domain, it modifies the orientation of the catalytic domain relative to the NAD/NADHbinding domain (Fig. 5c). This conformational change induced by the cofactor binding seems to be specific to AdhE. In homologous monofunctional AlDH enzymes, the cofactor binding site and catalytic residues adopt the same topology in the absence and in the presence of cofactor.

AdhE filamentation is essential for AlDH activity in vitro. The activities of AlDH and ADH domains within the purified spirosome fractions were recently reported in vitro by using absorbance properties of NADH at 340 nm. We performed similar activity assays either in physiological conditions (Ethanol production) (at pH 7 in the presence of acetyl-CoA and NADH) and in conditions forcing the reverse reaction (from Ethanol to Acetyl-CoA) (pH 8.8 in the presence of ethanol, NAD + , and CoA). Our results confirm that both AlDH and ADH domains are functional in vitro within the spirosome fraction (Fig. 6c). However, the spirosome fraction contains spirosomes but is also contaminated with smaller AdhE assemblies (tetramers, dimers, etc.). In order to confirm that AdhE filaments are active, it is necessary to disrupt AdhE filamentation and monitor AdhE activity. The AlDH-AlDH and ADH-ADH interfaces are conserved across homologous monofunctional AlDH and ADH enzymes. The vast majority of these monofunctional proteins are active as dimer and the conservation of these interfaces along the filament could be necessary for the activity of the domains. Therefore, we decided to shorten the linker between the AlDH and ADH domain to perturb the dimerization of AdhE. Guided by the structure, we generated a Δ446-449 AdhE mutant. We showed that this deletion prevents the filamentation of AdhE (Fig. 6b and Supplementary Fig. 7a). Negative stain EM shows that the Δ446-449AdhE mutant does not assemble into long filament as Wild-Type AdHE (WT AdhE) (Fig. 6b). Size-exclusion chromatography experiments coupled to multi-angle laser light scattering (SEC-MALS) experiments determined that this mutant has a molecular mass of 200 kDa, suggesting that a dimer still formed (Supplementary Fig. 7b). The activities of the AlDH and ADH domains of Δ446-449 AdhE were also monitored. In physiological condition (pH 7), the Δ446-449 AdhE activity was drastically reduced compared with the WT AdhE activity (Fig. 6c). Indeed, the specific activities calculated for the WT AdhE and Δ446-449 AdhE were 0.112 and 0.035 mmol min -1 mg -1 , respectively. In reverse condition, the Δ446-449 AdhE activity (0.351 mmol min -1 mg -1 ) was not affected compared with WT AdhE (0.336 mmol min -1 mg -1 ). These results show that the activity of the AlDH domain was strongly affected, while the ADH domain was still functional when AdhE filamentaion is disrupted. Filamentation is essential for AdhE activity in vivo. The importance of AdhE filamentation on AdhE activity was established in vitro (this work and ref. 15 ). However, it was still unclear whether it was essential for AdhE activity in vivo during fermentation. To test the activity of the full-length AdhE and the Δ446-449 AdhE in vivo, complementation assays were carried out using a ΔAdhE K12 strain 24 as described in Methods section. ΔAdhE E. coli was transformed with plasmid PKG116 expressing full-length AdhE, leading to restoration of bacterial growth in minimal medium in a low concentration of oxygen. In contrast, the strain transformed with PKG116 expressing Δ446-449 AdhE was not able to grow under the same conditions (Fig. 6d). These results show that the filamentation of AdhE and functional AlDH enzymatic activity is not only necessary for ethanol production during fermentation but also for regenerating NAD + pool required for bacterial growth. The presence of the full-length AdhE and the Δ446-449 AdhE was confirmed by western blotting using anti-AdhE antibody (Supplementary Fig. 8a,b).

Discussion

Recently, a study reported the structure of the compact conformation of the AdhE spirosomes 15 in the absence of any cofactor bound. Here we present the structure of the spirosomes in the presence of cofactors in their compact and extended conformation. We reveal the specific organization between AdhE monomers with three different interfaces along the filament as follows: AlDH-AlDH interface, ADH-ADH interface, and AlDH-ADH interface. Although the AlDH-AlDH and ADH-ADH interfaces are conserved in the corresponding monofunctional enzymes, the AlDH-ADH interface is specific to AdhE homologs. At this interface, specific loops inserted in the ADH domain (loops 2 and 3) define a channel connecting directly the active sites of one ADH domain to its adjacent AlDH domain. During alcoholic fermentation, this substrate channeling prevents the acetaldehyde produced by the AlDH domain, a highly toxic intermediate, to be released in the cytoplasm. In addition, this channeling is probably important for an efficient coupling of the AlDH and ADH enzymatic activities. Interestingly, bacteria evolved other strategies to confine multi-enzymatic reactions and prevent the release of toxic intermediates 25 . In Salmonella, bacterial microcompartment resembling viral capsids encapsulate aldehyde and ADH activities to optimize catalysis and prevent the release of toxic intermediates 26 .

Early studies showed that the addition of cofactors triggers a conformational change in E. coli spirosomes. Although these results were recently confirmed, the molecular basis of this phenomenon was not understood. In our study, we show that cofactor binding to both the ADH and AlDH domains of AdhE is required to trigger AdhE filament extension. Therefore, only extended filaments are bound to cofactors and are able to catalyze the two-step reaction of conversion of acetyl-CoA to ethanol. Our structural data show that, in the compact AdhE filament, the AlDH domain is in a conformation that is not favorable for cofactor binding and for catalysis. We show that cofactor binding to this domain triggers an induced-fit conformational change, which activates this active site and induces the closure motion of the AlDH n catalytic domain onto the cofactor binding domain in the AlDH domain. In turn, this motion is transmitted to the ADHn-ADH n + 1 dimer, which behaves as a rigid body moving towards the center and extremity of the filament. As they allow a tight contact between the AlDH and ADH domains, the ADH loops 2 and 3 are essential to the transmission of this motion. The AlDH n + 1 domain contacting the ADH n + 1 is in turn affected by the motion and switches its active site into an active conformation without the need of cofactor binding. We propose that, in the extended filament, a fraction of the AlDH domains bound to cofactors maintains the other AlDH active sites in an active conformation, optimal for substrate and cofactor binding and for catalysis. The observation that AlDH activity is impaired when AdhE filamentation is prevented supports the hypothesis of a cooperative behavior between AlDH active sites within the AdhE filament. In the mutants generated by us and others, the AlDH active site or dimerization interface is not directly affected by the mutation(s). However, each AlDH active site of the AdhE dimer needs to convert from inactive to active conformation at each catalytic cycle. Interestingly, we do not observe any filament extension in the presence of NADH. While this cofactor is bound to the ADH domain, its binding to the AlDH domain could be dependent on the covalent binding of the acetyl moiety to the catalytic cysteine residue or on the binding of NAD + to other AlDH domains within the filament. Furthermore, it has already been reported for the betaine aldehyde dehydrogenase 27 that the kinetic of NADH binding could be affected by the prior binding of NAD + or the aldehyde. More than 20 enzymes self-assemble as polymers (as reviewed in ref. 28 ). For the vast majority of them, polymer formation is essential for enzyme activity. For AdhE, we confirm the previous observation that AdhE filamentation is necessary for its activity in vitro. In addition, we show that this filamentation is essential in vivo during fermentation and anaerobic growth in minimal medium. However, until now, the exact role of filamentation in AdhE function was unclear. Indeed, the recent structure of the spirosome in their compact conformational state 15 did not bring any clear answer to this question.

We propose that the conformational switch from compact to extended conformation could be used by the bacteria to regulate AdhE activity. The compact conformation would be a storage form of inactive AdhE protomers. In low oxygen conditions, the pool of NADH to be oxidized to NAD + to be used in the glycolytic process increases in the bacterial cell. NADH binding to some AdhE subunits within the spirosomes would have a cooperative effect to convert the whole spirosome from compact to extended conformation and quickly activate all the AdhE subunit within the whole filament. The acetyl-CoA binding would be required for binding of the NADH to the AlDH domain. As long as the pool of NADH stays high within the cell, the AdhE filaments would stay extended and highly active. Interestingly, native spirosomes isolated from other bacteria or unicellular eukaryotic organisms all display an extended conformation. For example, we previously showed that spirosomes isolated from Clostridium difficile or streptococcal species, all grown in fermentative conditions, are in an extended conformation. It remains to establish whether these filaments are intrinsically extended or share the same conformational switch with E. coli spirosomes. Nevertheless, molecules that would prevent or stimulate the extension of AdhE filaments would be valuable tools to impair fermentation in pathogens or optimize the alcoholic fermentation process in biotechnology.

Methods

Spirosome expression and purification. The gene encoding AdhE from E. coli (AdhE E.coli ) was amplified from E. coli K12 genomic DNA (Supplementary Fig. 9) and cloned into the pET15 expression vector resulting in a sequence coding for a N-terminal hexahistidine tagged AdhE (His 6 -AdhE). Plasmids from a single clone were isolated, checked by sequencing and transformed into E. coli BL21(DE3) bacteria. Bacteria were grown in Luria-Bertani (LB) media at 37 °C, 180 r.p.m. to exponential phase. From OD 600 = 0.6-0.7, overexpression was induced by adding 1 mM isopropryl β-D-1 thiogalactopyranoside for 16 h at 16 °C. Then, cells were pelleted at 6000 × g for 30 min and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, cOmplete Protease Inhibitor Cocktail, DNase (1.25 μg ml -1 ), lysozyme (100 μg ml -1 )). Cells were sonicated and debris were pelleted at 11,000 × g for 1 h. The supernatant was loaded on a nickel-NTA affinity column (GE Healthcare) and His 6 -AdhE was eluted with 300 mM imidazole. Fractions containing AdhE were loaded on a Superdex200 (GE Healthcare) sizeexclusion column. Purity of AdhE was confirmed with 12% SDS-polyacrylamide gel electrophoresis. The presence of spirosomes was checked by negative stain electron microscopy. Five microliters of sample was applied on a glow-discharged carbon-coated grids (300 mesh Cupper, EMS). After two washes with water and one wash with a drop of 0.75% uranyl formate, the sample was stained for 1 min with one drop of uranyl formate and blotted with Whatman paper. Micrographs were collected using CM120 microscope (FEI) operated at 120 kV equipped with charge-coupled device camera.

Cryo-electron microscopy. Freshly purified protein was incubated with 5 mM NAD + , 5 µM CoA, 0.3 mM Fe(NH 4 )(SO 4 ) 2 , and 3 mM MgSO 4 , and applied to cryoEM grids. Samples were vitrified with a Vitrobot (ThermoFisher) at 4 °C at 100% humidity. Four microliters of sample was applied onto glow-discharged (Elmo Cordouan) grids. The excess of sample was immediately blotted away (4 s) with Whatman paper and plunged into liquid ethane cooled by liquid nitrogen. Movies were recorded on Talos Arctica (ThermoFischer) operated at 200 kV equipped with K2 direct electron detectors at ×36,000 of magnification and a pixel size of 1.13 Å per pixel using SerialEM 29 . Micrographs were collected in a defocus range of -0.4 μm to -2 μm and with a dose of 0.77 electrons per Å 2 per frame.

Image processing and 3D reconstruction. Movies were aligned for beam-induced motion using MotionCor2 and CTF parameters were assessed using GCTF. The following steps were performed using RELION 3.0 software. Details and statistics about each dataset are provided in the Table 1.

For SPA approach, non-overlapping segments of the AdhE filaments were manually picked and particles were extracted using a box size of 260 pixels. These particles were 2D classified in ten classes. The 2D classes corresponding to distinct orientations of the filaments were selected and used as references to automatically pick segments in all the micrographs After extraction, several rounds of 2D classification were performed. From a subset containing 10,000 particles, an initial 3D map was reconstructed without imposing symmetry. The whole dataset was then used to refine this 3D map. A homology model of E. coli AdhE was generated with SWISS-MODEL 19 using the crystal structure of Vibrio parahaemolyticus acetaldehyde dehydrogenase (PDB code 3MY7) of the V. parahaemolyticus AdhE and the structure of Geobacillus thermoglucosidasius ADH domain (PDB code 3ZDR) 16 of G. thermoglucosidasius AdhE. The generated AdhE model was fitted in the cryoEM map using UCSF Chimera 30 . A 3D mask was generated in Chimera to contain only one AdhE dimer and the adjacent ADH domains. This 3D mask was then used for focused refinement in RELION. After CTF refinement and Bayesian polishing, the 3D map was further refined by focused refinement. The densities outside of the 3D mask used above for focused refinement were subtracted and a final refinement was performed. For the map of the extended AdhE filament, C2 local symmetry was applied during this refinement to obtain the final map. The final resolution was calculated with two masked half-maps, using 0.143 Fourier shell correlation (FSC) cut-off criterion. Local resolution was estimated using RELION (Supplementary Fig. 3). Map visualizations were prepared using UCSF Chimera.

For HR, the procedure was similar with the following differences. During manual and automatic picking, overlapping segments of the filaments were used. No initial model was generated. Instead, a cylinder of 150 Å in diameter was used. A first refinement without any symmetry imposed was performed. The helical twist and rise were determined in real space on this initial model using relion_helix_toolbox (Rotational search and correlation along Z) Then, these helical parameters were used and refined in further refinements.

Model building and refinement of atomic models. For the model of AdhE in its extended state, a homology model of AdhE E.coli was fitted in the refined SPA cryoEM map with C2 local symmetry. Using these models as starting points, an initial 3D model of the AdhE dimer and adjacent ADH domains was manually built in Coot 31 . The map was sharpened in PHENIX 20,21 (phenix. autosharpen). The final model was refined by several rounds of manual refinement in Coot software and real-space refinement using phenix.real_spa-ce_refine with non-crystallographic symmetry (NCS) restrains. The model was validated using MolProbity 32 and phenix.validation_cryoem implemented in PHENIX software.

For the model of AdhE in its compact state, the models of the AlDH and ADH domains obtained for the extended state were docked in the cryoEM map obtained by SPA. The cryoEM map was sharpened in PHENIX (phenix.autosharpen). An initial 3D model of the AdhE dimer and adjacent ADH domains was manually built in Coot. The final model was refined by several rounds of manual refinement in Coot software and real-space refinement using phenix.real_space_refine with NCS and reference model restrains. The reference model used was the model obtained for AdhE in its extended state. The model was validated using MolProbity and phenix.validation_cryoem implemented in PHENIX software.

For the models of the AdhE filaments in their extended and compact states, the corresponding model of the AdhE dimer were docked into the cryoEM HR maps obtained. These maps were sharpened in PHENIX and symmetrized in RELION using the refined helical parameters. The model was refined using using phenix.real_space_refine with NCS and reference model restrains. The reference model used was the AdhE model in its extended state. The model was validated using MolProbity and phenix.validation_cryoem implemented in PHENIX software. Mutagenesis. His 6 -AdhE-pET15 plasmid was amplified using 5′-AACATGTTGT GGCACAAAC-3′ and 5′-AGCAACGGTTTTCTTGTTG-3′, to delete the coding region corresponding of 446-449 amino acids. After DpnI digestion, amplified fragment was phosphorylated using T4 polynucleotide kinase (NEB) and then ligated using T4 DNA ligase (NEB). DH5α were transformed with this plasmid. Plasmids from one single clone were extracted and checked by sequencing.

Multi-angle laser light scattering. SEC-MALS and refractometry were performed on a Superdex S200 5/150 GL increase column (GE Healthcare). Twenty-five microliters of AdhE mutant protein were injected at a concentration of 10 mg ml -1 in buffer 0.05 M Tris-HCl pH 7.5, 0.2 M NaCl. On-line MALS detection was performed with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).

Enzymatic assays. Enzymatic assays were performed at 37 °C by following the absorbance of NADH at 340 nm with infinite M1000 plate-reader (TECAN). Reactions were started by adding of 0.04-4 μM of purified enzyme. Reductase activities of AlDH-ADH were monitored using 0.2 mM acetyl-CoA and 0.4 mM NADH in Tris-NaCl buffer pH 7.5. Dehydrogenase activities of AlDH-ADH were monitored using 1% ethanol, 1.5 mM NAD + , and 0.2 mM CoA in Tris-NaCl buffer pH 8.8. Three independent experiments were performed each in triplicate.

Anaerobic growth of complemented Δadhe K12 strain. The Δadhe K12 strain was provided by the Keio Collection 24 . Two versions of the low copy PKG116 plasmid were constructed using AQUAcloning method: PKG116-AdhE WT and PKG116-AdhE Δ446-449 . Each plasmid was incorporated into the Δadhe K12 strain using electroporation technique. Bacteria were grown in LB medium containing 1 μM of sodium salicylate and then back diluted at OD 600 = 0.1 in minimum media. Anaerobic growth curves were monitored using optical density (OD 600 ).

Three independent experiments were performed each in triplicate using K12 WT as a positive control. The expression of AdhE was checked by western blot analysis using anti-AdhE antibody (AS10748, Agrisera) diluted 1/1000 coupled with a secondary anti-rabbit-HRP (AB_10015282, Jackson ImmunoResearch) antibody diluted 1/5000.

Reporting

Figure 1 : 28 Figure 2 : 30 Figure 3 : 31 Figure 4 : 32 Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :ResultsFigure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 26 :Figure 27 :Figure 28 :Figure 29 :Figure 30 :Figure 31 :Figure 32 :Figure 34 :Figure 35 :Figure 38 :Figure 39 :Figure 41 :Figure 43 :Figure 44 :Figure 45 :Figure 47 :Figure 48 :Figure 49 :Figure 50 :Figure 53 :Abbreviations

 12823033143256789101112131415161718192021222324252627282930313234353839414344454748495053 Figure 1: Louis Pasteur and his experiment about fermentation ............................... 28

Figure 1 :

 1 Figure 1: Louis Pasteur and his experiment about fermentation

Figure 2 :

 2 Figure 2: Fermentation pathways of E. coli using different sugars.

Figure 3 :

 3 Figure 3: Overview of different fermentation pathways of S. pneumoniae.From[START_REF] Gaspar | Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood[END_REF]

Figure 4 :

 4 Figure 4: Presentation of fermentation pathways of lactic acid bacteria. Homofermentation (left) involves lactate dehydrogenase (1) while heterofermentation

Figure 7 :

 7 Figure 7: Nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) coenzyme structures.

  PFL) or pyruvate ferredoxin oxidoreductase (POR) to convert pyruvate to acetyl Coenzyme A. Then, aldehyde (ALDH) and alcohol (ADH) dehydrogenases mediate the oxidation of acetyl-CoA into ethanol.

Figure 8 :

 8 Figure 8: Representation of the two-reaction pathway to the production of ethanol.

Figure 9 :

 9 Figure 9: Micrographs of examples of enzyme filamentation. (A) Filaments of acetyl-CoA carboxylase. (B) Filaments of CTP synthase (CTPS)

Figure 10 :

 10 Figure 10: Observations of spirosomes.

Figure 11 :

 11 Figure 11: adhE gene organization.

Figure 13 :

 13 Figure 13: Presence of spirosomes in C. reinhardtii (A) and E. histolytica (B) eukaryotic micro-organisms.([START_REF] Espinosa | The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact Cterminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity[END_REF][START_REF] Van Lis | Concerted up-regulation of aldehyde/alcohol dehydrogenase (ADHE) and starch in Chlamydomonas reinhardtii increases survival under dark anoxia[END_REF]

Figure 14 :

 14 Figure 14: Observation of conformation change depending on the cofactors incubated with. Compact (condensed) form is presented in A and extended (open) conformation is

Figure 15 :

 15 Figure 15: Representation of the number of released structures per year.

Figure 16 :

 16 Figure 16: First membrane protein model (bacteriorhodopsin) obtained using electron microscopy in 1975.(A) Overview of the transmembrane helices of the bacteriorhodopsin obtained using

Figure 17 :

 17 Figure 17: Evolution of the resolution capacity represented on the structure of the galactosidase enzyme.From (Veronica Falconieri, Sriram Subramaniam, National Cancer Institute, National

Figure 18 :

 18 Figure 18: Sample preparation work flow for automated cryo-electron microscope.

Figure 19 :

 19 Figure 19: Schematic representation of a transmission electron microscope.

Figure 20 :

 20 Figure 20: Electron/sample interactions.

Figure 21 :

 21 Figure 21: Detection quantum efficient in function of spatial frequency.

  Part I: Structural study of Escherichia coli spirosomes As explained before, Escherichia coli spirosomes could be found in different conformational states, in either compact (closed) or extended (open) conformation in function of ligands incubated with them. In order to understand this conformational change, we decided to solve the structure of the two different conformations using cryo-electron microscopy.

Figure 22 :

 22 Figure 22: Purified compact Escherichia coli spirosomes. (A) SDS-PAGE of purified AdhE enzyme. (B) Negative stain micrograph of purified

  The compact structure reveals the spatial arrangement of the two domains composing the bifunctional acetaldehyde -alcohol dehydrogenase enzyme. The E. coli AdhE (1-891) is composed of a N-terminal acetaldehyde dehydrogenase (ALDH) domain (1-449) linked to a C-terminal alcohol dehydrogenase (ADH) domain. This linker (440-451) is a short flexible region interconnecting the two domains allowing the formation of AdhE dimers (Figure24B). AdhE dimer is a symmetric unit forming two acetaldehyde -alcohol dehydrogenase (ALDH -ADH) interfaces and one acetaldehyde-acetaldehyde dehydrogenase (ALDH -ALDH) interface. The oligomerization of AdhE dimers (named n, n+1, n-1, …) assembles the spirosome thanks to the interaction of two alcohol-alcohol dehydrogenase (ADH -ADH) domain in a tail-to-tail manner. The compact filament is a right-handed helix which has a rise of 34.7Å and helical twist of 154.4Å (Figure23B).

Figure 23 :

 23 Figure 23: Determination of the E. coli spirosomes structure in compact conformation. 2D classes (A) used to reconstruct the final map of compact spirosome (B). Model of

Figure 24 :

 24 Figure 24: Details of the architecture of the compact spirosome. AdhE dimer associated to two other ADH domains. (A) AdhE monomer. (B)

Figure 25 :

 25 Figure 25: Determination of the E. coli spirosomes structure in extended conformation.

Figure 26 :

 26 Figure 26: Model of the asymmetric unit of the E. coli spirosomes structure in extended conformation.

Figure 27 :

 27 Figure 27: Comparison of ALDH-ALDH and ADH-ADH interface of bifunctional AdhE and monofunctional homologous. (A) Dimer of the propionaldehyde dehydrogenase from Rhodopseudomonas

Figure 28 :B

 28 Figure 28: Nucleotide binding domain in ALDH and ADH domains in extended (A) and compact (B) conformation. Amino-acid conservation analysis of homologous bifunctional AdhE enzyme.

Figure 29 :

 29 Figure 29: ALDH and ADH active sites linked by a channel in extended conformation.

Figure 30 :

 30 Figure 30: Multiple sequence alignments of E. coli bifunctional AdhE versus monofunctional enzymes or homologous bifunctional enzymes. The bifunctional AdhE sequence alignments are composed of AdhE sequence from

Figure 31 :

 31 Figure 31: Comparaison of the ALDH-ADH interface between the extended and compact conformation Representation of the ALDH-ADH interface in the extended (left) and compact (right)

Figure 32 :

 32 Figure 32: Conformational change along the E. coli spirosome Surface representation of the Escherichia coli spirosome in its extended conformation

Figure 33 :

 33 Figure 33: Intra-domains conformational change analysis. Local alignments of separated ALDH (A) and ADH (B) domains of the compact (blue) and

B

  . Intra-dimer interface a. Δ446 -449AdhE mutant As (α-ALDH)n/(β-ALDH)n interface and the two (α-ALDH)n/(β-ADH)n and (α-ADH)n/(β-ALDH)n interfaces were stabilized by a vast network of bonds as explained before. We made the choice to weaken the dimer AdhE by shorten the length of the linker between the ALDH and the ADH domain of AdhE. We tested 3 different lengths of linker by deleting 442 -449, 444 -449 or 446 -449. The goal was to prevent the formation of ALDH -ALDH interface without affecting the active site of the ALDH domain. These mutants were constructed as explained in Materials & Methods. After expression and purification, AdhE mutants were analyzed using gel-filtration chromatography and negative stain electron microscopy (Figure34). We observed that these mutants do not assemble into long filament as WT AdhE. The Δ446 -449AdhE mutant was selected to perform further analyses.

Figure 34 :Figure 35 :

 3435 Figure 34: Negative stain electron micrograph of purified WT AdhE filaments (A) and Δ 446-449 mutant AdhE (B).Scale bar 100 nm (left) and 50 nm (right). Gel filtration profile of WT AdhE filaments

Figure 36 :

 36 Figure 36: ADH-ADH interface stabilized by the 669 glutamic acid. (A) Elution profile of E669L (blue) and E669K (gray) AdhE mutants in Superdex200 column. (B)

Figure 37 :

 37 Figure 37: Interactions between the ALDHn and ADHn+1 domains.

Figure 38 :

 38 Figure 38: Extra density surrounded by positively-charged amino-acids.

Figure 39 :

 39 Figure 39: Negative stain electron micrograph of purified R772L,R773L,R774L,R790I AdhE mutant.

Figure 40 :

 40 Figure 40: S. pneumoniae spirosome structure

Figure 41 :

 41 Figure 41: Comparison between E. coli spirosomes (colored) in extended conformation and S. pneumoniae spirosomes (light gray).

Figure 42 :

 42 Figure 42: Extra-densities in nucleotide binding sites of ADH and ALDH domain.

  Part I: Study of the filamentation Based on the obtained structure of the extended and compact conformation of the Escherichia coli spirosomes, we could consider different targeted mutations in order to study the filamentation of AdhE.

  Based on the alcoholic fermentation pathway under anaerobic conditions, we decided to name the reductase activities as physiological reaction and the dehydrogenase activities as reverse reaction. The in vitro physiological (at pH 7 in presence of acetyl-CoA and NADH) and the reverse (pH 8.8 in presence of ethanol, NAD + and CoA) activities of purified AdhE WT and AdhE Δ446-449 were monitored using the specific absorbance properties of NADH at 340nm as described in Materials & Methods (Figure43).

Figure 43 :

 43 Figure 43: Spectrophotometric absorption spectrum of NAD+ and NADH. Salt form of NAD presents one peak of absorption at 260nm while the reduced form

Figure 44 :

 44 Figure 44: Enzymatic assays monitoring AdhE activities of the WT and D446-449 mutant AdhE.

Figure 45 :

 45 Figure 45: Growth curves of E. coli K12 strains in fermentative conditions in minimal medium.

Figure 46 :

 46 Figure 46: Growth curves of ALDHn -ADHn+1 interface mutants in fermentative conditions in minimal media.

Figure 47 :

 47 Figure 47: Enzymatic assays monitoring AdhE activities of the WT AdhE (black line) and R772L,R773L,R773L,R790I mutant AdhE (dotted line).

Figure 48 :

 48 Figure 48: Growth curves of positively-charged pocket mutants in fermentative conditions in minimal media.The anaerobic growth curve of K12 WT E. coli strain is shown with black line,

Figure 49 :

 49 Figure 49: Comparison of WT AdhE filaments and R772L, R773L, R774L, R790I AdhE filaments extension.

Figure 50 :

 50 Figure 50: Observation of compact filaments after enzymatic reaction performing dehydrogenase activities.

Figure 52 :

 52 Figure 52: Halo-tag and SNAP tag description

Figure 53 :

 53 Figure 53: Growth curves of Halo-AdhE and SNAP-AdhE mutants in fermentative conditions in minimal media.The anaerobic growth curve of K12 WT E. coli strain is shown with black line,

  change of conformation according to different ligands in Escherichia coli. This work has been focused on spirosomes from two different species: E. coli and S. pneumoniae bacteria.After solving the structure of E. coli spirosomes in different conformational states, mutated AdhE proteins were designed in order to study the role of the filamentation for the AdhE activity. This work shows that the filamentation is essential for the acetaldehyde dehydrogenase activity of AdhE enzyme in vitro and for the bacterial growth in minimal media in anaerobic condition.I.AdhE are active into the spirosome filament I. Spirosome formation and channeling During this project, Kim et al. published the structure of compact E. coli spirosomes in absence of any ligand bound (G. Kim et al., 2020). In this study, we present the structure of E. coli spirosomes in their compact and extended conformation. By adding cofactors: NAD + , Fe 2+ and/or not CoA, we could confirm the extension of spirosomes as shown by Kessler and colleagues. We reveal the specific organization between AdhE monomers with three different interfaces along the filament: ALDH-ALDH interface, ADH-ADH interface, ALDH-ADH interface. The high-resolution structure of spirosomes in their extended conformation state has uncovered the channeling between the ALDH-ADH interface. During the two consecutive NAD + -dependent reactions, the acetaldehyde is generated into the ALDH active site and oriented to the ADH active site to be converted into ethanol.The ALDH-ADH interface is unique compared to the ALDH-ALDH or ADH-ADH interface which are also found in dimers or tetramers of homologous monofunctional aldehyde or alcohol dehydrogenases, respectively.

  present in the iron-dependent ADH domain. Extended filaments present the active conformation of the spirosome. Our structural data reveal the channel between the two catalytic domains which is only present in extended filaments. Compact AdhE filaments present an ALDH domain in a non-favorable conformation for the catalysis. The binding of cofactors in the ALDH domain is required in order to trigger the conformational change. This motion enfolds the cofactor bound into the ALDH domain which could activate the active site. The adjacent ADH domain is then activated through the flexible ADH loops 2 and 3 which pass the motion on the ADH active site. The activation of this ADHn domain allows the activation of the ADHn+1 forming the ADH -ADH dimer.

  The cryo-EM map was sharpened in PHENIX (phenix.autosharpen). An initial 3D model of the AdhE dimer and adjacent ADH domains was manually built in Coot. The final model was refined by several rounds of manual refinement in Coot software and real-space refinement using phenix.real_space_refine with NCS and reference model restrains. The reference model used was the model obtained for AdhE in its extended state. The model was validated using MolProbity and phenix.validation_cryoem implemented in PHENIX software. For the models of the AdhE filaments in their extended and compact states, the corresponding model of the AdhE dimer were docked into the cryo-EM HR maps obtained. These maps were sharpened in PHENIX and symmetrized in RELION using the refined helical parameters. The model was refined using phenix.real_space_refine with NCS and reference model restrains. The reference model used was the AdhE model in its extended state. The model was validated using MolProbity and phenix.validation_cryoem implemented in PHENIX software.

Annex 4 :Annex 8 :

 48 Protein sequence of bifunctional AdhE enzyme from Gram-positive bacteria. Pony, P., Rapisarda, C., Terradot, L., Marza, E., Fronzes, R. Filamentation of the bacterial bi-functional alcohol/aldehyde dehydrogenase AdhE is essential for substrate channeling and enzymatic regulation. NatCommun 11, 1426Commun 11, (2020) ) .

Fig. 1

 1 Fig. 1 CryoEM analysis of the AdhE spirosomes in their compact and extended forms. a Spirosomes were incubated with different cofactors as indicated in the panel. The condition apo corresponds to the spirosomes as they were purified from E. coli. For each condition, five representative 2D classes are displayed. b CryoEM map of the extended spirosomes (incubated with NAD + , Fe 2+ , and CoA) obtained by single-particle analysis. An AdhE dimer is colored with each AdhE protomer in blue and light blue, respectively. Adjacent ADH domains are colored in orange and pink. c CryoEM map of the compact spirosomes (incubated with NADH and Fe 2+ ) obtained by single-particle analysis. The same color codes as in b are used. d CryoEM map of the extended spirosomes obtained by helical reconstruction. Representative 2D classes of the helical segments are displayed and the refined helical parameters are indicated. The same color codes as in b are used. e CryoEM map of the compact spirosomes obtained by helical reconstruction. Representative 2D classes of the helical segments are displayed and the refined helical parameters are indicated. The same color code as in b are used.

  of AdhE in extended and compact filaments. a Structure of the AdhE monomer in the presence of NAD + and Fe 2+ . The AlDH and ADH domains are identified. In each domain, the electron density corresponding to the NAD + and Fe 2+ is displayed in green mesh, as it is visible in the cryoEM map. In the lower part of the panel, larger views of the cofactor-binding sites are provided. b Structure of the AdhE dimer in its extended conformation. The AdhE n dimer is made of the α (in light blue) and β (in blue) protomers. Each protomer is composed of ADH and AlDH domains. The AdhE n dimer is in interaction with the ADH n -1 and ADH n + 1 . The AdhE α and AdhE β are related by C2 symmetry. c Structural model of the AdhE filament in its extended conformation. This model was obtained by docking the structure of the AdhE dimer shown in b, in the cryoEM map obtained by HR and by duplicating this dimer along the filament axis using its helical symmetry. d Structure of the AdhE monomer in the presence of NADH and Fe 2+ . The AlDH and ADH domains are identified. In the ADH domain, the electron density corresponding to the NADH and Fe 2+ is displayed in green mesh, as it is visible in the cryoEM map. No density corresponding to the NADH is visible in the AlDH domain active site. In the lower part of the panel, larger views of the ADH cofactor-binding site are provided. e Structure of the AdhE dimer in its compact conformation. The AdhE n dimer is made of the α (in light blue) and β (in blue) protomers. Each protomer is composed of ADH and AlDH domains. The AdhE n dimer is in interaction with the ADH n -1 and ADH n + 1 . The AdhE α and AdhE β are related by C2 symmetry. f Structural model of the AdhE filament in its compact conformation. This model was obtained by docking the structure of AdhE dimer shown in d, in the cryoEM map obtained by HR and by duplicating this dimer along the filament axis using its helical symmetry. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15214-y 4 NATURE COMMUNICATIONS | (2020) 11:1426 | https://doi.org/10.1038/s41467-020-15214-y | www.nature.com/naturecommunications

Fig. 3

 3 Fig. 3 Comparison of the structure of AdhE in its extended conformation with monofunctional ADH and AlDH enzymes. a The structures of the monofunctional enzyme propionaldehyde dehydrogenase from R. palustris (left) and AdhE AlDH domain (right) are compared. The monomers share the same overall fold. The root-mean-square deviation of atomic positions (RMSD) between the two structures is indicated in the figure. Both enzymes assemble as dimers, which are superimposable with each other. b The structures of the monofunctional enzyme lactaldehyde : 1,2-propanediol oxidoreductase from E. coli and AdhE ADH domain are compared. The monomers share the same overall fold. The RMSD between the two structures is indicated in the figure. Both enzymes assemble as dimers, which are superimposable with each other. c Localization and conservation of three loops specific to AdhE. These loops are not present in the monofunctional enzymes but are conserved in AdhE homologs.

Fig. 4 A

 4 Fig. 4 A continuous channel between the ADH and AlDH active sites. a A conserved channel crosses the monofunctional propionaldehyde dehydrogenase from R. palustris (left panels). This channel is conserved in the AlDH domain in AdhE (right panel). Top, cut-out slice through the AlDH domain of the bottom, surface representation of the AlDH domain showing the substrate entrance/exit channel (opposite of the NAD-binding channel). b A conserved channel crosses the monofunctional lactaldehyde : 1,2-propanediol oxidoreductase from E. coli (left panels). This channel is conserved in the ADH domain in AdhE (right panel). Top, cut-out slice through the ADH domain of the bottom, surface representation of the ADH domain showing the substrate entrance/exit channel (opposite of the NAD-binding channel). c Surface representation of ADH α (in light blue) and AlDH β (in blue). The loops 2 and 3 from ADH α are colored in red. d A channel connects directly the active sites of ADH α (in light blue) and AlDH β (in blue). The domains are represented as transparent surfaces and ribbons with the same color code than above. A surface representation of channel is colored in yellow. Surface representations of NAD + molecules bound to ADH and AlDH domains are colored in green.

Fig. 5

 5 Fig. 5 Conformational changes between the AdhE compact and extended states. a Sub-domain motion in the AlDH domain. Ribbon representation of the AlDH domain from AdhE. The NAD-binding domain (residues 1-214) is colored in green. The catalytic domain (residues 215-448) is represented in blue. The NAD-binding domains are represented in the same position for the compact and extended state. The catalytic domain moves relative to the NADbinding domain between AdhE compact and extended states. The axis of the catalytic domain is represented in red for the compact conformation (C) and yellow for the extended conformation (E). Top, side view of the AlDH domain. Bottom, view from the catalytic domain. b Zoomed view of the AlDH catalytic site of compact and extended AdhE and monofunctional propionaldehyde dehydrogenase from R. palustris. The proteins are represented as blue ribbons. The catalytic histidine, glutamate and cysteine are represented as sticks and volumes. They are colored in yellow. The NAD + molecules are represented as sticks. c Surface representation of the AdhE filament in its extended (left) and compact (right) conformation. The ADH domains are colored in beige and the AlDH domains are colored in pink. The NAD + molecules are colored in green.

Fig. 6

 6 Fig. 6 Functional analysis of AdhE in vitro and in vivo. a Negative stain electron micrograph of purified Wild-Type AdhE filaments. Scale bar 100 nm. b Negative stain electron micrograph of purified Δ446-449 AdhE filaments. Scale bar 50 nm. c Enzymatic assays monitoring AdhE activities of Wild-Type and Δ446-449 mutant AdhE. The conditions of each assay are indicated in the legend of the graph. The errors bars show SEM (3). d Growth curves for K12 E. coli strains in fermentative conditions in minimal medium. The strains used are indicated in the legend of the graph. The errors bars show SEM (3).

  NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15214-y ARTICLE NATURE COMMUNICATIONS | (2020) 11:1426 | https://doi.org/10.1038/s41467-020-15214-y | www.nature.com/naturecommunications

7 .

 7 Expression test and purification of AdhE WT and AdhE Δ446-449 . a. Size-exclusion chromatography profile of purified AdhE WT (dark line) and AdhE Δ446-449 (dashed line) on a HiLoad 16/600 Superdex 200 column (left) and SDS-PAGE (12%) of AdhE WT and AdhEΔ446-449 (right). Molecular weight markers are indicated on the left. b. Size-exclusion profile of purified AdhE Δ446-449 on a Superdex S200 5/150 column coupled with MALS and RI.

  En fonction des ligands présents, les spirosomes d'Escherichia coli sont retrouvés dans 2 états distincts. La structure atomique à haute résolution de la conformation étendue a été obtenue en présence de NAD + , Fe 2+ et/ou Coenzyme A et la structure de la conformation compacte a été obtenue en présence de la forme apo de l'enzyme ou de l'enzyme incubée avec du NADH. Ces structures ont révélé l'organisation de l'enzyme AdhE au sein du filaments. Deux AdhE s'entrelacent formant un dimère stabilisé par une interface acétaldéhyde-acétaldéhyde déshydrogénase (ALDH-ALDH)

	Lors de l'analyse de la structure étendue, nous avons identifié un canal dans le spirosome qui serait responsable de la canalisation de l'intermédiaire toxique qu'est l'acétaldéhyde. Ce canal relie les deux sites actifs des deux domaines lorsqu'ils sont produits en condition aérobie. Ceci pourrait être expliqué par les différences des séquences des deux protéines homologues. Cependant, il est nécessaire d'étudier plus en détails cette observation et comprendre si cela est liée au déshydrogénases contenant une molécule de coenzyme NAD semblent être naturellement stabilisés en conformation étendue et donc active, même métabolisme des bactéries, à l'évolution divergente des bactéries Gram-négative et
	Gram-positive ou à encore d'autres facteurs.

et deux interfaces acétaldéhyde-alcool déshydrogénase (ALDH-ADH). Enfin le filament est généré par l'association de plusieurs dimères d'AdhE au niveau des domaines alcools déshydrogénases créant des interfaces alcool-alcool déshydrogénases (ADH-ADH). De façon intéressante, les interfaces ALDH-ALDH et ADH-ADH générées au sein du filaments sont hautement superposables avec les structures des dimères d'acétaldéhydes et d'alcools déshydrogénases monofonctionnelles homologues déjà connues. A partir de ces structures, des mutants d'AdhE ont pu être générés afin d'empêcher la filamentation et ainsi d'étudier son rôle. Dans un premier temps, l'espace permettant de physiquement éloigner les deux domaines déshydrogénases d'AdhE a été raccourci dans le but de prévenir la formation du dimère d'AdhE. Ce mutant d'AdhE n'est plus capable de former de longs filaments d'AdhE et ne présente plus d'activité in vitro dans le sens de production d'éthanol lors de la fermentation alcoolique. Il a en effet perdu l'activité acétaldéhyde déshydrogénase mais conserve son activité alcool déshydrogénase. Cela nous a permis de montrer que les spirosomes contiennent des monomères d'AdhE actifs et que la filamentation est essentielle à son activité in vitro. De plus, nous avons montré que ce mutant d'AdhE est incapable de complémenter une souche n'exprimant plus la protéine AdhE et d'ainsi restaurer la croissance en milieu minimum en absence d'oxygène. La filamentation d'AdhE est donc également nécessaire au métabolisme bactérien in vivo en milieu minimum en absence d'oxygène. + . Comme d'autres exemples de filamentation d'enzymes ou de micro-compartiments enfermant des déshydrogénases, les spirosomes sont une forme de compartimentation bactérienne. Dans le but de l'étudier plus en détails, des expériences d'immuno-marquages ont été réalisées afin de localiser les filaments d'AdhE à l'intérieur de la bactérie. Ces observations ont révélé une localisation périphérique de la protéine sauvage d'AdhE alors que la protéine mutante non-filamentaire présente une localisation diffuse au sein de la bactérie. Même si cette observation ne peut encore être expliquée, elle pourrait suggérer une différence de fonction entre la forme oligomérique et monomérique. Toujours basé sur la structure atomique obtenue, une densité supplémentaire a été identifiée dans la structure atomique en conformation étendue. Cette densité est localisée à l'interface acétaldéhyde-alcool déshydrogénase correspondant à l'interface cruciale pour la réaction enzymatique. Pour le moment, il nous a été impossible de précisément identifier la molécule correspondant à cette densité. Des expériences approfondies sont nécessaires afin d'identifier cette molécule et de comprendre son rôle dans la régulation ou de la stabilisation du changement de conformation et de l'activité enzymatique des spirosomes. La structure des spirosomes de Streptococcus pneumoniae a aussi été résolue par cryo-microscopie électronique. Cette structure est hautement similaire à celle obtenue pour E. coli dans leur conformation étendue. Les spirosomes de S. pneumoniae La détermination des structures des spirosomes d'E. coli et S. pneumoniae à haute résolution puis l'étude de la fonction révèlent que la formation de filaments d'AdhE est nécessaire à l'activité enzymatique de l'enzyme et par conséquent indispensable à physiologie bactérienne en absence d'oxygène. Un des principaux intérêts qu'apporte la filamentation de AdhE est la canalisation de l'acétaldéhyde, intermédiaire de réaction hautement toxique pour la cellule. De plus, le rapprochement physique des deux domaines comportant les activités catalytiques pourrait être bénéfique afin d'accroitre la vitesse de réaction donc de régénération du NAD + en milieu dépourvu d'oxygène. Enfin, la filamentation d'AdhE pourrait présenter un intérêt dans la localisation de la protéine active puisque nous avons observé une différence de localisation entre la protéine sauvage capable de former des filaments et la protéine mutante incapable de former des spirosomes fonctionnels.

Cependant, l'étude approfondie des mécanismes de régulation de l'activité des spirosomes est encore à mener et pourrait aboutir à la découverte de molécules stimulant ou inhibant le changement de conformation des filaments d'AdhE de E. coli.

  The study of diffraction patterns of DNA molecule by Cochran, Crick, Van and Stokes[START_REF] Cochran | The structure of synthetic polypeptides. I. The transform of atoms on a helix[END_REF][START_REF] Sachse | Single-particle based helical reconstruction -how to make the most of real and Fourier space[END_REF] helped in development the latter used technique on the tail of

Bacteriophage T4, described for the first time in 1968 by D. J. Rosier and A. Klug

[START_REF] Rosier | Reconstruction of Three Dimensional Structures from Electron Micrographs[END_REF]

. Because helical assemblies can be compared to 2D crystals, the cylinder composing the helix can be converted to a flat object. The Fourier transform of a helix represents multiple lines called layer lines. Then, the layer lines are characterized by Fourier coefficients composed of Miller indices and Bessel order corresponding to Bessel functions. Two different lattices are represented is a diffraction pattern of a helical symmetry corresponding to two faces of the helix.

Table :

 : Plasmid list

	Organism Plasmid Cloned gene / relevant features	Resistance Reference
	E. coli	pET15b	6his tag in N-terminal -IPTG inducible AmpR	Commercial
		pET21b	6his tag in C-terminal -IPTG inducible AmpR	Commercial
		pKG116 Sodium salicylate inducible	CmR	The lab
		pPP002 E. coli AdhE in pET15b	AmpR	This study
		pPP005 E. coli AdhE D446-449 in pET15b	AmpR	This study
		pPP006 E. coli AdhE D444-449 in pET15b	AmpR	This study
		pPP007 E. coli AdhE D442-449 in pET15b	AmpR	This study
		pPP012 S. pneumoniae AdhE in pET21b	AmpR	Previously cloned in the
					lab
		pPP013 E. coli AdhE in pKG116	CmR	This study
		pPP014 E. coli AdhE D446-449 in pKG116	CmR	This study
		pPP024 E. coli AdhE R772I-R773L-R774L-R790L in	AmpR	This study
			pET15b

Table 1 :

 1 Data collection and processing

		Extended (Fe 2+ -NAD + -CoA)	Contracted (Fe 2+ -NADH)
	Data collection
	Microscope -Camera	Talos Arctica -K2 summit	Talos Arctica -K2 summit
	Voltage (kV)	200		200
	Magnification	36000		36000
	Electron exposure (e-/Å 2 )	0.77		0.77
	Pixel size (Å)	1.13		1.13
	Decofus range (um)	0.4 -2.4		0.4 -2.4
	Processing	Helical	Single particle	Helical	Single particle
		reconstruction	analysis	reconstruction	analysis
	Symmetry imposed	C1	C1	C1	C1
		Helical	C2	Helical
		parameters		parameters
		(Rise 57 Å,		(Rise 34.7 Å,
		Twist 163.1°)		Twist 154.5°)
	Initial particle images (no.)	221 351		3 051 336
	Final particle images (no.)	138 927		98 677
	Map resolution (Å) -(FSC	4.4 -(0.143)	3.2 -(0.143)	5 -(0.143)	3.8 -(0.143)
	threshold model)
	Refinement & Validation
	Map sharpening (B-factor)	121.41	82.58	246.65	206.89
	(Å-2)
	Model composition
	# chains		6		6
	Atoms (no.)		39 781		39760
	Residues (no.)		2 576		2578
	Ligands (no.)		Fe 2+ (2) -		Fe 2+ (2) -
			NAD + (4)		NADH (2)
	Bond lengths (Å)		0.011		0.006
	Bond angles (°)		0.989		1.587
	Ramachandran favored %		87.46		85.02
	Ramachandran allowed %		12.54		14.05
	Ramachandran outliers %		0.00		0.93
	Rotamers outliers %		1.45		3.67

  Structure and Function of Bacterial Nanomachines -Institut Européen de Chimie et Biologie, University of Bordeaux, 2 rue Robert Escarpit, 33600 Pessac, France. 2 Microbiologie fondamentale et pathogénicité, UMR 5234, CNRS, University of Bordeaux, 2 rue Robert Escarpit, 33600 Pessac, France. 3 UMR 5086 Molecular Microbiology and Structural Biochemistry, Institut de Biologie et Chimie des Protéines, CNRS-Université de Lyon, Lyon, France. Present address: UCB Celltech, Slough SL1 3WE, UK. ✉ email: esther.marza@u-bordeaux.fr; remi.fronzes@u-bordeaux.fr NATURE COMMUNICATIONS | (2020) 11:1426 | https://doi.org/10.1038/s41467-020-15214-y | www.nature.com/naturecommunications

	https://doi.org/10.1038/s41467-020-15214-y	OPEN
	1234567890():,;
	1 1

4

  summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

	a
		ALDH	ADH	BOND
			TYR 848	H
		GLU 63
			ILE 851	H
		HIS 70	ARG 572	H
		GLU 74	MET 574	H
		ARG 229	SER 764	H
	AlDH -ADH	THR 239	LYS 759	H
	interface	LYS 317	PRO 757	H
		ASP 44	ARG 572	SB
		ARG 46	ASP 838	SB
		LYS 51	GLU 834	SB
		ASP 64	LYS 759	SB
		ARG 229	ASP 767	SB
		AlDH	AlDH	BOND		ADH	ADH	BOND
		GLN 375	ARG 447	H		LEU 452	PHE 462	H
		GLY 386		H		HIS 454	ILE 460	H
			LYS 442
		MET 389		H		LYS 455	TYR 461	H
		ALA 392	THR 443	H		GLU 669	SER 705	H
	AlDH -AlDH interface	ILE 394	ALA 445	H	ADH -ADH interface	PHE 670	GLN 674	H
		ILE 396	ARG 447	H		LYS 455	GLU 473	SB
		TYR 409	ALA 448	H		ARG 463	GLU 669	SB
		SER 430	LYS 441	H			LYS 455	SB
						GLU 473
		ASP 92	LYS 412	SB			ARG 577	SB

Cette enzyme est communément retrouvée sous sa forme oligomérique appelée spirosome. Les spirosomes sont de longs filaments d'AdhE en forme de hélicoïdale observés pour la première fois dans les années 1970 chez l'espèce Lactobacillus. Une étude a identifié que les spirosomes chez Escherichia coli peuvent être stabilisés dans deux états conformationels distincts. En absence de ligands autrement dit la forme apo de l'enzyme AdhE, les filaments sont retrouvés dans une conformation dite compacte. En présence de ligands, les filaments présentent une conformation dite étendue. Ce changement de conformation induit par les ligands de la réaction enzymatique suggère que les spirosomes présentent une activité et ne serait pas seulement une forme de stockage de l'enzyme inactive. Malgré l'intérêt porté au monomère d'AdhE, la structure et la fonction des filaments restent jusque-là inconnues. L'objectif de cette thèse est de déterminer la structure atomique des spirosomes de deux espèces bactériennes différentes Escherichia coli et Streptococcus pneumoniae afin d'étudier le rôle de ces filaments au sein de la cellule. Pour cela, les structures de ces assemblages ont été résolues par cryo-microscopie électronique. Puis des mutants de l'enzyme AdhE ont été réalisés afin comprendre le rôle de la filamentation de AdhE dans son activité. En plus de l'étude de son activité in vitro, des expériences de complémentation in vivo ont été menées en vue d'identifier l'importance de la filamentation au sein du métabolisme fermentaire.

NATURE COMMUNICATIONS | (2020) 11:1426 | https://doi.org/10.1038/s41467-020-15214-y | www.nature.com/naturecommunications

Acknowledgements

This work was supported by University of Bordeaux, CNRS, and ERC CoG TransfoPneumo. P.P. holds a PhD fellowship from University of Bordeaux. We thank Claire Stines-Chaumeil for advice in Enzymology and Armel Bezault from the cryoEM facility at IECB.

Data availability

Acetaldehyde is converted to ethanol by an alcohol dehydrogenase which is associated to the NAD + regeneration (Figure 5). From [START_REF] Guragain | Fuel Alcohol Production[END_REF] We suggested that this arginine-rich domain is not responsible for the stabilization of the spirosome filamentation but could be involved either in the conformational change or activity regulation.

Expressed full-length AdhE and D446-449 AdhE proteins were immunolabelled using anti-AdhE antibody and a secondary antibody coupled with an Alexa 488 fluorophore as described in Materials & Methods. Full-length AdhE enzyme was preferentially detected in the periphery of the bacteria as the native AdhE. Rather, the Δ446-449 AdhE seemed to be distributed uniformly in the bacteria (Figure 51). These observations suggest a specific intracellular localization of the AdhE oligomeric form.

However, due to the poor resolution using confocal microscopy technique, we can't confirm if the spirosome form of AdhE is attached to the membrane, if an intermediate is present or just localized near the membrane.

b. Super resolution microscopy

In order to go further in our investigation, we started to use a super-resolution technique called DNA points accumulation for imaging in nanoscale topography (DNA-PAINT). This powerful method uses the capacity of DNA molecules to specifically bind a complementary sequence. The docking strand attached to the target protein is complementary to the imager strand which is associated to a fluorophore [START_REF] Nieves | DNA-Based Super-Resolution Microscopy: DNA-PAINT[END_REF]. Fluorophores are ligands which can easily diffuse in the medium. Two different tag systems have been tested SNAP-tag which is a derivative of the O 6 -alkylguanine-DNA alkyltransferase (hAGT) enzyme [START_REF] Keppler | A general method for the covalent labeling of fusion proteins with small molecules in vivo[END_REF] and

Halo-tag which is a modified haloalkane dehalogenase [START_REF] Los | HaloTag: A novel protein labeling technology for cell imaging and protein analysis[END_REF]) (Figure 52).

So, using the two complementary single strands, the SNAP and Halo-tagged proteins can be correlated to the fluorescence emitted by the fluorophore. The transient association of the two oligomers allow to characterize association and dissociation rate which depend on the length of the complementarity between the two strands. The blinking induced by the binding and the separation of the two strands leads to specifically localized the fluorescence spot, so the protein of interest.

DISCUSSION & PERSPECTIVES

respectively [START_REF] Erbilgin | The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle[END_REF]. It prevents the release of highly volatile acetaldehyde into cytoplasm. (A) [START_REF] Erbilgin | The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle[END_REF] Electron micrograph of micro-compartments (carboxysome in a cyanobacterium) (B) [START_REF] Kerfeld | Protein Structures Forming the Shell of Primitive Bacterial Organelles[END_REF] Spirosomes is another example of the compartmentalization of the bacterial cytoplasm.

By encapsulating the toxic intermediate, filaments protect the cell from the diffusion of the acetaldehyde. Enzyme filament is know well reported, more than 20 enzymes selfassemble as polymers (Park & Horton, 2019).

Another advantage to clustering different enzymatic activities is to maximize the reaction efficiency. Because small molecules can easily diffuse into the cell, clustering enzymes presents advantages for two-step metabolic reactions. Firstly, high local catalytic sites are concentrated and so substrate can rapidly be converted into intermediate of reaction. Secondly as mentioned before, the diffusion of this intermediate is prevented, and it can be quickly processed by the other enzyme. They also hypothesized that clustering enzymes might be responsible for regulating flux, A

V. Other roles

The bifunctional AdhE enzyme seems to not only be involved in alcoholic fermentation under anaerobic environment. The E. coli adhE expression is repressed by air and nitrate and so present in basal level in aerobic condition [START_REF] Leonardo | Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli[END_REF]. The resulted expressed protein is inactivated by metal-catalyzed oxidation (Echave, Tamarit, Cabiscol, & Ros, 2003a). Echave and colleagues have shown that the deletion of adhE gene in E. coli interrupts the bacterial growth in aerobic condition in minimal media with cell division dysfunction. Furthermore, the mutant strain is significantly affected by oxidative stress as H2O2. They suggested that AdhE may have a protective role for preventing oxidative stress under aerobic condition.

AdhE enzyme has been studied in another context that energy metabolism. This versatile bifunctional enzyme seems to also be involved in different pathways of bacterial pathogenicity.

In Listeria monocytogenes the Listeria adhesion protein (Lap), a homologous AdhE is secreted into the extracellular medium through the SecA2 system and mediate the paracellular translocation of the bacteria. (H. Kim & Bhunia, 2013). Further investigations could be done to understand how the homologous AdhE is secreted and if other homologous AdhE are also secreted in order to stimulate bacterial virulence.

The elucidation of the role of the filamentation could be crucial to interpreting result experiments done on the involvement of AdhE in bacterial pathogenicity.

In addition, these glycolytic enzymes which are localized on cell surface are also reported as moonlighting proteins and could be secreted by many bacteria. These

MATERIALS & METHODS

All the strains used in this study are listed in the following table.

Table: Strain list

Organism Strains

Genotype Reference

Competing interests

The authors declare no competing interests.

Supplementary information

Filamentation of the bacterial bi-functional alcohol/aldehyde dehydrogenase AdhE is essential for substrate channeling and enzymatic regulation

Pony et al. domain. This bifunctional AdhE enzyme is well-conserved in all bacterial kingdoms and also in more phylogenetically distant microorganisms such as green microalgae. AdhE is well-studied for its involvement in biofuel production, but as well for its role in bacterial pathogenicity. This enzyme is commonly found as an oligomeric form called spirosomes. Despite the great interest in the AdhE enzyme, the structure and function of these filaments remain unknown since their discovery in the 1970's. Depending on ligands incubated with, Escherichia coli spirosome are found as two different conformational states. During this thesis, the structure of the extended and the compact conformation have been solved at high-resolution using cryo-electron microscopy.

Based on these structures, AdhE mutants have been designed in order to disrupt the filamentation and study its role. We have shown that spirosomes contain active AdhE monomers and the AdhE filamentation is essential for its activity in vitro and in vivo. Again based on the obtained structure of the extended conformation, we identified that a channel within the filament which could be responsible for the channeling of the toxic acetaldehyde intermediate. Like other examples of metabolic enzyme filamentation or micro-compartments encapsulating dehydrogenases, spirosomes are a form of bacterial compartmentalization. In order to look deeper in this compartmentalization, immunolabelling experiments have been performed in order to localize AdhE filaments inside the bacteria. Observations revealed a peripheral localization for E. coli AdhE wild-type and a diffuse localization for the nonfilamentous AdhE. These differences are not understood but might suggest a difference of function between oligomeric and monomeric form.

The structure of the Streptococcus pneumoniae spirosome has also been solved using cryo-EM. Compared to E. coli spirosomes, AdhE filaments from S. pneumoniae have always been purified as extended conformation. S. pneumoniae spirosomes seem to be naturally found in extended conformation and so active form. This could be due to differences present in the two homologous AdhE primary sequences.

The determination of the E. coli and S. pneumoniae structures at high-resolution and the analysis of the function reveal that these filaments are required for bacterial physiology in absence of oxygen. The study of the regulation of spirosome activities might lead to the discovery of molecules stimulating or inhibiting conformation change in E. coli AdhE filaments. These molecules would present a great interest in biotechnology or to prevent pathogenic anaerobes.