Dissertationen zum Thema „Ruminococcus“

The xylanases and xylanase genes in the rumen cellulolytic bacterium <i>Ruminococcus flavefaciens</i> were studied in this thesis. Based on the experiments carried out in this thesis, the following conclusions can be drawn: At least seven multiple xylanases are revealed in <i>R.flavefaciens</i> strain 17 and strain 007, and nine in strain FD-1 by the method used here for activity detection in gels. A im54 kDa constitutive xylanase appears in both strains 17 and 007, but not in FD-1. Straw and xylan are the best substrates for production of most xylanases in <i>R.flavefaciens</i>. Sequencing one of the four xylanase genes isolated from <i>R.flavefaciens</i> 17, <i>xynA</i>, shows a 2862 bp open reading frame initiating from a TTG start codon which is preceded by a Gram-positive Shine-Dalgarno sequence (robosome binding site) of AAAGGAG. The enzyme encoded by <i>xynA</i> (XYLA) is predicted to have 954 amino acids including a probable signal sequence at the amino terminus. XYLA is novel in its structure having two dissimilar catalytic domains (domain A, 248 amino acids; domain C, 332 amino acids) linked by a highly repetitive region (domain B, 374 amino acids) extremely rich in asparagine and glutamine residues. The two catalytic domains can be active independently and act on xylan differently, with domain C producing smaller end products than domain A from oat-spelt xylan. Amino acid sequence comparisons with other enzymes show that domain A and domain C are related to two different families of xylanases, G and F, respectively. Therefore, this thesis provides the first evidence of a bifunctional hemicellulase comprising two different catalytic domains. Antibodies raised separately against domains A and C of XYLA recognize common enzyme bands in <i>Ruminococcus</i>, ranging in apparent molecular mass from 110 kDa to 200 kDa. This tends to confirm that XYLA is produced as a high molecular weight polypeptide in <i>R.flavefaciens</i>, as predicted from the sequence. <i>R.flavefaciens</i> has strong preference of codon usage for several amino acid residues, eg. glutamate (GAG); glutamine (CAG); and lysine (AAG).

Les spondyloarthrites (SpA) sont desrhumatismes inflammatoires chroniques fortementassociés à l'allèle HLA-B27 du complexe majeurd'histocompatibilité de classe I. La preuve du rôlepathogène du HLA-B27 a été apportée par plusieurslignées de rats transgéniques pour le HLA-B27 et lab2 microglobuline humaine (rats B27). Ces rats B27développent spontanément des manifestationscomparables à la SpA humaine. Dans ce modèle, lescellules hématopoïétiques HLA-B27+, leslymphocytes T CD4+ et un microbiote conventionnelsont nécessaires pour développer la maladie. Le rôledu microbiote a également été étayé par la mise enévidence d'une dysbiose intestinale au cours desSpA. Ainsi, une corrélation positive entre l'activitédes SpA et l'abondance de l'espèce bactérienneanaérobie Ruminococcus gnavus dans les selles a étédémontrée.Mon premier objectif de thèse a été de déterminerles mécanismes immunologiques impliqués dans ledéclenchement de la SpA du rat B27 par l'étude despopulations productrices des cytokines clefs de laSpA : l'IL-17 et le TNF. Parallèlement, je me suisintéressée au rôle des souches de R. gnavus aucours de la SpA. Mon premier travail a démontréque les LT CD4+ conventionnels exprimant lerécepteur de chimiokines CCR6 étaient lesprincipales cellules productrices d'IL-17 et de TNFau cours de la SpA et qu'elles étaient capablesd'induire la SpA après transfert à des ratsathymiques nude B27 habituellement protégés. Lesecond axe de ma thèse m'a permis d'isoler dessouches de R. gnavus de patients atteints de SpA etde témoins sains. Des expérimentationscomplémentaires sont nécessaires pour étayer leshypothèses concernant la pathogénicité de R.gnavus<br>Spondyloarthritis (SpA) is a chronicinflammatory rheumatic disease strongly associatedwith the HLA-B27 major histocompatibility complexclass I allele. Proof of the pathogenic role of HLA-B27was provided by lines of transgenic rats for HLA-B27and human β2 microglobulin (B27 rats). These B27rats spontaneously develop manifestationscomparable to human SpA. In this model, HLA-B27+hematopoïetic cells, CD4+ T lymphocytes and aconventional microbiota are required for diseasedevelopment. The role of the microbiota is alsosupported by evidence of intestinal dysbiosis in SpA.A positive correlation between SpA activity and theabundance of the bacterial anaerobic speciesRuminococcus gnavus in stools has beendemonstrated. The first aim of my thesis was todetermine the immunological mechanismsinvolved in triggering SpA by studying thepopulations producing the key SpA cytokines IL-17and TNF in B27 rat. In parallel, I examined thepotential role of R. gnavus strains in SpA. My firstwork demonstrated that conventional CD4+LTexpressing the chemokine receptor CCR6 are themain IL-17 and TNF-producing cells during SpAand are able to induce SpA after transfer to usuallyprotected nude B27 athymic rats. In the secondpart of my thesis, I isolated R. gnavus strains fromSpA patients and healthy controls. Furtherexperiments are required to substantiate thepathogenic hypotheses of R. gnavus in SpA

O celulossomo é um complexo multienzimático extracelular utilizado por bactérias anaeróbias para a degradação de biomassa vegetal. Ele é composto por escafoldinas, estruturas alongadas que abrigam diversos módulos cohesina, às quais se ligam dockerinas, seus parceiros de interação específica de alta afinidade, fusionados às enzimas celulolíticas. Os módulos cohesina e dockerina compõem o elemento central da interação entre todos os componentes que integram o celulossomo. Esses módulos são divididos em tipos, de acordo com sua sequência primária. Essa divisão reflete efeitos funcionais distintos, sendo o tipo I responsável pela ligação de enzimas às escafoldinas, enquanto o tipo II medeia a ligação de escafoldinas à célula. O celulossomo de Ruminococcus flavefaciens é o mais complexo conhecido, e na classificação por tipos, suas sequências divergem, formando o tipo III, que foi posteriormente subdividido em 6 grupos para significância funcional. Nesse sistema, o principal responsável pela integração de enzimas ao sistema é a escafoldina primária ScaA, a qual interage com escafoldina adaptadora ScaB. A especificidade dessa ligação - dockerina de ScaA (Rf-DocA) com cohesinas de ScaB (Rf-CohB1-7) - é classificada como único membro do grupo 5, na divisão de grupos que compõem o tipo III. Assim, essa interação é de suma importância para a organização do celulossomo desse organismo, tendo sido estudada por meio de experimentos biofísicos e bioquímicos. Porém a falta de uma estrutura cristalina resolvida desses componentes limita a compreensão que podemos ter sobre a interação. 1-2 Nesse trabalho, apresentamos as estruturas cristalográficas de Rf-DocA, em complexo com a Rf-CohB4, além da estrutura dessa cohesina isolada, e ainda, a Rf-CohB1, e alguns de seus mutantes pontuais. Com isso, esclarecemos aspectos estruturais desses módulos, como a presença de dois sítios funcionais de ligação a cálcio em Rf-DocA. Também é observável pelos modelos gerados, detalhes da ligação entre eles, como os resíduos participantes da interação. Estudos de afinidade entre esses módulos foram conduzidos para a elucidar algumas propriedades da ligação entre esses módulos, de forma que descobrimos que ela ocorre de uma única maneira, e que há um loop na cohesina cuja flexibilidade afeta a afinidade da ligação. Isso sugere um mecanismo de alteração conformacional que regula a ligação à dockerina. Adicionalmente, buscamos o emprego desses módulos em uma aplicação tecnológica, desenhando redes automontáveis de proteínas, visando a construção de um nanomaterial. Essas redes são formadas por características intrínsecas das proteínas que os compõem, sendo o principal fator considerado sua simetria rotacional.3 Nesse sentido, as dockerinas e cohesinas foram utilizadas para ligação entre proteínas de diferentes simetrias. Utilizamos proteínas de simetrias C3, C4 e C6 com fusão a dockerinas, que se conectam às cohesinas fusionadas a proteínas de simetria C2, as quais formam o elemento linear da ligação entre os diferentes módulos. Esse desenho experimental permite a expressão e purificação independentes dos componentes, o que facilita a obtenção das redes, a partir da mistura dos dois componentes. Através de análises preliminares por microscopia eletrônica de transmissão, observamos a formação de filmes bidimensionais extensos e nanotubos com a construção testada.<br>The cellulosome is an intricate multienzyme extracelular complexes evolved by anaerobic bacteria for degradation of cellulosic biomass. It is composed of scaffoldins, elongated structures, which bare numerous cohesin modules, which bind to dockerin modules, their high affinity and specificity partners, borne by cellulolytic enzymes. The cohesin and dockerina modules constitute the central element of the interaction between every component of the cellulosome. These modules are categorized in types, according to their primary sequence. That distribution reflects distinct functions, in which the type I is responsible for integration of enzymes to scaffoldins, while type II mediates anchoring of scaffoldins to the cell wall. The cellulosome of Ruminococcus flavefaciens is the most intricate known to date, which is categorized into a third type of cohesins and dockerins, due to sequence diversion. The type III was further divided into 6 groups to impart functional significance. In that system, the main enzyme integrating component is the primary scaffoldin ScaA, which interacts to the adaptor scaffoldin ScaB. The specificity of this interaction - dockerina of ScaA (Rf-DocA) to ScaB cohesins (Rf-CohB1-7) - is sorted as a single member of group 5, in the subtypes of type III. Thus, this interaction is essential for cellulosome organization, having been studied by biophysical and biochemical experiments. However, the lack of a solved crystalline structure of these components narrows our understanding on this interaction. In the present study, we present the structures of Rf-DocA, complexed to Rf-CohB4, besides the structure of this isolated cohesin, and also Rf-CohB1 and its point mutants. Due to these data, we clarify structural aspects of these modules, such as the occurrence of two functioning calcium binding sites in Rf-DocA. We also identified details of their binding, such as the interacting residues. Through binding affinity studies, we concluded that the interaction between these modules occurs in a single mode, and that there is a loop in the cohesin module whose flexibility has direct effects on the binding affinity to dockerin. Additionally, we sought to utilize these modules in a downstream application, by designing self-assembling arrays of proteins, aiming for the construction of a nanomaterial. These arrays are constructed from the intrinsic properties of its constituent proteins, in which the main factor is rotational symmetry. In this context, dockerina and cohesin modules were used of binding different symmetry proteins. We utilized C3, C4 and C6 point symmetry proteins fused to dockerin modules, which bind to the cohesin modules fused to C2 point symmetry proteins, which establish the linear connection between the distinct components. This experimental design allows for the independent expression and purification of the components, which facilitates the achievement of the arrays, by simple mixture of the two components. Through preliminary analysis by transmission election microscopy, we observed the construction of two-dimensional films and nanotubes.

Here we report the enzymatic characteristics and regulation of expression for two GH36 a- galactosidases. Agu 1 and Aga,'. li'0111 R. gnavus El. Bioinforrnatics analysis of their respective genetic environment showed a different organisation. Aga 1 having a simple organisation while Aga2 is organised as part nf an ()p~wn. They were heterologously expressed in Escherichia coli. puri tied to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern (If the strain in minimum media demonstrates a preference tor complex substratcs (melibiose and raffinose) that require the expression of the a-galactosid.rscs for their utilisation and assimilation. Keywords: a-galactosidase. Ruminococcus gnavus E I. characterisation. transglycosylation. regulation. metabolism

Ruminococcus gnavus E1, une bactérie à Gram positif, anaérobie stricte, isolée du microbiote dominant d'un homme sain. Un ORF d'environ 6kb, nommé radA, a été identifié sur le chromosome de la souche E1, à proximité des clusters génétiques impliqués dans la biosynthèse des bactériocines RumA et RumC qui sont actives contre le pathogène Closthdium perfringens. RadA présente de fortes homologies avec des gènes de Staphylococcus aureus, Bacillus cereus et C. Perfingens codant pour des protéines des adhésines de la famille des MSCRAMMs. La partie du gène radA codant pour les 414 acides aminés situés à l'extrémité N-terminale de la protéine putative mature, sans, a été clonée dans le vecteur pGEXT4, exprimée chez Escherichia coli. Les tests réalisés par la méthode ELISA montrent ce fragment de RadA est impliqué dans l'adhésion au collagène de type I. Afin de localiser plus précisément la région responsable de l'adhésion, le fragment du gène radA codant pour les 218 acides aminés localisés à l'extrémité N-terminale de RadA a été clone dans le vecteur pGEXT4 et exprimé chez E. Coli. La protéine fusion GST-RadA218 présente une adhésion au collagène nettement plus forte que RadA414. Ll a été montré par RT-PCR que le gène radA est fortement exprimé in vivo, quand la souche E1 colonise le tube digestif d'animaux monoxéniques, et peu transcrit in vitro. Les expériences complémentaires montrent que radA est largement disséminé chez différentes souches isolées du microbiote dominant de l'Homme du cluster phylogénétique Clostridium coccoides qui comprend l'espèce R. Gnavus. Les résultats suggère que RadA pourrait jouer un rôle important dans la colonisation de l'écosystème digestif<br>Ruminococcus gnavus E1 is a Gram positive strict anaerobic bacterium that was isolated from the dominant faecal microbiota of a healthy adult. A 6kb-long open reading fragment called radA was identified on the E1 chromosome, next to the genetic clusters involved in the biosynthesis of the RumA and RumC bacteriocins which are active against pathogenic Clostridium perfringens. RadA shares a high sequence homology with genes of Staphylococcus aureus, Bacillus cereus and C. Perfingens encoding adhesins of the MSCRAMMs family. The gene fragment coding for the 414 amino acids located at the N-terminus of the mature protein was cloned in the pGEXT4 vector and expressed in Escherichia coli. ELISA-based tests showed that this fragment of RadA is involved in adhesion to type I collagen. To localize more precisely the region responsible for adhesion, the gene fragment coding for the 218 amino acids located at the N-terminus was cloned in the pGEXT4 vector and expressed in E. Coli. The fusion protein GST-RadA218 exhibited a stronger adhesion to collagen than RadA414. RT-PCR experiments demonstrated that the radA gene was strongly expressed in vivo, when the E1 strain colonized the digestive tract of monoxenics animals, while little transcription occured in vitro. Complementary experiences showed that radA was widely spread among various strains isolated of the human dominant microbiota that belonged to the phylogenetic duster Clostridium coccoides that includes the R. Gnavus species. Taken together, these results suggest that RadA could play an important role in the colonization of the digestive ecosystem

Ruminococcus gnavus E1 appartient au groupe des Firmicutes, l’un des deux groupes dominants du microbiote intestinal humain. Les a-galactosidases sont des glycosides hydrolase (GH) actives sur des substrats contenant des galactoses liés en a. Elles sont très largement distribuées dans tous les domaines du vivant, bactéries, champignons, plantes et animaux, mais sont absentes du tractus digestif humain. Ces travaux portent sur les caractéristiques enzymatiques et la régulation de l’expression de deux &#61537;-galactosidase, Aga1 et Aga2, de R. gnavus E1. L’analyse bioinformatique de leur environnement génétique respectif indique une organisation simple pour Aga1 tandis qu’Aga2 est organisée en opéron. Elles ont été exprimées en système hétérologue chez E. coli, purifiées et leurs propriétés biochimiques ainsi que leurs spécificités de substrat ont été analysées. Le profil de croissance de la souche indique une préférence pour des substrats complexes (raffinose et mélbiose) faisant intervenir les a-galactosidase pour leurs utilisations ainsi que leur assimilation<br>Ruminococcus gnavus E1 belongs to the Firmicutes, one of the two dominant groups in the human gut microbiota. a-galactosidases are glycoside hydrolases (GH) active on a-galactoside containing substrates. They are widely distributed through all the domains of life: bacteria, fungi, plants, and animals, but are absent from the human gastro-intestinal tract.Here we report the enzymatic characteristics and regulation of expression for two GH36 &#61537;-galactosidases, Aga1 and Aga2, from R. gnavus E1. Bioinformatics analysis of their respective genetic environment showed a different organisation, Aga1 having a simple organisation while Aga2 is organised as part of an operon. They were heterologously expressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. The growth pattern of the strain in minimum media demonstrates a preference for complex substrates (melibiose and raffinose) that require the expression of the a-galactosidases for their utilisation and assimilation

The Gram-positive, strictly anaerobic bacterium, <I>R. flavefaciens</I>, plays an important role in the degradation of plant cell wall polysaccharides in the rumen. There is a paucity of information available, however, regarding the multiplicity and organisation of <I>R. flavefaciens </I>cellulolytic and xylanolytic enzyme systems. A technique involving PCR amplification of DNA with primers designed from conserved sequences, followed by hybridisation of the PCR products to chromosomal DNA, has led to an estimate of xylanase gene multiplicity in <I>R. flavefaciens</I>. The xylanase-specific primers were also useful in the isolation and sequencing of a partial xylanase gene, <I>xynC</I>. Although <I>R. flavefaciens</I> 17 appears to produce a cellulose-binding enzyme-complex, none of the individual enzymes examined was found to bind cellulose in isolation. However, a 210 kDa protein which is present in the complex was shown to bind cellulose after isolation from a renatured SDS-gel. In order to look for genetic evidence for a cellulose-binding mechanism, sequencing of the <I>R. flavefaciens</I> 17 endoglucanase gene, <I>endA</I>, was completed from PCR products. The carboxy-terminus of the predicted <I>endA</I> product consists of a domain which is similar to dockerins found in <I>Clostridium thermocellum </I>polysaccharidases. Homologous domains are also found in the <I>R. flavefaciens </I>xylanases, XynB and XynD. As the <I>C. thermocellum</I> dockerin domains mediate binding to the 210 kDa scaffolding protein in the cellulosome complex, it is likely that the <I>R. flavefaciens</I> domains play a similar role in assembly of a cellulosome-like complex (Lamed and Bayer, 1994). A gene which maps approximately 1.5 kb downstream from <I>endA</I> on the <I>R. flavefaciens</I> 17 chromosome was sequenced and found to be homologous to <I>nifS</I> genes from nitrogen-fixing bacteria (Zheng <I>et al, </I>1993). The <I>R. flavefaciens</I> NifS product catalyses the production of sulphur from cysteine, and is suspected to partake in the assembly of iron-sulphur clusters. The precise role of NifS is not yet known, but may be related to the degradation of crystalline cellulose.

The mesophilic Ruminococcus flavefaciens FD-1 (NCDO 2215) is a Gram-positive obligate anaerobic bacterium. The aim of this thesis was to clone, sequence and analyze a cellodextrinase gene (celA) and a carboxymethylcellulase gene (celE), and study their regulation and induction at the transcriptional level. The sequence of the celA gene from FD-1 was determined and the amino acid sequence of the CelA enzyme (336 amino acid residues) deduced. It showed 40% identity with endoglucanase C of Clostridium thermocellum and 27.4% identity with endoglucanase 3 of Fibrobacter succinogenes. These three enzymes are grouped into subfamily "A3". The ATG start codon of celA is preceded by a GAGG sequence, predicted to be a ribosome binding site. The derived amino acid sequence corresponded to a protein of Mᵣ 38686. SDS-PAGE analysis of in vitro and in vivo translational products showed that CelA has a molecular mass of ca 39 kDa and was secreted into the Escherichia coli periplasmic space. Although CelA has activity on carboxymethylcellulose, further study on the enzyme showed that it degraded cellopentaose and other cellodextrins to predominantly cellobiose. Thus CelA is a cellodextrinase. It also has high activity against p-nitrophenyl-β-D-cellobioside. A gene, expressing a protein with both carboxymethyl cellulase and xylanase activity, was cloned from R. flavefaciens FD-1 using an E. coli/Bacillus subtilis shuttle vector, pEBl. The 3.6 kb DNA insert on the plasmid pWFl, which carried the gene, celE, contained an open reading frame of 963 bp encoding 320 amino acid residues with a caculated Mᵣ of 35937. Homology analysis showed 11.6% identity and 55.3% similarity with the N-terminal catalytic region of the cellulase gene of alkalophilic Bacillus sp. strain 1139. In order to obtain expression in E. coli, the gene had to be transcribed from the lambda Pᵣ promoter. To determine whether cellulase genes of R. flavefaciens FD-1 were regulated at the level of transcription, celA and celE were used as probes against RNA isolated from R. flavefaciens FD-1 grown on cellobiose, cellulose or cellotriose. Transcription of both genes was induced when cellulose was added to cells growing in cellobiose. This induction continued after cellulose depletion and after cell division had ceased. Transcription of both genes was also induced by cellotriose although not to the same extent as by cellulose. This suggests that cellotriose and possibly ether dextrins may act as key inducers to trlgger celA and eels gene expression in R. flavefaciens FD-1.

Le microbiote humain est constitué de milliers d’espèces bactériennes qui synthétisent de nombreux métabolites secondaires. Cependant, notre connaissance des produits naturels dérivés du microbiome est encore limitée. Parmi eux, les RiPPs (Ribosomally Synthesized and Post-translationally modified Peptides) apparaissent comme une famille majeure de produits naturels possédant diverses structures et fonctions biologiques dont des propriétés antibiotiques, en faisant une famille de molécules d’intérêt majeur pour la santé publique. La biosynthèse des RiPPs commence par la traduction d’un peptide précurseur, qui est ensuite maturé par l’action d’une ou plusieurs enzymes avant l’excision d’une séquence signal et l’export du produit naturel actif. La diversité structurale et fonctionnelle des RiPPs démontre la nécessité de la compréhension des voies de biosynthèse de ces produits naturels, de l’étude systématique des mécanismes de modification et de la caractérisation des maturases associées. En particulier, une famille de métallo-enzymes, les enzymes à radical S-adénosyl-L-méthionine (SAM), a récemment été impliquée dans la biosynthèse de nombreux RiPPs. Ces enzymes catalysent un large éventail de réaction, via un mécanisme de chimie radicalaire, aboutissant à une grande variété de modifications post-traductionnelles. Néanmoins, les voies de biosynthèse de nombreux RiPP restent mal comprises.En 2011, il a été montré que Ruminococcus gnavus, un membre important du microbiote humain, produisait un peptide actif contre Clostridium perfringens, la Ruminococcin C (RumC). Le séquençage de l’opéron de biosynthèse de RumC montre la présence de cinq gènes codants des peptides précurseurs (RumC1-5) et deux gènes codant des enzymes (RumMC1 et RumMC2).L’objectif de ma thèse est de mieux comprendre les voies de biosynthèse des produits naturels au sein du microbiome humain. Nous avons démontré l’appartenance des protéines RumMC1 et RumMC2 à la famille des enzymes à radical SAM, ainsi que leurs implications dans la formation de quatre modifications post-traductionnelles (ponts α-thioether) essentielles à l’activité antibiotique de RumC1 et RumC2. Ces études nous ont permis de proposer un mécanisme catalytique pour la maturation de la Rummonicoccin C et ainsi de mieux documenter cette famille d’enzymes émergentes<br>The human microbiota consists of thousands bacterial species which synthesize numerous secondary metabolites. However, our knowledge of microbiome-derived natural products is still limited. Among them, RiPPs (Ribosomally synthesized and Post-translationally modified Peptides) are emerging as a major family of natural products possessing diverse structures and biological functions including antibiotic properties, making them a major family of molecules of interest for public health. The biosynthesis of RiPPs occurred by the translation of a precursor peptide, which is then matured via the action of one or more enzymes before the excision of a signal sequence and the export of the active natural product. The structural and functional diversity of RiPPs demonstrates the need for understanding the biosynthetic pathways of these natural products, the systematic study of the modification mechanisms and the characterization of associated maturases. In particular, a family of metallo-enzymes, the S-adenosyl-L-methionine radical (SAM) enzymes, has recently been implicated in the biosynthesis of many RiPPs. These enzymes catalyze a wide range of reactions, via a mechanism of radical chemistry, resulting in a wide variety of post-translational modifications. Nevertheless, the biosynthetic pathways of many RiPPs remain poorly understood.In 2011, it was shown that Ruminococcus gnavus, a major member of the human microbiota, produced an active peptide against Clostridium perfringens, Ruminococcin C (RumC). Sequencing of the RumC biosynthesis operon shows the presence of five genes encoding precursor peptides (RumC1-5) and two genes encoding enzymes (RumMC1 and RumMC2).The aim of my thesis is to understand the biosynthetic pathways of natural products within the human microbiome. We have demonstrated that the RumMC1 and RumMC2 proteins belong to the radial SAM enzyme family, as well as their involvement in the formation of four post-translational modifications (α-thiother bridges) essential for the antibiotic activity of RumC1 and RumC2. These studies allowed us to propose a catalytic mechanism for the maturation of Rummonicoccin and thus to better document this family of emerging enzymes

L'objectif du groupe Im3i est d'étudier les mécanismes moléculaires impliqués dans la relation symbiotique entre l'hôte et le microbiote intestinal afin de proposer des solutions pour le traitement de certaines pathologies humaines dans le contexte de la nutrition et de la sécurité alimentaire. Nous avons choisi une bactérie à Gram positif, Ruminococcus gnavus E1 , comme modèle d'étude. Cette bactérie, anaérobie stricte, a été isolée à partir du microbiote d'un adulte sain et son génome a été séquencé . L'étude in vivo a été menée sur des groupes de souris mono-colonisées par R. gnavus E1. Nos résultats montrent clairement une surexpression des gènes codant les fucosyltransférases et sialyltransférases intestinales au niveau du côlon. Les observations réalisées en microscopie confocale à l'aide de lectines ont confirmé ces résultats. L'étude in vitro nous a permis d'analyser l'impact des facteurs solubles présents dans le surnageant de R. gnavus E1 sur la glycosylation des cellules épithéliales intestinales. La caractérisation préliminaire de cette fraction soluble indique qu'elle est thermorésistante, de nature non lipidique et qu'elle possède une faible masse moléculaire (&lt; 3 kDa). Nous pouvons conclure que les variations d'expression des ARNm des différentes glycosyltransférases des cellules en gobelet et des entérocytes entrainant un programme de réinitialisation des glycoprotéines du mucus impacté par R. gnavus E1. Cela ouvre des perspectives considérables dans le dialogue moléculaire qui s'établit entre l'hôte et les bactéries endogènes<br>The aim of IM3I group is study molecular mechanisms involved in the symbiotic relationship between the host and the intestinal microbiota, accounting for both partners, in order to propose solutions to human health problems, especially in the context of nutrition and food safety. We have chosen a Gram positive bacteria, Ruminococcus gnavus E1, as a model. This bacterium, in strict anaerobia, is isolated from the dominant microbiota of healthy human for which the genome is currently being sequenced. In vivo, using animals that are mono-associates with R. gnavus E1, our RT-qPCR studies clearly show an up regulation of the expression of genes coding for intestinal fucosyltranferases and sialyltransferases, in the colon. This is also confirmed by confocal microscopy using lectins. Thus, R. gnavus E1 is able to reestablish the glycosylation profile. In vitro, we have studied the impact of soluble factors from the R. gnavus E1 supernatant. We found that the soluble supernatant fraction of R. gnavus E1 cultures differentially modulates the intestinal glycosylation process in HT29-MTX, Caco-2, independently or coculture 75% Caco-2 and 25% HT29-MTX cell lines. Preliminary characterization of this soluble fraction indicates that it is heat resistant, is not affected by elimination of lipids, and has a low molecular weight form (&lt; 3 kDa). We conclude that the variation of expression of mRNAs coding for different glycosyltransferases in goblet cells and enterocytes proves the re-initiating program of glycoproteins and mucus protection layer by the dominant bacteria R. gnavus E1 and opens the prospect to a new host-indigenous bacteria molecular crosstalk in the intestine

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Produção Animal<br>Protein-protein interactions play a vital role in many cellular processes as exemplified by the assembly of the cellulosome, a bacterial multi-enzyme complex that efficiently degrades cellulose and hemicellulose. Cellulosome assembly involves the high-affinity binding of type I enzyme-borne dockerins to repeated cohesin modules located on non-catalytic structural proteins termed scaffoldins. In addition, the complex is anchored into the bacterial surface through the binding of a scaffoldin type II dockerin to cell-bound cohesins. Initially, the architecture and organization of cellulosomes was thought to rely uniquely on type I and type II cohesin-dockerin interactions. It was recently suggested that cellulosomes from rumen bacteria are organized through different mechanisms involving a third type of cohesin-dockerin complexes. Thus, the genome of the major ruminal bacterium Ruminococcus flavefaciens FD-1 revealed a particularly elaborate cellulosome system that is assembled from a library of more than 200 different components through divergent cohesin-dockerin pairs. Providing structural insights for the specificity displayed by the increasing repertoire of cohesin-dockerin interaction is not only of fundamental importance but essential for the development of novel cellulosome based tools. The present work aimed to identify the molecular basis for the organization of R. flavefaciens cellulosome by dissecting the structural basis of cohesin-dockerin specificity in cellulosomes of rumen bacteria. The data revealed a collection of unique cohesin-dockerin interactions, supporting the functional relevance of dockerin classification in groups based on primary sequence similarity. In addition, R. flavefaciens cellulosome is assembled through a mechanism involving single but not dual-binding mode dockerins. This contrasts with the majority of the cellulosomes described to date where dockerins generally present two similar cohesin-binding interfaces, supporting a dual-binding mode. To illustrate this, the structures of two cohesin-dockerin complexes containing an Acetivibrio cellulolyticus dual-binding mode dockerin were solved. Finally, structural information was used to engineer a dockerin presenting a dual cohesin specificity, revealing the plasticity of the cohesin-dockerin platform to design novel protein-protein interactions.<br>RESUMO - Arquitetura celulossomal divergente em bactérias ruminais: estudos de estrutura e função em complexos coesina-doquerina do Ruminococcus flavefaciens - As interacções proteína-proteína desempenham um papel essencial em vários processos celulares, sendo exemplo disso a estruturação do celulosoma, um complexo bacteriano multienzimático altamente eficiente na degradação da celulose e hemicelulose. A montagem do celulosoma envolve interações de alta afinidade entre doquerinas do tipo I, presentes em enzimas, e os módulos coesina presentes em proteínas estruturais não catalíticas denominadas de escafoldinas. Adicionalmente, todo o complexo é ancorado à superfície bacteriana através da ligação de uma dockerina do tipo II, presente numa escafoldina, a coesinas ligadas à célula. Inicialmente, pensava-se que a arquitectura e organização dos celulosomas assentava exclusivamente em interacções coesina-doquerina do tipo I e II. Recentemente, foi sugerido que a microbiota ruminal contém bactérias produtoras de celulossoma com diferentes mecanismos de organização, envolvendo um terceiro tipo de complexos coesina-dockerina. O genoma da bactéria ruminal Ruminococcus flavefaciens FD-1, revelou um sistema celulossomal particularmente elaborado, montado a partir de uma biblioteca com mais de 200 componentes, através de complexos coesina-doquerina do tipo III. Estabelecer uma base estrutural para a especificidade exibida pelo crescente repertório de pares coesina-doquerina é não só fundamentalmente importante mas também essencial para o desenvolvimento de novas ferramentas com base no celulossoma. O presente trabalho teve como objetivo identificar a base estrutural para a especificidade coesina-doquerina do R. flavefaciens, permitindo descortinar os mecanismos por detrás da montagem dos celulosomas ruminais. Os dados obtidos revelaram uma colecção de interacções coesina-doquerina única, suportando a relevância funcional da classificação das doquerinas em grupos com base na homologia da sua estrutura primária. Mostraram ainda que o celulossoma do R. flavefaciens é montado através de um mecanismo envolvendo doquerinas com modo de ligação único mas não duplo. Isto contrasta com a maioria dos celulosomas descritos até à data, em que as doquerinas geralmente apresentam duas interfaces semelhantes de ligação à coesina, suportando um modo de ligação dupla. Tal é ilustrado pela estrutura de dois complexos coesina-doquerina do Acetivibrio cellulolyticus, envolvendo uma doquerina com modo de ligação dupla. Finalmente, esta informação estrutural foi usada para desenhar uma doquerina com dupla especificidade, mostranto a plasticidade da plataforma coesina-doquerina para o desenvolvimento de novas interações proteína:proteína.<br>N/A

Nous avons comparé quatre méthodes de détermination du pourcentage de bactéries fixées à la cellulose Avicel à partir de cultures pures de bactéries cellulolytiques du rumen, Fibrobacter succinogenes et Ruminococcus flavefaciens. Nous avons ensuite étudié l'influence de quelques facteurs physico-chimiques sur ce phénomène et enfin comparé la charge énergétique et la teneur en nucléotides adényliques des bactéries fixées et des bactéries libres. L'ensemble des résultats laissent supposer que les mécanismes d'adhésion sont différents pour ces deux espèces bactériennes. L'adhésion de F. Succinogenes, contrairement à celle de R. Flavefaciens, est sensible à la température, aux concentrations élevées de glucose et cellubiose, au potentiel redox, aux variations de pH, à la carence en sodium et à certains inhibiteurs métaboliques. L'attachement de R. Flaveciens exige la présence de cations divalents. L'adhésion des deux espèces n'est pas affectée par la carboxyméthylcellulose et les inhibiteurs des chaînes de transfert d'électrons. Les cellules adhérentes de R. Flavefaciens présentent une très forte teneur en ATP et une charge énergétique élevée par rapport aux bactéries non fixées. Cette différence est moins marquée chez F. Succinogenes. L'adhésion de F. Succinogenes semble donc nécessiter l'intégrité des fonctions métaboliques cellulaires et les cellulases bactériennes pourraient jouer un rôle important dans ce phénomène. Plusieurs mécanismes sont vraisemblablement impliqués lors de l'attachement de R. Flavefaciens, le principal étant une liaison entre le glycocalyx bactérien et les cations divalents Mg++ et Ca++. La présence de liaisons hydrophobes ainsi qu'une participation ne sont cependant pas exclues.

Ruminococcus albus est une des bactéries cellulolytiques majeures du rumen. L'adhésion de cette bactérie à la cellulose est une première étape indispensable dans le processus de dégradation des fibres végétales. L'objectif de ce travail est d'élucider les mécanismes impliqués dans l'adhésion de R. Albus à la cellulose. Ce travail a permis de caractériser une glycoprotéine de 25 kDa (GP25) sous-produite par un mutant non adhérent de la souche 20 de R. Albus. GP25 est la sous-unité majeure des pili de type IV observés à la surface de R. Albus 20. Ces pili sont nécessaires à l'adhésion de la bactérie à la cellulose. En aval du gène gp25, deux gènes pilA2 et secD-F codant une deuxième protéine homologue aux pilines de type IV et des protéines impliquées dans la sécrétion, sont identifiées. Deux protéines affines pour la cellulose (CBP1 et CBP2) sous-produites par le mutant ont également été caractérisées par électrophorèse bidimensionnelle et spectrométrie de masse (MALDI-TOF). Ces protéines homologues à deux endoglucanases (Cel9E et Cel48A) de R. Albus 8 seraient impliquées dans l'adhésion. Ainsi, le système d'adhésion de R. Albus est multi-factoriel et ferait intervenir des pili de type IV et des glycosyl-hydrolases

Les α-galactosides sont des glucides non digestibles constitués d'unités galactose liées en α(1,6). Les α-galactosides de la famille du raffinose (RFO) sont, avec le saccharose, les principaux oligosaccharides des légumineuses. Cependant, aucune activité α(1,6)-galactosidase n'est retrouvée au niveau de l'épithélium intestinal humain, les RFO sont donc exclusivement fermentés par les enzymes microbiennes. Ces travaux introduisent une enzyme bifonctionnelle de Ruminococcus gnavus E1, un membre majoritaire du microbiote intestinal humain, présentant une activité α(1,6)-galactosidase/ saccharose kinase (AgaSK). L'analyse de la séquence peptidique montre qu'AgaSK présente deux domaines : un domaine homologue aux α-galactosidases GH36, et un autre contenant un motif de fixation des nucléotides (motif A de Walker). La caractérisation des paramètres biochimiques d'AgaSK met en évidence cette bifonctionnalité puisqu'elle est capable d'hydrolyser les α(1,6)-galactosides solubles, et parallèlement en présence d'ATP de phosphoryler le saccharose spécifiquement sur la position C6 du glucose. La production directe de saccharose-6-phosphate à partir de l'hydrolyse du raffinose constitue une voie métabolique jamais décrite chez les bactéries. L'analyse de chacun des domaines montre que les domaines isolés d'AgaSK sont actifs mais la comparaison de leurs paramètres cinétiques montre qu'il y a des différences entre la protéine entière et les domaines isolés. La résolution de la structure du domaine α-galactosidase en complexe avec le galactose démontre que l'état oligomérique est nécessaire pour le bon repliement de la protéine et pour une fixation efficace du substrat<br>Α-galactosides are non digestible carbohydrates present in many leguminous plants. Soluble α-galactosides consist of galactose units α(1,6) linked to different carbohydrates. Among these, the raffinose family oligosaccharides (RFO) and sucrose, are the most abundant oligosaccharides found in legumes. However, no α(1,6)galactosidase activity exists in the human intestine mucosa and α-galactosides are exclusively fermented by microbial α(1,6)galactosidases (EC3.2.1.22). Here we introduce a bifunctional enzyme, the α(1,6)galactosidase/sucrose kinase (AgaSK) whose gene is highly transcribed in vivo by Ruminococcus gnavus E1, a major member of human dominant intestinal microbiota. Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide binding motif (Walker A motif). Its biochemical characterization showed that AgaSK is able to hydrolyze efficiently soluble α-galacosides. Furthermore, AgaSK it is able to bind nucleotide to phosphorylate specifically on the C6 position of glucose sucrose. The production of sucrose-6-P directly from raffinose brings out a glycolytic pathway in bacteria, not described so far. In addition, AgaSK isolated domains are active but the biochemical characterization has shown that there are differences in the activities between the whole protein and isolated domains. The crystal structures of the galactosidase domain in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding

Chez le rat monoxénique et sous la dépendance de trypsine, Ruminococcus gnavus E1, souche isolée de fèces humains, produit RumC, une bactériocine anti-Clostridium perfringens. LEM 9-17, capable de produire RumC in vitro, a été obtenue par mutagenèse aléatoire. RumC a été purifiée à partir de contenu caecal de rats monoxéniques pour E1 et de surnageant de culture de LEM 9-17. Dans les 2 cas, 2 fractions ont été identifiées : la 1ère contient un peptide de 4235 Da, la 2ème 2 peptides de 4324 et 4456 Da. Les séquences N-terminales des peptides contenus dans ces fractions sont très homologues, mais la réaction d'Edman est bloquée après le 11ème résidu. Trois gènes différents codant pour des pré-RumC, 2 pour des enzymes à radical SAM et 8 probablement impliqués dans la biosynthèse, la sécrétion, la régulation ou l'immunité ont été identifiés sur une région de 13,5 kb du génome de E1. De plus, les gènes rumC-like semblent disséminés au sein du microbiote fécal humain et sont très conservés<br>When colonizing the digestive tract of monoxenic rats, Ruminococcus gnavus E1, isolated from human feces, produced a trypsin-dependant anti-Clostridium perfringens bacteriocin, RumC. LEM 9-17, able to produce RumC in vitro, was generated by random mutagenesis. RumC was purified from E1-monocontaminated caecal content and LEM 9-17 culture supernatant. In both cases, 2 fractions, the 1st one containing a 4235Da peptide, and the 2nd one two peptides of 4324Da and 4456Da were identified. The N-terminal sequence of the peptides present in both fractions was highly homologous, but Edman degradation reaction was blocked after the 11th residue. Three non identical structural genes encoding pre-RumC, 2 coding for radical SAM enzymes and 8 with predicted functions in biosynthesis, secretion, regulation and immunity were identified on a 13,5 kb region of E1 chromosome. Moreover, the rumC-like genes were shown to be widely disseminated among the human fecal microbiota with a high conservation rate

Les antibiotiques sont des médicaments qui ont changé notre manière d’aborder les infections bactériennes et sont devenus l’un des symboles de la médecine moderne. Cependant leur utilisation massive a conduit à l'émergence de souches bactériennes multirésistantes. Ce problème est sans aucun doute un des grands défis que la médecine actuelle doit relever. Sachant que les bactéries évoluent à un rythme plus rapide que la production de nouveaux antibiotiques, il est urgent de trouver des approches alternatives. Il a été mis en évidence que ces mêmes bactéries sont capables de sécréter différents peptides antimicrobiens, ou bactériocines. Ces macromolécules présentent une grande diversité structurale et sont très efficaces pour combattre un grand nombre de souches pathogènes de façon spécifique. Les bactériocines ont un immense potentiel dans les domaines agroalimentaire et pharmaceutique. Notre projet s’intéresse aux bactériocines RumCs produites par une souche dérivée de Ruminococcus gnavus, une bactérie anaérobie stricte, membre dominant du microbiote intestinal humain. Le travail présenté dans ce manuscrit concerne la mise au point d’un système d’expression et de maturation hétérologue chez E. coli de la bactériocine RumC1. La caractérisation biochimique du peptide RumC1 montre que les bactériocines RumCs appartiennent à la famille des sactipeptides pour laquelle l’étape de biosynthèse fait intervenir une enzyme radical-SAM. Les sactipeptides présentent dans leurs séquences peptidiques un ou plusieurs ponts thioéther entre une cystéine et le carbone alpha d’un acide aminé partenaire. RumC1 renferme 4 ponts thioéther ce qui lui confère une structure originale en double épingle à cheveux. L’activité biologique de RumC1 montre que ce peptide est efficace contre un large spectre de bactéries à Gram positif incluant des pathogènes résistants tels que S.aureus et E. faecalis. Dans ces études nous n’avons pas noté de toxicité significative de RumC1 sur différentes lignées cellulaires humaine ni observé de phénomène de résistance. Les travaux en cours visent notamment à définir le mode d’action de RumC1 et à évaluer l’activité biologique de RumC1 dans un contexte d’infection in vivo chez la souris<br>Antibiotics are drugs that have changed the way we approach bacterial infections and have become one of the symbols of modern medicine. However, their widespread use has led to the emergence of multiresistant bacterial strains. This problem is undoubtedly one of the major challenges facing today's medicine. Knowing that bacteria evolve at a faster rate than the discovery of new antibiotics, it is urgent to find alternative approaches. It has been shown that these same bacteria are capable of secreting antimicrobial peptides, the bacteriocins. These macromolecules have a high structural diversity and are very effective in combating a large number of pathogenic strains in a specific way. Bacteriocins have immense potential in the agro-food and pharmaceutical sectors. Our project focuses on the bacteriocins RumCs produced by a strain derived from Ruminococcus gnavus, a strict anaerobic bacterium of the human intestinal microbiota. The work presented in this manuscript concerns the development of a heterologous expression and maturation system in E. coli of the bacteriocin RumC1. The biochemical characterization of the RumC1 peptide shows that the RumCs bacteriocins belong to the family of sactipeptides for which the biosynthesis step involves a radical-SAM enzyme. The sactipeptides have in their peptide sequences one or more thioether bridges between a cysteine and the alpha carbon of a partner amino acid. RumC1 contains 4 thioether bridges which gives it an original structure in double hairpin. The biological activity of RumC1 shows that this peptide is effective against a broad spectrum of Gram-positive bacteria including resistant pathogens such as S.aureus and E. faecalis. In these studies, we did not note any significant toxicity of RumC1 on different human cell lines nor observed resistance phenomena. Current work aims to define the mode of action of RumC1 and to evaluate the biological activity of RumC1 in an in vivo context of infection in mice

Tese de doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e Biomédicas<br>ABSTRACT - Plant cell wall polysaccharides offer an abundant energy source efficiently utilized by a large repertoire of micro-organisms, which thus play a central role in carbon re-cycling. Aerobic micro-organisms secrete Carbohydrate-Active Enzymes (CAZymes) as free-standing proteins, whereas anaerobic bacteria organize a diverse enzyme consortium in a multi-component complex, the cellulosome, which performs a more efficient deconstruction of this composite structure. CAZymes are modular enzymes containing, in addition to catalytic domains, non-catalytic Carbohydrate-Binding Modules (CBMs). CBMs direct the appended enzymes to their target substrates thus potentiating catalysis. Here we show that the CBMs of Eubacterium cellulosolvens endoglucanase 5A (EcCel5A), designated as CBM65A and CBM65B, display a significant preference for xyloglucan. The crystal structure of CBM65B in complex with a xyloglucan-derived oligosaccharide, in combination with mutagenesis studies on CBM65A, revealed the mechanism by which these proteins display a preference for xyloglucan by establishing hydrophobic interactions with xyloglucan xylose side chains (Chapter 2). The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 encodes a large number of putative novel cellulosomal proteins. Here, genes encoding cellulosomal modules of unknown function were cloned and their corresponding proteins expressed at high levels in Escherichia coli. Complementary techniques combining affinity gel electrophoresis, a microarray platform and isothermal titration calorimetry were used to identify novel CBMs in cellulosomal-modules of unknown function. This strategy allowed the identification of 8 novel CBM families. The structures of representative members of two of these families (CBM-A and CBM-B1) have been solved and detailed functional characterization of these CBMs was performed. CBM-A and CBM-B1 comprise β-sandwich folds. CBM-A binds decorated β-1,4-glucans at a shallow binding cleft and displays preference for xyloglucan. In contrast, CBM-B1 displays a flat surface complementary to an open cleft that allows binding to a range of β-glucans including insoluble cellulose recognition (Chapter 3). Finally, the structure of CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, was solved. BhCel5B is a multi-modular enzyme composed of a GH5_4 N-terminal catalytic domain, followed by an internal immunoglobulin-like module (Ig) and a C-terminal CBM46. BhCBM46 does not bind soluble or insoluble polysaccharides. However, the crystal structure of BhCel5B revealed that CBM46 is integral to the GH5_4 enzyme catalytic cleft and thus plays an important role in substrate recognition (Chapter 4).<br>RESUMO - Estrutura e Função de novas glucosil hidrolases (CAZymes) e de Módulos de Ligação a Hidratos de Carbono (CMBs) envolvidos na degradação da Parede Celular Vegetal - Os polissacarídeos da parede celular vegetal são uma fonte de energia abundante, eficientemente utilizada por um vasto número de microrganismos, os quais desempenham um papel central na recilagem do carbono. As enzimas secretadas pelos microrganismos aeróbicos, que promovem a hidrólise de hidratos de Carbono (CAZymes), funcionam de froma individualizada, ao passo que as bactérias anaeróbicas organizam essas enzimas num complexo multi-enzimático designado por Celulossoma, o qual efetua uma degradação mais eficiente da parede celular vegetal. As CAZymes são enzimas modulares que contêm, além de domínios catalíticos, módulos de ligação a hidratos de Carbono (CBMs) com função não catalítica. Os CBMs direcionam as enzimas a eles ligadas para os substratos-alvo, potenciando assim a catálise. Neste trabalho mostra-se que os CBMs associado à endoglucanase 5A (EcCel5A) da Eubacterium cellulosolvens designados por CBM65A e CBM65B, possuem uma significativa preferência por xiloglucano. A estrutura tridimensional do CBM65B, em complexo com um derivado oligossacárido do xiloglucano e os estudos de mutagenese realizados no CBM65A, revelaram que o mecanismo de preferência destas proteínas pelo xiloglucano se deve ao estabelecimento de interações hidrofóbicas com as cadeias laterais (xilose) deste substrato (capítulo 2). O genoma da bactéria celulolítica do rúmen Ruminococcus flavifaciens, estirpe FD1, codifica um vasto número de putativas proteínas celulosomais, ainda não estudadas. Neste estudo, os genes que codificam os módulos celulosomais de funções desconhecidas foram clonados e as proteínas por eles codificadas foram expressas em níveis elevados em Escherichia coli. Técnicas complementares, combinando eletroforese em gel nativo, uma plataforma de matriz de alta densidade (microarray) e calorimetria de titulação isotérmica, foram usados para identificar novos CBMs em módulos celulosomais de função desconhecida. Esta estratégia permitiu a identificação de 8 novas famílias de CBMs. Foram determinadas as estruturas tridimensionais representativas de duas destas famílias (CBM-A e CBM-B1), e efectuada a sua caracterização funcional detalhada. O CBM-A e o CBM-B1 apresentam um enrolamento em sanduiche β. O CBM-A liga-se ao β-1,4-glucano ramificado através de uma fenda superficial, revelando preferência por xiloglucano. Em contraste, o CBM-B1 revela uma superfície plana complementar a uma fenda aberta que permite a ligação a uma série de glucanos de tipo β, incluindo o reconhecimento de celulose insolúvel (capítulo 3). Por último, a estrutura do CBM46 derivado de uma endoglucanase do Bacillus halodurans designada por BhCel5B, foi determinada. A BhCel5B é uma enzima multi-modular composta por um domínio catalítico da família GH5_4 no terminal N, seguida por um módulo interno do tipo da imunoglobulina (lg) e o CBM46 no terminal C. O BhCBM46 não se liga a polissacarídeos solúveis ou insolúveis. Porém, a estrutura tridimensional da BhCel5B revelou que o CBM46 é parte integrante da fenda onde se alojam os resíduos responsáveis pela catálise da enzima GH5_4 e, por conseguinte, desempenha um papel importante no reconhecimento do substrato (capítulo 4)

碩士<br>國立臺灣大學<br>動物科學技術學研究所<br>96<br>Our research was set up to find more detailed characteristics of the bacteriocin from R. albus 7 and its potential as alternative of antibiotics. We also tried to establish reliable production and semi-purification procedures. In our results, R. albus 7 could be cultured in the medium without ruminal fluid to produce bacteriocin effectively against five pathogens (Enterobacter, Bacillus, Pseudomonas, Samonella, Staphylococcus). MIC50 and MIC90 of R. albus 7 bacteriocin were as low as 0.75 mg/mL and 6.5 mg/mL, respectively. The bacteriocin is highly released after 24 hr of incubation. Inclusion of 3μM 3-phenylpropanoic acid（PPA）and 0.2 % Tween 80 in culturing medium improved 2.5 times production of bacteriocin. Native-gel electrophoresis showed that bacteriocin of R. albus 7 had a molecular weight about 33 kDa. Semi-purification procedure resulted in 23% recovery rate of bacteriocin by using the DEAE column in FPLC system. The bacteriocin of R. albus 7 could be destroyed by pepsin, protease, and pancreatin. It also could be inactivated after heating at 65℃for 1 hr. Simulated in vitro avian digestion decreased the antagonistic activity by 74.7%. But, addition 1% BSA could retain 13% more antagonistic activity. In conclusion, the bacteriocin of R. albus 7 is a heat-labile small protein. PPA and Tween 80 are recommended to be included in its production culture medium. DEAE column was suitable for its semi-purification. The bacteriocin of R. albus 7 has the chance as an antibiotic alternative with protectant added.

Στόχος της παρούσας εργασίας ήταν η διερεύνηση της δυνατότητας παραγωγής υδρογόνου από καθαρές καλλιέργειες του μικροοργανισμού Ruminococcus albus εστιάζοντας κυρίως στο μηχανισμό της διεργασίας. Ο R. albus είναι ένα ετερότροφο, αυστηρά αναερόβιο βακτήριο που διαβιεί στο πρώτο διαμέρισμα του τετραμερούς στομάχου των μηρυκαστικών, τον κεκρύφαλο (rumen). Αναπτύσσεται καταναλώνοντας τους σύνθετους υδατάνθρακες που φτάνουν εκεί μέσω της πρόσληψης τροφής από το μηρυκαστικό, αφού πρώτα τους υδρολύσει μέσω εξωκυτταρικών ενζύμων που παράγει. Τα προϊόντα της υδρόλυσης είναι απλοί υδατάνθρακες που ζυμώνονται περαιτέρω προς παραγωγή λιπαρών οξέων, αιθανόλης, διοξειδίου του άνθρακα και υδρογόνου. Η ικανότητα παραγωγής υδρογόνου και η τελική απόδοση εξαρτώνται από τις συνθήκες υπό τις οποίες πραγματοποιείται η ανάπτυξη. Ο R. albus πιστεύεται ότι είναι πολλά υποσχόμενος για την παραγωγή υδρογόνου από αγροτικά υπολείμματα που είναι πλούσια σε λιγνοκυτταρινούχα υλικά καθώς και ενεργειακά φυτά , όπως είναι το γλυκό σόργο. Οι βλαστοί του γλυκού σόργου είναι πλούσιοι σε σάκχαρα, κυρίως σακχαρόζη σε ποσοστό έως και 55% επί ξηρής μάζας και γλυκόζη (3.2% επί ξηρής μάζας) που απομακρύνονται εύκολα μέσω της διεργασίας εκχύλισης με νερό. Οι βλαστοί του γλυκού σόργου περιέχουν επίσης μεγάλο ποσοστό κυτταρίνης (12.4%) και ημικυτταρίνης (10.2%). Προκειμένου να μελετηθεί ο μεταβολισμός του βακτηρίου και να υπολογιστούν οι κινητικές σταθερές πραγματοποιήθηκαν πειράματα σε αντιδραστήρες διαλείποντος έργου και αντιδραστήρες συνεχούς τροφοδοσίας (CSTR). Ως πηγές άνθρακα χρησιμοποιήθηκαν διάφορα απλά υδατανθρακικά υποστρώματα καθώς και βιομάζα από το ενεργειακό φυτό Sorghum bicolor (γλυκό σόργο). Τα κύρια προϊόντα που ανιχνεύτηκαν σε όλες τις περιπτώσεις ήταν τα οξέα οξικό και μυρμηκικό, η αιθανόλη και το υδρογόνο. Η απόδοση σε υδρογόνο ήταν γενικά μεγαλύτερη στα πειράματα διαλείποντος έργου. Ειδικότερα για τα πειράματα με γλυκόζη, η απόδοση κυμαινόταν μεταξύ των τιμών 2 και 2.6 mol H2/mol γλυκόζης στις καλλιέργειες διαλείποντος έργου, ενώ η βέλτιστη απόδοση από τις συνεχείς καλλιέργειες ήταν 1.07± 017 mol H2/mol γλυκόζης για υδραυλικό χρόνο παραμονής 42 h. Η τελική τιμή απόδοσης παρουσίαζε εξάρτηση από τη μερική πίεση υδρογόνου στην αέρια φάση των καλλιεργειών, το αναγωγικό μέσο για την εξασφάλιση αναγωγικών συνθηκών καθώς και την ποσότητα παραγόμενης αιθανόλης. Η κινητική μικροβιακής ανάπτυξης και παραγωγής υδρογόνου μελετήθηκε μέσω των πειραμάτων με γλυκόζη και η δεύτερη συνδέθηκε μέσω κινητικών εξισώσεων με την διάσπαση του μυρμηκικού οξέος και την παραγωγή αιθανόλης. Άλλα απλά υποστρώματα που μελετήθηκαν είναι οι πεντόζες D- και L-αραβινόζη και η D-ξυλόζη, οι δισακχαρίτες κελλοβιόζη και σακχαρόζη, για τα οποία υπολογίστηκαν οι κινητικές ανάπτυξης του μικροοργανισμού και βέβαια η βιομάζα σόργου, το εκχύλισμα σόργου και τα υπολείμματα σόργου μετά την εκχύλισή του. Οι αποδόσεις σε υδρογόνο ήταν πολλά υποσχόμενες σε όλες τις περιπτώσεις.<br>The aim of the present work was to investigate the process of hydrogen production using pure cultures of fibrolytic bacterium Ruminococcus albus, focusing mainly on the mechanism of the activity. R. albus is an important fibrolytic bacterium of the rumen, where it cohabits with other bacteria and protozoa. R. albus can ferment soluble sugars and also complex carbohydrates, such as cellulose and hemillulose, after breaking them down through the extracellular enzymes it produces. Regardless the initial substrate used a significant amount of hydrogen evolves from the fermentation process. Previous research with pure cultures of R. albus and whole sorghum, sorghum extract and lignocellulosic residues as substrate, lead to very promising hydrogen yields. Moreover, it was shown that sorghum biomass can be used for hydrogen production with high and similar final yields, independent on whether the process takes place in one stage, i.e. when both simple and complex carbohydrates are fermented in the same fermentor, or in two stages i.e. when sorghum extract and extraction residues are fermented separately. Therefore, it is believed that R. albus is very promising for the production of hydrogen from agricultural residues rich in lignocellulosic materials and from energy crops, such as sweet sorghum which contains soluble sugars and complex carbohydrates in almost equal amounts. Sweet sorghum is an annual C4 plant of tropical origin, well-adapted to sub-tropical and temperate regions and highly biomass-productive. Sweet sorghum stalks are rich in sugars, mainly in sucrose that amounts up to 55% of dry matter and in glucose (3.2% of dry matter). They also contain cellulose (12.4%) and hemicelluloses (10.2%). Extraction of free sugars from the stalks is easily achieved by extraction with water at 30°C. After the extraction process a liquid fraction, rich in sucrose, and a solid fraction, containing the cellulose and hemicelluloses, are obtained. The liquid fraction could be directly fermented to hydrogen, whereas the solid fraction should first be hydrolyzed in order to fully exploit the potential of the sorghum biomass for biohydrogen production In order to study the metabolism of bacterium and estimate growth and hydrogen production kinetics, batch and continuous experiments were carried out with glucose as carbon source. Besides glucose pentoses and disaccharides were tested as well, and the growth kinetics on these substrates were estimated.. The main products that were detected in all the cases were acetate, formate, ethanol and hydrogen. Hydrogen yield was generally higher in batch experiments. More specifically glucose experiments showed yields varying between the values 2 and 2.6 mol H2/mol of glucose in batch cultures, while the optimum yield in continuous cultures was 1.07± 017 mol H2/mol of glucose when the hydraulic retention time was 42h. The final hydrogen yield seemed to depend on hydrogen partial pressure, the reducing agent used and the final amount of ethanol.. The production of hydrogen was studied with glucose experiments and was connected via kinetic equations with formate breaking down acid and ethanol production. The other simple substrates that were studied were the pentoses D - and L-arabinose and the D-xylose, the disaccharides cellobiose and sucrose, for which the growth constants were calculated. Subsequently whole sorghum biomass, sorghum extract and lignocellulosic sorghum residues were tested and the experimental results were simulated. The simulations were sufficient in all cases, and hydrogen yields were very promising.

The plant cell wall is, in its majority, constituted by complex and structurally diverse polysaccharides that are valuable resources for industrial and biotechnological applications. Anaerobic microbial organisms are highly efficient for plant cell wall polysaccharide biodegradation and have evolved a multi-enzyme complex system, the Cellulosome, where catalytic enzymes have non-catalytic Carbohydrate Binding Modules (CBMs) appended that highly potentiate the enzymes’ catalytic efficiency. Deciphering at molecular level the mechanisms underlying plant cell wall carbohydrate recognition and deconstruction by different cellulolytic bacteria is crucial to elucidate these complex biological systems, as well as to further promote novel potential applications. The work developed in this Thesis focused on the unique approach of combining carbohydrate microarrays with X-ray crystallography, to uncover carbohydrate ligands for CBMs and to structurally characterize novel CBM-carbohydrate interactions of two anaerobic bacteria that reside in different ecological niches: Clostridium thermocellum, found in soils, and Ruminococcus flavefaciens FD-1, present in the rumen of herbivorous. To this end, microarrays featuring carbohydrate probes with polysaccharide and oligosaccharide sequences representative of the structural diversity found on plant cell walls, but also in fungal and bacterial cell walls, were developed and then used to screen the carbohydrate-binding and ligand-specificity of 150 CBMs of C. thermocellum and R. flavefaciens CBMomes. The groups of polysaccharides that are differentially recognised were revealed for 59 CBMs and novel CBM-ligand specificities were identified for 23 modules from C. thermocellum and 21 from R. flavefaciens. Overall, the two bacteria differentially expressed CBM families with different carbohydrate-binding specificities, which may reflect adaptation to substrate availability in their specific ecological niche or the complexity of their Cellulosome. Using the information derived from the high-throughput microarray analysis, and according to their biotechnological relevance or novelty, CBMs and the respective ligands were selected for further structural studies. The novel CBM structures solved, complemented with biochemical and biophysical data, enabled the characterization of the molecular determinants for the recognition of mixed-linked β1,3-1,4-glucans by C. thermocellum family 11 CBM, chitin and peptidoglycan-derived sequences by a novel LysM domain from C. thermocellum family 50 CBMs, and pectic arabinans by R. flavefaciens family 13 CBM. The results reported here allow to assign a functional role for these CBMs and CBM families and contribute to the classification of the novel CBMs identified in the genome of the two bacteria, particularly those from R. flavefaciens FD1. Furthermore, the information derived from this integrative study, can promote a better understanding of cellulolytic capabilities of these bacteria, as well as to potentiate biotechnological applications of CBMs.