Streptococcus pneumoniae is a commensal inhabitant of the human nasopharynx that can sometimes become pathogenic and cause diseases such as pneumonia, otitis media and meningitis. Carbohydrate metabolism is a critical component of S. pneumoniae virulence. Among the myriad of carbohydrate-specific pathways involved in the host-pneumococcus interaction, the N-glycan foraging pathway stands out because of its direct implication in numerous aspects of virulence such as fitness, adhesion/invasion and impairment of the host immune response. Much of the literature has been focussed on the importance of step-wise depolymerisation of N-glycans by the enzymes NanA, BgaA and StrH. However, the importance of the liberation of N-glycans from host glycoconjuguates and their intake by the bacterium has yet to be examined. We have identified a Carbohydrate Processing Locus (CPL) that is highly conserved throughout a large number of Firmicutes and whose individual components appear widespread in bacteria that we hypothesize is active on host N-glycans. This locus encodes for two putative α-mannosidases GH92 and GH38, a characterised α-mannosidase GH125, a putative β-hexosaminidase GH20C, a putative α-fucosidase GH29 and a ROK (Repressor, Open reading frame, Kinase) protein. The genomic context of CPL orthologues suggests that an endo-β-N-acetylglucosaminidase (EndoD) and an ABC transporter (ABCN-glycan) are functionally associated with this locus. Based on our bioinformatic analyses and known functions of these proteins we hypothesize that the CPL encodes a concerted pathway responsible for the liberation, transport, and processing of N-glycans. The objective of this research is to characterize the putative components of this pathway and assess their implication in virulence. Specific focus on ABCN-glycan demonstrated its specificity for a range of N-glycans liberated by EndoD, shedding light on a novel import system for branched N-glycans. Furthermore, we provided evidence that GH92 is an α-1,2-mannosidase that likely removes the terminal mannose residues found on high-mannose N-glycans. EndoD and GH92 are shown to participate in virulence in mice; however, their role in virulence has yet to be determined. This work will significantly advance the construction and validation of a model of N-glycan processing by S. pneumoniae. As the components of this model pathway are conserved amongst a wide variety of bacteria, this work is of fundamental relevance to understanding how microbes from various environments degrade and metabolize N-glycans. / Graduate

The degradation of carbohydrates is essential to many biological processes such as cell wall remodelling, host-pathogen defense, and energy synthesis in the form of ATP. Several of these processes utilize carbohydrate-active enzymes to accomplish these goals. Studying the degradation of polysaccharides by carbohydrate-active enzymes synthesized by microbes has allowed us to further understand biomass conversion. A portion of these polysaccharides consists of β-1,3-linked glucose (i.e. β-1,3-glucan), which is found in plants, fungi, and brown macroalgae. The hydrolysis of β-1,3-glycosidic linkages is catalyzed by β-1,3-glucanases, which are present in six different glycoside hydrolase (GH) families: 16, 17, 55, 64, 81, and 128. These enzymes play important biological roles including carbon utilization, cell wall modeling, and pathogen defense. This study focuses on a gene from Bacillus halodurans encoding for a multi-modular protein (BhLam81) consisting of a glycoside hydrolase from family 81 (BhGH81), a carbohydrate-binding module (CBM) from family 6 (BhCBM6), and a CBM from family 56 (BhCBM56). Previously, thorough structural and substrate-specific characterization has been carried out on BhCBM6. This CBM binds the non-reducing end of β-1,3-glucan. A member of CBM family 56 has been shown to recognize and bind the insoluble β-1,3-glucan, pachyman, however it is structurally uncharacterized. A glycoside hydrolase belonging to family 81 from Saccharomyces cerevisiae has been previously shown to degrade the β-1,3-glucans, laminarin and pachyman, however the structure of this enzyme was not determined. Recently, a member of GH family 81 has been structurally characterized; however, substrate-specificity was not determined in that study. Therefore, this study concentrated on two goals: Determining the substrate-specificity of BhGH81 and BhCBM56, and solving the structure of BhGH81 and BhCBM56 in order to gain insight into the molecular details of how they recognize and act on their substrate(s). The deoxyribonucleic acid (DNA) encoding for these modules were dissected by restriction digest from B. halodurans genomic DNA and recombinantly expressed in Escherichia coli (E. coli) as separate constructs. Both BhGH81 and BhCBM56 were purified and their crystal structures obtained. BhGH81 and BhCBM56 were solved to 2.5 Å resolution by single-wavelength anomalous dispersion (SAD) and to 1.7 Å resolution by multi-wavelength anomalous dispersion (MAD), respectively. In order to determine the substrate-specificity of BhGH81 and BhCBM56 and speculate on the molecular details of how they recognize and act on their substrate(s), substrate-specificity tests were combined with structural analysis for both of these modules. By using qualitative depletion assays, quantitative depletion assays, and affinity electrophoresis, it was revealed that BhCBM56 binds both insoluble and soluble β-1,3-glucan. The crystal structure of BhCBM56 revealed that it is a β-sandwich composed of two antiparallel β-sheets consisting of five β-strands each. By comparing BhCBM56 to a β-1,3-glucan binding protein from Plodia interpunctella (βGRP) a putative substrate-binding cleft on the concave side of the β-sandwich created by a platform of hydrophobic residues surrounded by several polar and charged residues was revealed. This comparison also allowed for speculation of the amino acids (W1015, H965, and D963) that are potentially essential for recognition of β-1,3-glucan substrates by BhCBM56. Activity of BhGH81 on β-1,3-glucans was confirmed by both thin-layer chromatography and product analysis using high performance anion exchange chromatography. The high performance anion exchange chromatography of BhGH81 hydrolysis suggested it has both exo and endo modes of action. The crystal structure of BhGH81 revealed that it consists of domains A, B, and C: A β-sandwich domain (A), a linker domain (B), and an (α/α)6-barrel domain (C). This structure revealed a putative substrate-binding cleft on one side of the (α/α)6-barrel with a blind canyon active site topology. It also revealed two putative catalytic residues, E542 and E546. All GHs from family 81 characterized so far, hydrolyze β-1,3-glucan in an endo acting manner. By comparing the structure of BhGH81 acquired in this study to a cellulase from Thermobifida fusca, which has an endo-processive mode of action, we can speculate that BhGH81 also has an endo-processive mode of action. The structural and biochemical analysis of BhGH81 and BhCBM56 in this study has aided in further understanding the molecular details both GH family 81 and CBM family 56 proteins, as well as the degradation of β-1,3-glucan by multimodular enzymes. Understanding these molecular details could be important for industrial applications such as, engineering a microbial platform for more efficient biofuel production. / Graduate

Background: Relatively little is known about the genomic basis and evolution of wood- feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. Results: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. Conclusions: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Le but de mon projet de doctorat est la recherche de chitosanases thermostables qui peuvent mener la réaction d'hydrolyse du chitosane à de hautes températures. La procédure mise au point pour isoler ces chitosanases était planifiée pour moduler l'effet antimicrobien du chitosane qui augmente avec son poids moléculaire. Les objectifs spécifiques de ce projet sont, mettre au point un nouveau dosage de Csn, purifier, caractériser et cloner le gène des chitosanases les plus thermostables sélectionnées et mettre au point un milieu de production de chitosanase. La première étape du projet est la recherche de nouvelles chitosanases thermostables, via un criblage ciblé de bactéries productrices de chitosanases. En effet, une nouvelle méthode d'enrichissement était utilisée par l'ajout de chitosane de différents poids moléculaires à notre source bactérienne, soit les composts. La deuxième étape, est la réalisation d'un dosage de l'activité chitosanase en utilisant le soluble-dyed Remazol Brillant Bleu-Chitosane (sRBB-C) qui a été mise au point pour détecter à grande échelle une activité chitosanase de manière facile et rapide. Enfin, la troisième étape est un test de thermostabilité en présence de substrat, appliqué à des chitosanases choisies, pour sélectionner les plus performantes à l'étape de la purification. Parmi le lot de chitosanases testées, la chitosanase notée Csn1794 s'est distinguée par sa thermostabilité à 70 degrés C, ainsi elle a été retenue pour des études plus approfondies. Les études biochimiques réalisées sur la Csn1794 après purification ont révélé qu'elle a un poids moléculaire de 40 kDa, un pH optimal de 4.8 et des K[indice inférieur m] et k[indices inférieurs cat] de 0.042 mg/ml et 7588 min[indices supérieurs -1] respectivement. Le temps de demi-vie de la Csn1794 en présence de chitosane est plus de 20 heures à 70 degrés C. L'activité de la Csn1794 varie légèrement avec le degré d'acétylation du chitosane, elle hydrolyse la carboxyméthyl-cellulose, mais pas la chitine. Le clonage du gène de la Csn1794 par génétique inverse a permis de déterminer sa séquence. Ce gène codé pour une protéine de 441 acides aminés. La Csn1794 appartient à la famille 8 des glycosides hydrolases (GH8). Le rang taxonomique de l'isolat produisant la Csn1794 a été déterminé par des méthodes classiques ainsi que par des tests de biologie moléculaire. Les résultats obtenus indiquent qu'il s'agit d'un isolat appartenant à une espèce non caractérisée appartenant au genre Paenibacillus qu'on a appelé Paenibacillus sp. 1794. Enfin, la méthode de plan d'expériences était utilisée pour mettre au point le milieu de production de la Csn1794. Les essais réalisés par les plans d'expériences Plackett-Burman ont permis non seulement de définir un milieu de base pour la production de la Csn1794, mais aussi les oligosaccharides et le sucrose se sont distingués comme facteurs à effet nettement positif sur la production de la Csn1794. Les essais par plans d'expérience Box-Hunter ont permis l'étude d'interactions entre les différents facteurs dont le niveau était déterminé par les plans Taguchi. Les résultats obtenus indiquent qu'en plus de milieu de base, l'ajout de 10g/l de glucosamine, 7g/l d'oligosaccharide et 4g/l de sucrose constitue la meilleure combinaison pour un milieu qui permet de produire une moyenne de 7U/ml de Csn1794 d'une manière constante. En conclusion, nous disposons d'une nouvelle chitosanase thermostable, facile à produire et à purifier, qui sera un outil adéquat pour l'application au niveau industriel. Ceci va non seulement permettre de mener le processus d'hydrolyse de chitosane à haute température, mais aussi d'utiliser de grandes concentrations de substrat sans que la viscosité ne devienne excessive. Au niveau de la recherche fondamentale, la Csn1794 peut nous apporter plus d'informations d'une part, sur la thermostabilité des enzymes et d'autre part, sur les enzymes de la famille GH8, notamment les chitosanases.

Ready-to-eat meat products such as dry-fermented sausages have been associated with foodborne outbreaks despite the multiple hurdles used in the manufacturing process to prevent growth of pathogens. As a result, new strategies such as natural products with antimicrobial activity are being used to control pathogens of importance like Escherichia coli O157:H7. This study investigated how different concentrations and sources of mustard can influence its antimicrobial activity against E. coli O157:H7 in dry-fermented sausage, as well as the contribution of residual myrosinase enzyme in mustard to this process. The genetic basis for the degradation of mustard glucosinolate by E. coli O157:H7, which is associated with the antimicrobial action of mustard, was also characterized. The ability of E. coli O157:H7 to withstand inhibitory allyl isothiocyanate (AITC) concentrations and the role of the two-component BaeSR system as a defense mechanism against AITC was also investigated. Results showed that 4% (w/w) deodorized yellow mustard powder was effective to control E. coli O157:H7 in dry-fermented sausage at 28 d. The presence of endogenous plant myrosinase in the mustard powder or meal enhanced E. coli O157:H7 reduction rates. Fully-deodorized, deoiled, yellow mustard meal as low as 2% (w/w) containing either 0.1% or 0.2% of residual plant myrosinase achieved the same results as 4% (w/w) mustard powder also containing similar residual myrosinase. Regardless of the type of mustard, the antimicrobial activity of yellow mustard derivatives were more pronounced than those of Oriental mustard. The initial genetic assessment through in silico analysis found similarity between plant myrosinase and enzymes encoded by genes (bglA, ascB, and chbF) from β-glucosidase families in E. coli O157:H7 strains. After disruption of these genes using lambda-red replacement, single (∆bglA, ∆ascB, ∆chbF) and double (∆bglAascB, ∆chbFascB, ∆chbFbglA) mutant strains were created and assessed for glucosinolate degradation. The comparison of the gene expression profiles and changes in the extent of sinigrin degradation by different mutants suggested that ascB have a prominent role in the degradation of this β-glucoside by E. coli O157:H7. E. coli O157:H7 did not develop resistance to AITC, the essential oil formed from sinigrin degradation that is responsible for the antimicrobial activity of Oriental mustard.

Although carbohydrates are a principal component of the human diet, the mechanism of the final stages of starch digestion is not fully understood. One approach to treating metabolic diseases such as type II diabetes, obesity, and congenital sucrase isomaltase deficiency is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals: the release of free glucose. MGAM and SI consist of two catalytic subunits: N-terminal and C-terminal, with overlapping, but variant substrate specificities. The objective of this thesis is to increase the understanding of the differential substrate specificity seen in the catalytic subunits of SI and MGAM. Through inhibitor studies, the structural and biochemical differences between the enzymatic subunits are explored, illustrating that each individual catalytic subunit can be selectively inhibited. In Chapter 3, homology models of ctSI and ctMGAM-N20 are presented, giving insight into the residues hypothesized to impact substrate specificity, enhancing our understanding of the functionality of these enzymatic subunits and overlapping substrate specificity. The structural implications of mutations seen in ntSI in CSID patients and the potential functional and structural implications are discussed in Chapter 4 in addition to the prevalence of SNPs in the SI gene in different populations. The mammalian α-glucosidases are compared to the 3 Å structure of CfXyl31, a Family 31 glycoside hydrolase from Cellulomonas fimi. Comparison to Family 31 glycoside hydrolases of known structure gives rise to possible mutations proposed to mimic ntMGAM α-glucosidase activity. Through inhibitor studies, homology models, examining mutations found in disease states such as congenital sucrase isomaltase deficiency, and investigating a bacterial family 31 glycoside hydrolase from Cellulomonas fimi, the active site characteristics and substrate specificities of SI and MGAM are better understood.

abstract: This project was completed to understand the evolution of the ability to digest wood in termite symbiotic protists. Lower termites harbor bacterial and protist symbionts which are essential to the termite ability to use wood as a nutritional source, producing glycoside hydrolases to break down the polysaccharides found in lignocellulose. Yet, only a few molecular studies have been done to confirm the protist species responsible for particular enzymes. By mining publicly available and newly generated genomic and transcriptomic data, including three transcriptomes from isolated protist cells, I identify over 200 new glycoside hydrolase sequences and compute the phylogenies of eight glycoside hydrolase families (GHFs) reported to be expressed by termite hindgut protists. Of those families examined, the results are broadly consistent with Todaka et al. 2010, though none of the GHFs found were expressed in both termite-associated protist and non-termite-associated protist transcriptome data. This suggests that, rather than being inherited from their free-living protist ancestors, GHF genes were acquired by termite protists while within the termite gut, potentially via lateral gene transfer (LGT). For example one family, GHF10, implies a single acquisition of a bacterial xylanase into termite protists. The phylogenies from GHF5 and GHF11 each imply two distinct acquisitions in termite protist ancestors, each from bacteria. In eukaryote-dominated GHFs, GHF7 and GHF45, there are three apparent acquisitions by termite protists. Meanwhile, it appears prior reports of GHF62 in the termite gut may have been misidentified GHF43 sequences. GHF43 was the only GHF found to contain sequences from the protists not found in the termite gut. These findings generally all support the possibility termite-associated protists adapted to a lignocellulosic diet after colonization of the termite hindgut. Nonetheless, the poor resolution of GHF phylogeny and limited termite and protist sampling constrain interpretation. / Dissertation/Thesis / Masters Thesis Biological Design 2019

Agarose and porphyran are related galactans that are only found within red marine algae. As such, marine microorganisms have adapted to using these polysaccharides as carbon sources through the acquisition of unique Carbohydrate Active enZymes (CAZymes). A recent metagenome study of the microbiomes from a Japanese human population identified putative CAZymes in several bacterial species, including Bacteroides plebeius that have significant amino acid sequence similarity with those from marine bacteria. Analysis of one potential CAZyme from B. plebeius (BpGH50) is described here. While displaying up to 30% sequence identity with β-agarases, BpGH50 has no detectable agarase activity. Its crystal structure reveals that the topology of the active site is much different than previously characterized agarases, while containing the same core catalytic machinery. It is unclear whether the enzyme has endo- or exo- activity; the large binding ‘groove’ is typical of an endo-acting enzyme, while a loop at one end of the groove may provide a terminal pocket for the substrate, which is suggestive of exo-activity. Furthermore, the enzyme contains a basic pocket that may dock a sulphated substrate, like porphyran. While no quantifiable porphyran activity was observed, properties of the putative active site suggest that this unusual enzyme may be specific on an unusual substrate, such as a porphyran-agarose hybrid. / Graduate