<P> Type II Secretion (T2S) and type IV pilus (T4P) systems in Gram-negative bacteria share many features that suggest a common ancestral origin. This study examined the role of the minor pilins FimU, PilV, PilW, PilX and PilE, as well as the putative adhesin PilYl in both the T4P and T2S systems, and elucidated the role of these proteins in pilus assembly. Genetic analysis of the major pilin cluster and the minor pilin operon revealed that the major pilin alleles are associated with a specific set of minor pilins, and that unrelated strains of the same major pilin type have identical minor pilin genes, suggesting that the two gene clusters were horizontally acquired as a 'pilin island'. We observed that the minor pilins required a specific stoichiometric ratio for proper assembly, as overexpression either completely abolished, or significantly reduced twitching motility in mutant backgrounds. We demonstrated that the minor pilins were incorporated into the pilus fibre, and that they were dependent on PilA for surface localization. The T4P minor pilins were also shown to play a role in the secretion of effectors through the T2S system, as elastase and haemolytic phospholipase C secretion was reduced in minor pilin mutants, while overexpression of FimU or PilX significantly increased secretion of T2S exoproteins. Therefore, the minor pilins may participate in T2S substrate recognition. We found that PilYl was not essential for assembly in the absence of retraction, but that its absence caused changes in the levels of other T4P biogenesis proteins, namely FimU, PilW, PilF and PilQ secretin multimers. Finally we show that the minor pilin, PilX functions as a strain-specific factor, potentially through specific interactions with non-conserved residues of PilQ that are necessary to induce opening of the secretin. </p> / Thesis / Doctor of Philosophy (PhD)

Type IV pili (T4P) are protein filaments found on the surface of a variety of bacterial species and mediate biofilm formation, adhesion, and flagellum-independent twitching motility. The biogenesis of T4P is dependent on a cell envelope-spanning, multiprotein complex that localizes to the poles in rod-shaped cells. How these proteins localize and cross the peptidoglycan (PG) layer in the absence of dedicated PG-hydrolyzing enzymes is unknown. In P. aeruginosa, PilMNOP interact to form the alignment subcomplex, connected via PilP to PilQ, which forms the outer membrane secretin. We hypothesized that polar localization and integration of the T4P machinery was driven by ordered recruitment to future sites of cell division, placing assembly system components at division septa in the correct position before daughter-cell separation. To determine which T4P components are essential for localization of the complex, we fused the T4P inner membrane assembly protein PilO to the fluorescent protein mCherry to monitor its localization. mCherry-PilO localized to the cell poles and midcell in wild type bacteria. However, it was delocalized in a strain lacking PilQ. A PilQ-mCherry fusion localized to the cell poles, likely through its putative septal PG binding AmiN domains, suggesting that PilQ binds PG and thus localizes its partners to future sites of cell division. In the absence of the associated pilotin protein (PilF), which is required for PilQ multimerization in the OM, T4P components were polarly localized, implying that localization is not dependent on secretin formation. The results of this research support a pre-installation mechanism for integration of protein complexes in the gram negative cell envelope without PG hydrolysis, which may be applicable to other systems. / Thesis / Master of Science (MSc)

HPr est une protéine du système phosphoénolpyruvate:sucre phosphotransférase impliquée dans le transport, la régulation d'enzymes et de transporteurs et la régulation de la transcription de certains gènes. La protéine HPr peut être retrouvée sous quatre formes différentes. Elle peut être phosphorylée sur un résidu histidyl par l'enzyme 1 pour former HPr(His"-'P). Sous cette forme, HPr peut, entre autres, contrôler par phosphorylation certains régulateurs transcriptionnels contenant des domaines PRD. Chez les bactéries à Gram positif, HPr peut être phosphorylée sur un résidu séryl par la HPr kinase/phosphorylase. Le produit formé, HPr(Ser-P), est impliqué dans la répression catabolique selon un mécanisme impliquant la formation d'un complexe entre HPr(Ser-P), la protéine CcpA et une séquence spécifique d'ADN située dans la région promotrice des opérons cibles, la séquence cre. HPr(Ser-P) est également impliquée dans le phénomène d'exclusion d'inducteur. Enfin, des taux importants de HPr(Ser-P)(His-P) ont été détectés chez Streptococcus salivarius, Streptococcus mutans et Streptococcus thermophilus. Afin de caractériser HPr(Ser-P)(His-P), nous avons vérifié si certaines bactéries de la classe des Bacilli pouvaient produire et maintenir des taux importants de HPr(Ser-P(His-P) et vérifié si HPr(Ser-P)(His-P) pouvait participer au transport des sucres. Les quantifications de HPr ont été effectuées par immunoélectrophorèse croisée et la capacité de HPr(Ser-P)(His-P) à phosphoryler les enzymes IIABMan et la glycérol kinase ont été étudiées. Des taux importants de HPr(Ser-P)(His-P) ont été détectés chez toutes les souches de streptocoquès et lactocoques testées mais pas chez Bacillus subtilis, Enterococcus faecalis et Staphylococcus aureus. HPr(Ser-P)(His-P) était en mesure de transférer son groupement phosphoryle aux enzymes IIABMan mais' pas à la glycérol kinase, suggérant que HPr(Ser-P)(His-P) participe au transport des sucres PTS mais ne serait pas impliquée dans le contrôle de l'activité de la glycérol kinase. Nous avons finalement étudié le rôle de HPr(His"-'P) dans la régulation de la transcription chez Streptococcus salivarius via une étude comparative des protéomes de la souche parentale ATCC 25975 et du mutant G71 , une souche Er, incapable de produire HPr(His-P). Les analyses protéomiques différentielles ont été réalisées à partir d' extraits provenant de cellules récoltées en phase exponentielle et stationnaire de croissance. Chez les cellules récoltées en phase stationnaire de croissance, l'analyse des profils protéiques a révélé que 1 % à 2% des protéines étaient exprimées différentiellement chez S. salivarius G71 suggérant que HPr(His-P) serait impliquée dans le contrôle de l'expression de gènes chez les streptocoques.

This thesis consists of two chapters, each of which is a unique research project. Chapter 1 is focused on the synthesis and biological evaluation of novel sideromycin antibiotics. Sideromycins are bifunctional “Trojan Horse” molecules that have an iron chelator “siderophore” moiety covalently bound to an antibiotic. Such molecules exploit existing bacterial mechanisms for obtaining iron from their environment. Antibiotics that would typically not pass Gram-negative membranes are allowed access via siderophore transporter proteins. This project utilized a siderophore that has not previously been used in this capacity. The synthesis and biological evaluation of multiple sideromycin conjugates is reported. Chapter 2 describes the chemical synthesis of coumarin natural products using a synthetic process recently developed in the Magolan laboratory that enables the efficient prenylation of phenols. These natural products are molecules of biological interest in various capacities but are rare and difficult to isolate from their plant sources. They have also previously been cumbersome to make via chemical synthesis. The chemistry described herein constitutes an inexpensive and efficient process to produce these compounds that is superior to previously known methods. / Thesis / Master of Science (MSc) / This thesis is divided into two chapters. The first chapter is focused on the development of new sideromycin antibiotics. Sideromycins are “Trojan Horse”-like antibiotics that exploit the mechanisms of Gram-negative bacteria for obtaining iron, an essential nutrient, to enable antibiotic entry. This chapter details the synthesis of molecules that attach functionalities called “siderophores” to antibiotics, enabling them to be “smuggled” into Gram-negative microbes. This project uses a siderophore not previously utilized in sideromycin research. The second chapter is focused on the chemical synthesis of rare natural products that are phenols with prenyl substituents. Many such compounds are plant-derived and have potential for biomedical use. However, difficulty in isolating them makes them prohibitively expensive in the purity and quantity required for research. They are also challenging to make synthetically. This chapter details the application of a recently discovered process in the Magolan laboratory to synthesize coumarin-containing prenylated phenolic natural products.

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Clostridium perfringens is a Gram-positive bacterium that secretes a biofilm matrix material. The goal of these experiments was to identify pilin mutants that are needed for secretion of the biofilm matrix and develop a functional model for a type II secretion system (T2SS) in C. perfringens. Protein tagging, western blot, and slot blot experiments were done to quantify protein secretion. After performing experiments using a CPE0515-FLAG construct, it was concluded from immunoblot densitometry data that, except for the pilA1 deletion mutant, none of the 18 tested pilin mutants had a statistically significant difference from the wild type (WT) with regard to protein secretion. From slot blot densitometry assays, it was concluded that the pilA1 and CPE2280 mutants showed statistically significant lower values than the WT but the pilA2 and CPE1841 mutants had values that were higher than the wild type. Testing the construct containing only CPE0514 and CPE0515-FLAG showed that CPE0516 and CPE0517 are not needed for secretion of the protein CPE0515. HA-tagged CPE0516 qualitative immunoblots showed that, unlike CPE0515, oligomerization of CPE0516 is not occurring, and that this protein likely forms a heat stable dimer. Overall, the data did not allow us to construct a T2SS model, since there were not enough proteins revealed to be involved to create a complete Type II secretion system. / Master of Science / The methods by which C. perfringens can persist and survive in environmental conditions is something that would be useful to learn more about. One of the methods that many bacteria use to survive is by creating a biofilm matrix material, which provides protection for the bacteria from environmental stresses. In this study, the goal was to determine which specific proteins are needed for the secretion of the biofilm matrix material. Using molecular biology techniques, the proteins thought to be involved in biofilm formation quantified. The results showed that while two proteins ultimately appeared to be needed for secretion, there were not enough proteins involved to create a complete model for a functional secretion system in C. perfringens.

L'objectif était de déterminer si et comment les populations microbiennes des laits crus pouvaient faire barrière à Listeria monocytogenes dans un fromage à pâte pressée non cuite. La comparaison des profils SSCP de fromages avec et sans développement de L. monocytogenes a permis d'identifier les pics de Lactococcus lactis, Lactococcus garvieae, Enterococcus saccharominimus, Enterococcus faecium, Chryseobacterium sp. et Corynebacterium flavescens dominants dans les profils des fromages inhibiteurs. Lc. lactis s'est révélée le plus inhibiteur en fromages par production d'acide lactique en début d'affinage. L'inventaire des populations du lait cru le plus inhibiteur a permis de reconstituer une communauté de 32 espèces. Par omission successive de groupes microbiens constituant cette communauté, il a été montré que les bactéries lactiques agissaient en synergie avec les bactéries à Gram positifs non lactiques dans l'inhibition. Le rôle des levures serait moindre et les bactéries à Gram négatif n'interviendraient pas. En cours d'affinage, l'inhibition serait plutôt attribuée à la production d'acides organiques, d'alcools et de certains esters

本研究目的在於結合關聯規則新詞發掘演算法來擴增詞庫，並藉此提高結斷詞句的精確度以及透過非監督式情感分析方法，從中央通訊社中抓取國民黨以及民進黨的相關新聞文本，建立主題模型與情緒傾向的標注。再藉由監督式學習方法建立分類模型並驗證其成果。 　　本研究藉由n-gram with a-priori algorithm來進行斷詞斷句的詞庫擴增。共有32007組詞被發掘，於這些詞中具有真正意義的詞共有28838筆，成功率可達88%。 　　本研究比較兩種分群方法建立主題模型，分別為TFIDF-Kmeans以及LDA。在TFIDF-Kmeans分群結果中，因為文本數量遠大於議題詞數量，造成TFIDF矩陣過於稀疏，造成分群效果不佳。在LDA的分群結果底下，因為LDA模型其多文章多主題共享的特性，主題分類的精準度更高達八成以上。故本研究認為在分析具有多主題特性之文本，採用LDA模型來進行議題詞分群會有較佳的表現。 　　本研究透過結合不同的資料時間區間，呈現出中央通訊社的新聞文本在我國近五次總統大選前後三個月間的新聞情緒傾向。同時探討各主題模型中各類別於大選前後三個月之情緒傾向變化。可以觀察到大致上文本的情感指數高峰值會出現於投票日的時候，而近三次總統大選的結果顯示，相關的政黨新聞情感值會於選舉過後趨於平緩。而從新聞文本的正負向情感統計以及以及整體情緒傾向分析可以看出，不論執政黨為何，中央通訊社的新聞對於國民黨以及民進黨皆呈現了正向且平穩的內容，大抵不會特別偏向單一政黨 / The purpose of this research is to combine association rules and new word mining algorithms to expand the lexicons so as to improve the accuracy of word segmentations, and by capturing the KMT and DPP news from the Central News Agency, it establishes the theme model and sentiment orientation through the unsupervised sentiment analysis method. Finally, by means of supervised learning methods, this research establishes classifications models and verifies its results. 　　This research uses n-gram with a-priori algorithm to segment words and sentences to expand the lexicons. A total of 32007 word are found, and among them, there have 28838 words with real meaning. The success rate is up to 88%. 　　In this research, we compare two different clustering methods to form the theme model, which are the TFIDF-Kmeans, and the LDA. From the results of TFIDF-Kmeans, the TFIDF matrix is too sparse, resulting in poor clustering because the number of texts is a lot larger than that of the issues. Unlike TFIDF-Kmeans, because of LDA model with more features of multi-topic sharing, the accuracy of topic classification is more than 80%. Therefore, this research suggests that it will have a better performance to analyze the multi-subjective texts with LDA model to classify the word clustering. 　　Through the combination of different data time interval, this research presents the sentimental tendencies of Central News Agency’s news in three months before and after the last five presidential elections in Taiwan. At the same time, it also explores the changes of the sentimental tendencies in the various theme models in the three months before and after the election. It can be observed the sentimental peak of the text will be appeared on the polling day, and nearly three times of the presidential election results show that the sentimental value of the relevant party’s news will become smooth after the election. From the positive and negative sentimental statistics of the news text and the analysis of the overall sentimental tendencies, no matter which the ruling party is, the news of the Central News Agency for the KMT and the DPP presents a positive and stable content, not particularly toward any political party.