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Dissertations / Theses on the topic 'Piwi proteins'

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Published: 4 June 2021
Last updated: 9 February 2022

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1

Bagijn, Marloes Pauline. "Genetic and functional characterization of the Piwi proteins and piRNAs of Caenorhabditis elegans." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609250.

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2

Kim, Iana [Verfasser], and Claus-Dieter [Akademischer Betreuer] Kuhn. "On piRNAs and PIWI proteins in Schmidtea mediterranea and their role in mRNA surveillance of adult stem cells / Iana Kim ; Betreuer: Claus-Dieter Kuhn." Bayreuth : Universität Bayreuth, 2020. http://d-nb.info/1210999617/34.

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3

Eckhardt, Stephanie. "Le complexe MILI/mHEN1 et études fonctionnelles des protéines DrTDRD1 et DrMOV10L." Phd thesis, Université de Grenoble, 2011. http://tel.archives-ouvertes.fr/tel-00601225.

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Les protéines Argonaute sont associées à de petits ARN et participent à la régulation de l'expression des gènes. Les protéines Piwi, sous-famille des protéines Argonaute, sont principalement exprimées dans les lignées germinales. Elles recrutent les piRNA (Piwi-interacting RNA) et assurent la stabilité du génome en inhibant les transposons. Une caractéristique des piRNA est la présence de groupes 2'-O-methyl à l'extrémité 3'. Les microARN et siRNA (small interfering RNA) de plantes, comme les siRNA de Drosophyle portent aussi cette modification qui est catalysée par l'ARN méthyl-tranférase HEN1. Son homologue murin, mHEN1, méthyle in vitro de petits ARN, mais son rôle dans la voie des piRNA n'avait pas encore été envisagé. Mon objectif était de relier mHEN1 à la voie des piRNA. J'ai démontré que mHEN1 interagit directement avec la partie N-ter de MILI mais pas avec les autres protéines Piwi de souris. La partie N-ter de MILI porte des arginines méthylées. J'ai démontré que l'interaction ne dépendait pas de la présence de cette modification, ce qui suggère que mHEN1 intervient avant la modification de MILI. Par imagerie cellulaire j'ai montré la compartimentation de HEN1 et des protéines Piwi dans des granules cytoplasmiques différents. Parallèlement, afin de caractériser les éléments de la voie piRNA, j'ai développé un nouveau modèle d'étude basé sur des embryons de poisson zèbre (Danio rerio). Ainsi, j'ai évalué le rôle de deux protéines interagissant avec les protéines Piwi, TDRD1 (Tudor-domain containing) et l'hélicase MOV10l décrits chez la souris mais pas chez le poisson zèbre. J'ai montré que l'expression de DrTDRD1, spécifique à la lignée germinale, dépend de sa partie 3'UTR. La réduction de l'expression de DrMOV10l, obtenue grâce à l'utilisation de morpholinos, entraîne la dérépression des éléments rétrotransposables des embryons en développement. Cette technique de Knock Down sera utilisée pour identifier de nouveaux éléments de la biogenèse des piRNA.
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4

Merrifield, Matthew, and University of Lethbridge Faculty of Arts and Science. "Radiation-induced deregulation of PiRNA pathway proteins : a possible molecular mechanism underlying transgenerational epigenomic instability." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Biological Science, c2011, 2011. http://hdl.handle.net/10133/2617.

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PiRNAs and their Piwi family protein partners are part of a germline specific epigenetic regulatory mechanism essential for proper spermatogenesis, silencing of transposable elements, and maintaining germline genome integrity, yet their role in the response of the male germline to genotoxic stress is unknown. Ionizing radiation (IR) is known to cause transgenerational genome instability that is linked to carcinogenesis. Although the molecular etiology of IR-induced transgenerational genomic instability is not fully understood, it is believed to be an epigenetically mediated phenomenon. IR-induced alterations in the expression pattern of key regulatory proteins involved in the piRNA pathway essential for paternal germline genome stability may be directly involved in producing epigenetic alterations that can impact future generations. Here we show whole body and localized X-irradiation leads to significant altered expression of proteins that are necessary for, and intimately involved in, the proper functioning of the germline specific piRNA pathway in mice and rats. In addition we found that IR-induced alterations to piRNA pathway protein levels were time and dose dependent.
ix, 123 leaves : ill. (some col.) ; 29 cm
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5

Vargas, Aguilar Stephanie. "A novel mammalian PIWI protein regulates self-renewal and lifespan of macrophages." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20073.

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PIWI Proteine sind die zentralen Darsteller eines RNA-basierten Mechanismus, der die Mobilisierung transponierbarer Elemente im Genom unterdrückt, um genetische Stabilität zu gewährleisten. Demzufolge sind PIWI-Proteine für die langfristige Erhaltung verschiedener Stamzellpopulationen notwendig. Beispiele dafür sind verschiedene adulte somatische Stammzellen in Drosophila und die Stammzellen der Keimbahn aller bisher untersuchten Tierarten. Bei Säugetieren sind die beschriebenen Funktionen von PIWI Proteinen strikt auf die männliche Keimbahn beschränkt. Trotz Andeutungen auf eine Rolle von PIWI-Proteinen in somatischen Zellen von Säugetieren, wurde eine Funktion bisher nicht beschrieben. Ähnlich wie Stammzellen, können sich Makrophagen in verschiedenen Geweben selbst-erneuern, um ihre Populationen zu erhalten. Diese Selbsterneuerung beruht auf der geringen Expression der Transkriptionsfaktoren MafB und cMaf, was die Aktivierung eines stammzell-ähnliches Gen-Netzwerk, das die Proliferation vorantreibt. Makrophagen mit einer genetischen Deletion von MafB und cMaf (MafDKO-Makrophagen) oder Makrophagen mit natürlich niedriger Expression von MafB oder cMaf, wie z.B. alveoläre Makrophagen, weisen dementsprechend eine erweiterte Kapazität zur Selbsterneuerung auf. Wie haben festgestellt, dass eine kurze Isoform des Maus- Gens Piwil2, die wir ‚Piwito’ genannt haben, in MafDKO und alveolären Makrophagen exprimiert wird. Die Expression von Piwito ist für die normale Selbsterneuerung der untersuchten Makrophagen notwendig, wie die in vitro und in vivo Untersuchungen darlegen. Eine Abwesenheit von Piwito in alveolären Makrophagen führt zu einer Verkürzung derer Lebenspanne in Kultur. Außerdem beweisen wir, dass Piwito von MafB in nicht-proliferierenden Makrophagen gebunden und unterdrückt wird. Diese Studie ist somit der erste Bericht über eine somatische Funktion von PIWI-Proteinen in nicht transformierten Zellen von Säugetieren.
PIWI proteins are the main players of an RNA-based gene regulatory machinery that represses transposable elements in the genome to prevent their mobilization and ensure genetic stability. PIWI proteins have thus highly conserved stem-cell functions. They are indispensable for the long-term maintenance of the somatic stem cells that drive regeneration in invertebrates, of various adult somatic stem cells in Drosophila and, most prominently, of the germline of all species studied so far. In mammals, their described functions are strictly restricted to the male germline. Despite suggestive observations for a role of PIWI proteins in the mammalian soma, robust evidence remains absent. Similar to stem cells, tissue macrophages can locally self-renew to maintain their populations. Mechanistically, their self-renewal relies on low expression of the macrophage transcription factors MafB and cMaf, since it allows the induction of a stem cell-like network of genes that drives proliferation. Macrophages with a genetic deletion of MafB and cMaf (MafDKO macrophages) acquire therefore the capacity to self-renew, defined by an indefinite growth in culture that does not comprise their identity and does not involve cancerogenic transformation. Similarly, macrophages with naturally low levels of MafB or cMaf, such as alveolar macrophages, display an extended self-renewal capacity in vivo and in vitro. We have found that a short isoform of the murine Piwil2 gene, that we named ‘Piwito’, is expressed in MafDKO and alveolar macrophages. Piwito expression is necessary for the unaltered self-renewal of macrophages, as shown by in vitro and in vivo assays. To highlight is the fact that Piwito deficiency limits the extended lifespan of alveolar macrophages in culture. Additionally, we show that Piwito is bound and repressed by MafB in quiescent macrophages. This study thus represents the first report of a somatic function for mammalian PIWI proteins in non-transformed cells.
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6

Balaratnam, Sumirtha. "BIOGENESIS AND FUNCTIONAL APPLICATIONS OF PIWI INTERACTING RNAs (piRNAs)." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1531753741509242.

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7

Berry, Jamie. "Structural characterization of type IV pilus biogenesis proteins." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/structural-characterization-of-type-iv-pilus-biogenesis-proteins(1e0d7119-58d5-4e5d-839d-daef8deb76ab).html.

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Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.
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8

Mösinger, Carina [Verfasser], Hermann M. [Akademischer Betreuer] Behre, Jürgen [Akademischer Betreuer] Dittmer, and Holger [Akademischer Betreuer] Herlyn. "Die Rolle der Proteine Piwi-like 1 und Piwi-like 3 für die Spermatogenese des Menschen und die männliche Fertilität / Carina Mösinger ; Hermann M. Behre, Jürgen Dittmer, Holger Herlyn." Halle, 2016. http://d-nb.info/1117028240/34.

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9

Selim, Khaled [Verfasser]. "Structural and Functional Characterization of PII and PII-like Proteins and their Network of Interactions / Khaled Selim." Tübingen : Universitätsbibliothek Tübingen, 2021. http://d-nb.info/1233678507/34.

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10

Shirdel, Mariam. "Probing protein - Pili interactions by optical tweezers and 3D molecular modelling." Thesis, Umeå universitet, Institutionen för fysik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-68747.

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11

Karlsson, Katarina Flemmer. "Synthesis, conformational analysis, and biological evaluation of peptides from E. coli P pilus proteins." Lund : Organic Chemistry 2, Lund Institute of Technology, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39777038.html.

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12

Leutz, Achim [Gutachter], Michael [Gutachter] Sieweke, and Marco [Gutachter] Prinz. "A novel mammalian PIWI protein regulates self-renewal and lifespan of macrophages / Gutachter: Achim Leutz, Michael Sieweke, Marco Prinz." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1189213729/34.

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13

Nikraftar, Sarah. "Localization of Type IV Pilin Polymerization Proteins in Clostridium perfringens." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/71742.

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Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS. We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT. In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells.
Master of Science
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14

Brown, Daniel Robert. "Systematic analysis in Neisseria meningitidis of proteins that fine-tune functions mediated by type IV pili." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6834.

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Type IV pili (Tfp) are widespread virulence factors whose multifunctional ability sets them apart from other pili. Tfp mediate adhesion as well as aggregation, competence for DNA transformation and twitching motility (a non-­flagellar form of locomotion) in many bacterial pathogens. The molecular mechanisms of these functions and the biogenesis of these pili are not yet fully understood. To better understand Tfp biology, our group started a systematic characterization of all of the proteins involved in Tfp biology in a well-­defined genetic background, Neisseria meningitidis strain 8013. Screening of a large library of mutants in this strain followed by an in-­depth mining of the genome resulted in the discovery of 23 genes that may be involved in Tfp biology. Seven of these genes encode proteins deemed “accessory” for Tfp biogenesis as the corresponding mutants are piliated. Since previous analysis of one these genes (pilX) led to the finding that it modulates one Tfp-­linked function (aggregation), the first aim of my project was to phenotypically characterize mutants in the remaining genes along with double mutants in which filament retraction is abolished by a concurrent mutation in the pilT gene, and strains overexpressing the corresponding proteins with regards to levels of piliation, adhesion, aggregation and DNA competence. This was achieved using a battery of quantitative and qualitative methods well-­established in our research group. Results from this study showed that each of the seven proteins plays a role in fine-­tuning of Tfp-­linked function(s). This completed our systematic study of mutants in N. meningitidis Tfp biology, and gave us a complete picture of the roles of these proteins in Tfp function. This provides us with a platform for further in-­depth study of these proteins. In parallel, we attempted to further characterize one of these proteins, PilZ, by determining its localization and protein structure, however, we have been unsuccessful in both efforts so far. Finally we attempted to improve upon the current pilus purification method using a His-­tagged version of the major pilus subunit PilE to purify fibres by affinity chromatography. This could be a very useful tool for future studies.
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Fokina, Oleksandra Verfasser], and Karl [Akademischer Betreuer] [Forchhammer. "From Metabolite Sensing to Protein Regulation by Synechococcus elongatus PCC 7942 PII Protein / Oleksandra Fokina ; Betreuer: Karl Forchhammer." Tübingen : Universitätsbibliothek Tübingen, 2011. http://d-nb.info/1162699329/34.

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Deng, L. W. "Infection mechanism of filamentous bacteriophage fd : interaction between E. coli F-pilus and phage protein pIII." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598493.

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Gene 3 protein (pIII), a minor coat protein located at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. An introduction and the experimental materials and methods are described in Chapters 1 and 2, respectively. Chapter 3 describes the construction and expression of the N-terminal di-domain and individual domains of pIII protein. The plaque-forming assay in vivo was used to investigate the ability of the isolated pIII domains to interact with F-pilus. Chapter 4 presents the investigation of the structure of F pilus, including displaying fragments of F pilus subunit (pilin) by means of phage display technology, expression of pilin subunit and X-ray fibre-diffraction of F pilus. Site-directed alanine mutagenesis of the second N-terminal domain of pIII in the phage as well as the evaluation of their infectivity are described in Chapter 5. Chapter 6 describes the establishment of a competitive ELISA assay in vitro in order to analyse all the mutated phages. Combining the results from two assays and mapping out the affected residues on the 3D structure, a region located at the outer rim of pIII-D2 domain was implicated as the F pilus binding epitope. Second generation mutagenesis and evaluation of these mutants were performed in order to define the interface region more closely (Chapter 7). Furthermore, several pIII proteins each containing an alanine mutation were isolated to investigate whether any major disruption of protein integrity occurs. Finally, Chapter 8 places this work in perspective and offers ideas for further work.
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Pedro, Roig Laia. "GlnK regulatory proteins and their role in Haloferax mediterranei nitrogen metabolism." Doctoral thesis, Universidad de Alicante, 2012. http://hdl.handle.net/10045/27319.

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Chellamuthu, Vasuki Ranjani Verfasser], and Karl [Akademischer Betreuer] [Forchhammer. "Novel structures of PII signal transduction proteins from oxygenic phototropic organisms / Vasuki Ranjani Chellamuthu ; Betreuer: Karl Forchhammer." Tübingen : Universitätsbibliothek Tübingen, 2014. http://d-nb.info/1162970812/34.

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Chellamuthu, Vasuki Ranjani [Verfasser], and Karl [Akademischer Betreuer] Forchhammer. "Novel structures of PII signal transduction proteins from oxygenic phototropic organisms / Vasuki Ranjani Chellamuthu ; Betreuer: Karl Forchhammer." Tübingen : Universitätsbibliothek Tübingen, 2014. http://d-nb.info/1162970812/34.

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Larsson, Andreas. "Antiadhesive agents targeting uropathogenic Escherichia coli : Multivariate studies of protein-protein and protein-carbohydrate interactions." Doctoral thesis, Umeå : Dept. of Chemistry, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-314.

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Tan, Li. "VIRULENCE MECHANISM OF THE NEMATODE PHASMARHABDITIS HERMAPHRODITA AND ITS ASSOCIATED BACTERIUM MORAXELLA OSLOENSIS TO THE GRAY GARDEN SLUG DEROCERAS RETICULATUM." The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1038718955.

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Krieger, Natalie [Verfasser], and Klaus [Akademischer Betreuer] Harter. "Expression, localization, and interaction studies of the plastidic PII protein from Arabidopsis thaliana / Natalie Krieger ; Betreuer: Klaus Harter." Tübingen : Universitätsbibliothek Tübingen, 2020. http://d-nb.info/1212025156/34.

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GONZALEZ, MAYA LETICIA. "Etude de la transduction du signal impliquant la phospho-proteine pii et des ser/thr-kinases chez les cyanobacteries." Université Louis Pasteur (Strasbourg) (1971-2008), 2000. http://www.theses.fr/2000STR13092.

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Des genes codant pour une famille de ser/thr-kinases chez la cyanobacterie filamenteuse et fixatrice d'azote anabaena sp. Pcc 7120, ont ete isoles dans notre laboratoire. Ces genes sont impliques dans la croissance ou developpement cellulaire. Les substrats de cette famille de ser/thr-kinases sont encore inconnus. Un des substrats potentiels de ces kinases est la proteine pii, connue pour etre phosphorylee sur un residu ser. Cette proteine est impliquee dans la regulation de l'assimilation de l'azote, et dans la coordination des metabolismes azote et carbone. Le gene glnb codant pour pii a ete clone chez anabaena sp. Pcc 7120. La cinetique de modification de pii pendant le developpement des heterocystes est de facon dynamique, ce qui incite a poursuivre l'etude du role eventuel de pii dans la differenciation cellulaire induite par la carence en azote combine. L'une des ser/thr-kinases de cette bacterie, pknc, peut phosphoryler la proteine pii recombinante sur un residu ser in vitro. L'introduction d'une mutation dans le domaine catalytique elimine l'activite de proteine-kinase de pknc. La phosphorylation de pii par pknc est stimulee par l'atp, mais l'-cetoglutarate, glutamate ou glutamine n'ont pas d'effet sur le niveau de phosphorylation de pii in vitro. Un essai de mutagenese suggere que pii est probablement essentielle chez anabaena sp. Pcc 7120. Afin de dissequer la fonction biologique de pii, nous avons construit des mutants ponctuels sur le residu ser qui constitue la cible de phosphorylation. Les resultats d'analyses seront presentes. L'inactivation du gene pknc n'influence pas le niveau de phosphorylation de pii chez anabaena sp. Pcc 7120. De nombreuses observations experimentales suggerent que pii joue des roles differents chez les souches unicellulaires non fixatrices d'azote et chez les souches filamenteuses fixatrices d'azote. Plusieurs hypotheses seront discutees afin de comprendre et d'expliquer le role de pii et pknc chez anabaena sp. Pcc 7120.
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Hisbergues, Michael. "Implication des protéines Hat et PII dans la régulationdes métabolismes carbone et/ ou azote chez la Cynobacterie Synechocystis PCC6803." Aix-Marseille 2, 1999. http://www.theses.fr/1999AIX22010.

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La regulation des metabolismes carbone et azote est primordiale a l'adaptation des cyanobacteries a leur environnement. Deux proteines, hat et pii, engagees respectivement dans l'un ou les deux processus regulateurs ont ete etudiees chez synechocystis pcc6803. La proteine hat, originale chez les procaryotes, contient dans sa partie c-terminale 11 repetitions-wd rencontrees essentiellement dans une famille de proteines d'eucaryotes de fonctions diverses. L'organisation de cette region en 7 et 4 repetitions separees par 159 acides amines est aussi originale. Elle a conduit a etablir trois modeles structuraux theoriques, dont la validite a ete analysee. Des anticorps polyclonaux diriges contre les parties n- et c-terminales ont permis de localiser la proteine au niveau des membranes thylacoidales. Cette localisation, ainsi que l'analyse phenotypique de trois mutants formant des proteines diversement tronquees, ont amene a supposer que l'implication de hat dans la regulation du systeme de transport a forte affinite du bicarbonate est indirecte. La proteine pii (glnb), dont le role dans la regulation du metabolisme azote est etabli chez plusieurs bacteries, intervient aussi dans la co-regulation des metabolismes carbone et azote chez la cyanobacterie synechococcus pcc7942. L'etude biochimique de pii chez synechocystis pcc6803 a permis de determiner ses caracteristiques de multimerisation (trimere) et de modification (phosphorylation). L'etude d'un mutant nul de pii a conduit a etablir un mode d'action distinct dans la regulation des transports de nitrate et de bicarbonate. L'implication de pii dans la detection du niveau redox des cellules ainsi que dans la regulation de l'activite de transfert des electrons au niveau du photosysteme i, a ete etablie. Ces travaux proposent donc une intervention de la proteine pii a plusieurs niveaux cles dans la co-regulation des metabolismes carbone et azote, au moins chez synechocystis.
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Black, Wesley P. "Regulation of Exopolysaccharide Production in Myxococcus Xanthus." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/30250.

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The surface gliding motility of Myxococcus xanthus is required for a multicellular developmental process initiated by unfavorable growth conditions. One form of the M. xanthus surface motility, social (S) gliding, is mediated by the extension and retraction of polarly localized type IV pili (Tfp). Besides Tfp, exopolysaccharides (EPS), another cell surface associated component, are also required for M. xanthus S motility. Previous studies demonstrated that the Dif chemotaxis-like signal transduction pathway is central to the regulation of EPS production in M. xanthus. Specifically, difA, difC and difE mutants were found to be defective in EPS production and S motility. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA, respectively, are therefore positive regulators of EPS. This study, undertaken to better understand the regulation of EPS production, led to a few major findings. First, DifD and DifG, homologous to CheY and CheC, respectively, were found to be negative regulators of EPS production. Both DifD and DifG likely function upstream of the DifE kinase in EPS regulation. DifB, which has no homology to known chemotaxis proteins, was found not to be involved in EPS production. Secondly, this study led to the recognition that Tfp likely function upstream of the Dif pathway in the regulation of EPS production. Extracellular complementation experiments suggest that Tfp may act as sensors instead of signals for the Dif chemotaxis-like pathway. We propose a regulatory feedback loop that couples EPS production with Tfp function through the Dif signaling proteins. Lastly, we sought to identify additional genes involved in EPS production. Our efforts identified a mutation in a separate chemotaxis gene cluster as a suppressor of difA mutations, suggesting potential cross-talks among the multiple chemotaxis-like pathways in M. xanthus. In addition, we identified twenty-five previously uncharacterized genes that are predicted to be involved in M. xanthus EPS production. These genes appear to encode additional EPS regulators and proteins with biosynthetic function.
Ph. D.
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LEE, HYUN-MI. "Role de la proteine pii dans la coordination de l'assimilation de l'azote et du carbone chez la cyanobacterie synechococcus sp. Pcc 7942." Paris 7, 1998. http://www.theses.fr/1998PA077087.

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La cyanobacterie unicellulaire synechococcus sp. Pcc 7942 possede une proteine homologue a la proteine pii d'e. Coli (produit du gene glnb), transmetteur du signal bien connu pour son role dans l'assimilation de l'azote chez les enterobacteries. Chez synechococcus, cette proteine est un homotrimere dont chaque monomere peut etre phosphoryle en presence de nitrate ou en carence d'azote, ainsi qu'en presence d'une forte concentration en co#2 dans l'environnement. Dans le present travail, nous avons etudie le role de la proteine pii dans l'assimilation de l'azote chez synechococcus pcc 7942 selon deux axes principaux : 1) etude du role et de l'importance de son etat de phosphorylation en utilisant un mutant glnb et trois autres mutants, glnb#s#e#r#4#9#a#l#a, glnb#s#e#r#4#9#a#s#p et glnb#s#e#r#4#9#g#l#u obtenus par mutagenese dirigee in vitro ; 2) etude de la regulation de l'expression de cette proteine. La comparaison du phenotype de la souche de type sauvage et d'un mutant glnb a montre que pii est impliquee dans un controle negatif de l'entree du nitrate et du nitrite par l'ammoniaque. Les resultats de l'analyse des mutants glnb#s#e#r#4#9#a#l#a, glnb#s#e#r#4#9#a#s#p et glnb#s#e#r#4#9#g#l#u ont suggere que la proteine pii regule l'entree du nitrate et du nitrite en interagissant avec un second effecteur (probablement l'-cetoglutarate) et qu'elle module la reduction du nitrate et du nitrite par son etat de phosphorylation. Cette proteine aurait ainsi un double role dans la regulation de l'assimilation de l'azote pour maintenir en equilibre les differents metabolites produits au cours de ce processus. Les etudes de l'expression et de la modification de la proteine pii ont egalement montre que cette proteine est regulee aux niveaux transcriptionnel et post-traductionnel, de facon coordonnee et en reponse aux changements de la disponibilite en azote et en carbone dans la cellule. Cette regulation fait intervenir la proteine ntca. L'ensemble de nos resultats a permis d'eclaircir certains mecanismes de la perception du signal par la proteine pii, et ainsi que le systeme de regulation qui permet a cette proteine de coordonner l'assimilation de l'azote et celle du carbone chez synechococcus sp. Pcc 7942.
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Watzer, Björn Verfasser], and Karl [Akademischer Betreuer] [Forchhammer. "Regulation of the carbon/nitrogen storage polymer cyanophycin by the signal transduction protein PII in Synechocystis sp. PCC 6803 / Björn Watzer ; Betreuer: Karl Forchhammer." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1187724629/34.

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Watzer, Björn [Verfasser], and Karl [Akademischer Betreuer] Forchhammer. "Regulation of the carbon/nitrogen storage polymer cyanophycin by the signal transduction protein PII in Synechocystis sp. PCC 6803 / Björn Watzer ; Betreuer: Karl Forchhammer." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1187724629/34.

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29

Worst, Emanuel Gregor [Verfasser], and Albrecht [Akademischer Betreuer] Ott. "Evolution wachsender, sich reproduzierender Polymere : Zellfreie Genexpression zum Einbau nicht-kanonischer Aminosäuren in Proteine sowie zur Analyse des epigenetischen Escherichia-coli-Pili-Phasenvariationsmechanismus. / Emanuel Gregor Worst ; Betreuer: Albrecht Ott." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2018. http://d-nb.info/1159569088/34.

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30

Konto-Ghiorghi, Yoan. "Biosynthèse, régulation et rôle(s) du pilus chez Streptococcus agalactiae." Paris 6, 2009. http://www.theses.fr/2009PA066274.

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31

Santos, Moreno Javier. "Molecular mechanism of pseudopilus assembly in the Klebsiella oxytoca type II secretion system." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC216/document.

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Le système de sécrétion de type II (SST2) permet la sécrétion de protéines repliées à travers la membrane externe chez les bactéries à Gram-négatif. Le SST2 est une nano-machine enchâssée dans l’enveloppe bactérienne, proche par sa composition et structure aux systèmes d’assemblage des pili de type IV (PT4) impliqués, entre autres, dans d’adhésion et motilité. Chez Klebsiella oxytoca, la surexpression des gènes pul codant le SST2 permet l’assemblage de pili composées des sous-unités PulG. Ceci suggère qu’en conditions physiologiques l’assemblage d’un pseudopilus périplasmique permet la sécrétion du substrat spécifique du SST2, la pullulanase. Dans ce projet nous avons exploré le mécanisme moléculaire de l’assemblage du pseudopilus en se focalisant sur les interactions de PulG avec les composants du SST2 dans la membrane interne. En utilisant l’approche de double-hybride bactérien, nous avons établi le réseau d’interactions de PulG avec les pseudopilins mineures PulH, I, J et K et avec la plateforme d’assemblage (PA). Pour valider ces interactions, nous avons combiné des techniques de biochimie (co-purification par affinité, pontage cystéine et chimique) avec des analyses fonctionnelles de sécrétion et de formation du pseudopilus. Nous avons mis en évidence des interactions entre PulG et les protéines de la PA, PulF et PulM, et nous avons analysé en détail l’interface PulG-PulM. Les résultats suggèrent la formation d’un complexe PulK-I-J-H-G dans la membrane interne impliqué dans des étapes précoces de la formation du pseudopilus, à travers les interactions de PulG et PulH avec PulM et PulF. Nos données expérimentales suggèrent un rôle majeur de PulM dans la sécrétion, vraisemblablement durant l’assemblage du SST2 et l’élongation du pseudopilus. Nos travaux collaboratifs mettant en jeu l'analyse par spectroscopie de masse et en dynamique moléculaire in silico révèlent le rôle essentiel des résidus conservés Glu5 et Thr2 de PulG, requis pour l’interaction avec PulM. Ces données suggèrent que Glu5 participe à l'extraction de PulG de la membrane, en neutralisant la charge positive de son peptide N-terminal par des interactions intramoleculaires. Ces résultats permettent d'établir un modèle détaillant les étapes initiales de l’assemblage des pseudopili dans la membrane interne, relevant pour de futures études sur le SST2 et nanomachines homologues. sécrétion de protéinespili de type 4 assemblage de fibres complexes protéiques membranairesinteractions protéine-protéinemicroscopie à immuno-fluorescence simulations en dynamique moléculairedouble-hybride bactérien spectrométrie de masse nanomachines bacteriennes
The type II secretion system (T2SS) drives the translocation of folded, periplasmic proteins across the outer membrane in Gram-negative bacteria. Secretion is carried out by an envelope-spanning nanomachine that is similar to the apparatus that builds type IV pili (T4P), bacterial surface filaments involved in adhesion, motility and other functions. In the Pul T2SS of Klebsiella oxytoca, overexpression of pul genes in plate-grown bacteria allows the assembly of T4P-like surface fibres made of PulG subunits, suggesting that a periplasmic pseudopilus fibre plays a role in the secretion of the type II substrate pullulanase under physiological conditions. In this project, we explored the molecular mechanism of pseudopilus assembly by focusing on the interaction between PulG and the T2SS inner membrane and pseudopili components. The network of interactions of PulG with the minor pseudopilins PulH, I, J and K and the assembly platform (AP) components was established using bacterial two-hybrid analysis. To validate these interactions, we combined biochemical approaches (affinity co-purification, chemical or cysteine cross-linking) with functional assays of secretion and pseudopilus formation. We provide evidence of the interaction between PulG and the AP proteins PulF and PulM, and delve into the PulG-PulM interface. Our results point to the formation of a PulK-I-J-H-G complex in the plasma membrane involved in early steps of fibre assembly, with a determinant role for PulG and PulH interaction with PulM and PulF. We obtained experimental evidence supporting a major role for PulM in pseudopilus assembly and protein secretion, probably by intervening in the assembly of the T2SS apparatus and in pseudopilus elongation. The results of experimental and in silico studies in collaboration with experts in mass spectrometry and molecular dynamics support the essential role of the highly conserved PulG residues Glu5 and Thr2, which participate in PulM binding. In addition, Glu5 probably favours PulG membrane extraction by neutralising its N-terminal positive charge through intra-molecular interaction. These findings shed new light on early membrane events during fibre assembly, and open new and exciting avenues in research on T2SSs and related nanomachines.protein secretiontype 4 pilifibre assemblymembrane protein complexprotein-protein interactionsimmunofluorescence microscopymolecular dynamics simulationsbacterial two-hybrid assaymass spectrometrybacterial nanomachines
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Le, Doan-Thanh-Lam. "Identification et caractérisation des déternimants physico-chimiques et biologiques mis en jeu dans l'adhésion de Lactococcus lactis à la mucine modèle PGM." Thesis, Toulouse, INSA, 2011. http://www.theses.fr/2011ISAT0018/document.

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Dans le tractus gastro-intestinal, l'adhésion des bactéries commensales à l’épithélium permet leur maintien, ce qui aide à contrôler l’implantation de germes indésirables par des mécanismes de concurrence (effets nutritionnels, sites spécifiques d'adhésion ...). Bien que le rôle de la couche du mucus (principalement composée de glycoprotéines à haut poids moléculaire, appelées mucines) recouvrant la muqueuse soit connu et décrit depuis de nombreuses années, notamment pour sa fonction de barrière protectrice, l'intérêt pour décrypter les mécanismes précis d’interaction(s) avec le microbiote (bactéries commensales, pathogènes ou probiotiques) n’a que récemment émergé. Dans ce cadre, l'objectif de cette thèse, menée en collaboration entre le Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés de Toulouse et le Laboratoire d’Analyse et d’Architecture des Systèmes de Toulouse, est de caractériser in vitro le comportement muco-adhésif de Lactococcus lactis, le modèle des bactéries lactiques, en utilisant des approches de quantification multi-échelles (du niveau moléculaire à l’échelle multicellulaire) sur un large panel de souches naturelles et recombinantes. Une attention particulière est accordée au rôle des mucines, en utilisant le modèle PGM (mucine gastrique de porc ou « Pig Gastric Mucin »).La première partie du travail a porté sur la quantification à l'échelle de la cellule unique des interactions entre L. lactis et une surface abiotique (polystyrène) recouverte de PGM, en utilisant la microscopie à force atomique (AFM). La faisabilité de la méthode a tout d'abord été démontrée sur la souche modèle L. lactis ssp. cremoris MG1820. La couche de PGM a été caractérisée en utilisant des méthodes analytiques complémentaires (AFM, XPS - spectroscopie de photoélectrons induits par rayons X, QCM-D - Microbalance à Quartz à mesure de Dissipation...). En parallèle, les bactéries L. lactis ont été immobilisées sur la pointe AFM et utilisées comme « sonde de force », en considérant la souche naturelle IBB477 (L. lactis ssp. cremoris), d’origine laitière et présentant une forte persistance dans le tractus digestif du rat lors d’essais réalisés in vivo (collaboration avec l’Institut de Biochimie et de Biophysique de Varsovie, Pologne). Comparé aux conditions contrôle (i.e., surface de polystyrène sans PGM), les niveaux de force d'adhésion enregistrés entre L. lactis et PGM sont inférieurs, ceci en raison des répulsions électrostatiques, hydrophiles et stériques s’établissant entre la couche de PGM et la paroi cellulaire. La forme des courbes représentant l’évolution de la force au retrait en fonction de la distance est également différente. Une analyse détaillée souligne la contribution, conjointe et différente selon les souches testées, d’événements (i) non adhésifs, (ii) adhésifs non spécifiques (interactions électrostatiques, hydrophobes, de van der Waals) et (iii) adhésifs spécifiques (liaison de type ligand/récepteur). La contribution spécifique a ensuite été explorée plus finement en termes de constantes cinétiques d’association et de dissociation. Nous avons, par ailleurs, poursuivi notre exploitation de la biodiversité naturelle chez les lactocoques en étudiant la souche TIL448 (L. lactis ssp. lactis) d’origine végétale, en collaboration avec l’Institut MICALIS de Jouy-en-Josas. Nous avons ainsi démontré, pour la première fois chez L. lactis, le rôle combiné des protéines à domaine(s) MUB (« Mucus-Binding ») et des pili, à travers l'analyse approfondie des données AFM (force d'adhésion, répartition des événements adhésifs spécifiques/non spécifiques, distances d'interaction...). Le rôle des pili a été confirmé sur des souches recombinantes piliées (L. lactis ssp. lactis IL1403), toujours en partenariat avec l’Institut MICALIS de Jouy-en-Josas. En parallèle, en collaboration avec l’Unité de Glycobiologie Structurale et Fonctionnelle de Villeneuve d'Ascq, nous avons cherché à identifier les O-glycannes de PGM (fractions neutre et acide), impliqués dans le processus d'interaction avec la surface bactérienne. Pour confirmer l’ensemble des résultats obtenus à l'échelle de la cellule unique et en mode statique par AFM (effet anti-adhésif de PGM, comportement muco-adhésif différent selon les souches de L. lactis), la deuxième partie du travail a été consacrée à des expérimentations à l’échelle de l’ensemble de la population bactérienne, en conditions dynamiques (QCM-D, chambre à écoulement cisaillé). Nous avons évalué par QCM-D chez les souches IBB477 et MG1820 les propriétés viscoélastiques des dépôts bactériens, en relation avec le comportement bio-adhésif vis-à-vis de la couche de PGM. Les données obtenues par AFM et chambre à écoulement cisaillé sur ces mêmes souches ont été confrontées pour accéder plus finement au mode d’interaction avec PGM (densité de liaisons sur la surface bactérienne). Enfin, nous avons évalué chez IL1403 l’effet des pili sur la dynamique de détachement et d’orientation sous cisaillement contrôlé.En conclusion, la combinaison des échelles d'observation et d’analyse, aussi bien au niveau de la cellule unique qu’à celui de l’ensemble de la population bactérienne, nous permet désormais de disposer de nouvelles connaissances sur les mécanismes diversifiés d'interaction entre L. lactis et PGM, visant à une meilleure compréhension des fonctionnalités de cette bactérie au niveau du tractus gastro-intestinal
In the gastrointestinal tract, adhesion of commensal bacteria to epithelial cells allows their retention, which helps to control the implementation of unwanted germs through mechanisms of competition (nutritional effects, specific sites of adhesion ...). Indeed, bacterial adhesion to the intestinal epithelium seems to be important for the balance of intestinal microbiota. Although the role of the mucus layer lining the mucosa, which is mainly composed of large glycoproteins termed mucins, is known and described for many years, particularly for its protective barrier function, the interest for unraveling precise mechanisms of interaction with bacteria (commensal, pathogens or probiotics) has just recently emerged. In this framework, the aim of the PhD thesis was to characterize in vitro muco-adhesive behavior of Lactococcus lactis, the model of Lactic Acid Bacteria, using multi-scale approaches (from molecular to multicellular levels) on a large set of natural and recombinant strains. A particular attention was paid to the role of mucins, using the PGM model (Pig Gastric Mucin).The first part of the work was focused on the quantification at nanoscale of interactions between L. lactis and adsorbed PGM, using AFM. The feasibility of the method was first demonstrated on the reference strain MG1820. PGM coating was characterized using a complementary set of analytical methods (AFM, XPS, quartz crystal microbalance with dissipation monitoring…). In parallel, L. lactis cells were immobilized onto the AFM tip and used as a living force probe, considering the natural strain IBB477 (L. lactis subsp. cremoris), isolated from Polish artisanal dairy products and previously shown to display in vivo retention in the rat gut (collaboration with the Institute of Biochemistry and Biophysics of Warsaw, Poland). Compared to control conditions (i.e., no PGM coating), adhesion force levels recorded for PGM were lower, due to the interplay of electrostatic, hydrophilic and steric repulsions. The shape of retraction force-distance curves for L. lactis/PGM interactions was also different. The detailed analysis of curve shape highlighted the contribution of non-adhesive, non-specific (electrostatic, hydrophobic, van der Waals interactions) and specific adhesive events (ligand/receptor bonding), depending on the strain under study. Specific forces were analyzed in terms of dissociation/association kinetic constants.We then explored the natural biodiversity among lactococci by studying the natural strain of L. lactis (subsp. lactis) TIL448 from plant origin, in collaboration with MICALIS (Jouy-en-Josas). We demonstrated, for the first time for L. lactis, the combined role played by “MUB-like” domain-containing protein and pili, through the thorough analysis of AFM data (adhesion force, repartition of specific/non-specific adhesive events and distances of interaction…). The role of pili was also confirmed with recombinant piliated strains (L. lactis subsp. lactis IL1403), in partnership with MICALIS (Jouy-en-Josas). In parallel, in collaboration with the “Unité de Glycobiologie Structurale et Fonctionnelle de Villeneuve d'Ascq”, a first attempt was done to identify the O-glycans of PGM (neutral and acid fractions), involved in interactions with the bacterial surface.To confirm these results achieved at single-cell scale and under static mode by AFM (anti-adhesive of PGM, different muco-adhesive properties among several strains of L. lactis), the second part of the work was devoted to experiments at multicellular scale under dynamic conditions (quartz crystal microbalance with dissipation monitoring – QCM-D, shear stress flow chamber). We evaluated by QCM-D, for MG1820 and IBB477 strains, the viscoelastic properties of the cell layers, in relation with the bio-adhesive behavior towards PGM. The data obtained by AFM and shear stress flow chamber were combined to access more precisely to the interaction mode with PGM (density of bonds over the cell surface). Finally, using the recombinant piliated strain (IL1403), we focused on the effect of pili on detachment and re-orientation dynamics under shear flow
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33

Silva, Ricardo Andrés Zacarias 1988. "PIWI proteins in mammals: a cow's perspective." Master's thesis, 2011. http://hdl.handle.net/10451/4533.

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Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2011
The integrity of DNA is under constant threat from both exogenous and endogenous agents. As a response, the cell has evolved numerous mechanisms to protect its genetic information from such dangers, especially in the germline, where unwanted changes in the genome can be transferred to the offspring. One major peril to genomic stability are transposable elements, mobile genetic fragments that can insert themselves at new locations in the genome. The mechanisms involved in controlling these elements are poorly understood. However, recent breakthroughs have provided insight into a germ cell-specific mechanism that silences transposons through RNA interference. Piwi, a subclass of the Argonaute protein family, associates with a novel class of small RNAs termed piRNAs. Interestingly, these piRNAs exhibit sequences complementary to those of transposable elements and, together with Piwi proteins, are able to thwart the mobility of such elements. Although the importance of Piwi proteins in germ cells has been established in many different organisms, in mammals Piwi function seems to be male-specific. Interestingly, phylogenetic examination of the Argonaute protein family revealed an additional Piwi gene (PIWI-LIKE 3 or PIWIL3) that is present in numerous mammals, including humans and bovine, but absent from rodents. Until a thorough analysis of PIWIL3 and other Piwi-like genes is performed in other animals, we cannot exclude a function for these genes in female mammals. Here, we show that PIWIL3 mRNA and protein are expressed in bovine oocytes. Using quantitative PCR we have determined the relative expression pattern of PIWIL3 transcripts during oocyte maturation and early embryo development. Furthermore, immunofluorescence studies localized Piwi-like 3 to the meiotic process of bovine oocytes. Lastly, preliminary experiments were made in an effort to adapt a known transfection mechanism to its use in bovine oocytes.
A protecção do conteúdo genético das células é essencial para manter a integridade da informação responsável pela vida. Existem vários factores que podem afectar a estabilidade do genoma, tais como: radiação ultravioleta, compostos mutagénicos e erros de replicação do DNA. Contudo, um dos maiores perigos que a célula enfrenta encontra-se no próprio código genético. Uma grande proporção dos genomas eucarióticos corresponde a sequências repetidas denominadas transposões. Os transposões são elementos genéticos móveis que têm a capacidade de inserir-se em novos locais do genoma, podendo interferir com a estrutura e função de genes essenciais para a célula. O perigo iminente da transposição genética merece especial atenção em células da linha germinal, onde qualquer alteração no genoma pode ser transferida à descendência. Por esta razão, torna-se necessário controlar a mobilidade destes elementos. RNA interferência (RNAi) é um mecanismo de regulação da expressão génica que tem sido implicado no silenciamento de elementos móveis. Esta ferramenta celular utiliza uma proteína da família Argonaute associada a uma sequência de RNA de pequenas dimensões como motor de pesquisa, localizando sequências mensageiras de RNA (mRNA) que lhe sejam complementares. Uma vez encontradas, esses mRNAs são clivados pelo complexo proteico (proteína Argonaute) acoplado ao RNA guia, impedindo a sua tradução. A família de proteínas Argonaute pode ser dividida em dois grupos: o grupo Ago, que são expressas ubiquitamente; e o grupo Piwi, cuja expressão é restrita às células da linha germinal. Para além do seu padrão de expressão, estes grupos de proteínas também diferem na classe de RNAs aos quais se associam. Membros do grupo Ago interagem com miRNAs e siRNAs, enquanto que proteínas Piwi interagem com uma nova classe de RNAs, denominados piRNAs (piwi-interacting RNAs). O estudo de proteínas Piwi e piRNAs tem avançado consideravelmente nos últimos anos com base em experiências realizadas em Drosophila, C. Elegans, zebrafish e ratinho. Estas experiências têm convergido para um papel das proteínas Piwi no desenvolvimento e sobrevivência das células da linha germinal. No genoma do ratinho existem três genes da família Piwi: miwi (Piwi-like 1), mili (Piwi-like 2) e miwi2 (Piwi-like 4). A produção de indivíduos mutantes para qualquer uma destas proteínas resulta na esterilidade de ratinhos machos, mas em fêmeas nenhum defeito é visível. Contrariamente, noutros animais modelo o conhecimento sobre a biologia das proteínas Piwi surgiu do estudo das células germinais de ambos os sexos. Ainda não foi possível identificar um papel para estas proteínas na linha germinal feminina de mamíferos. O avanço no conhecimento das proteínas Piwi deve-se principalmente à identificação e sequenciação dos pequenos RNAs a que se associam, os piRNAs. A sua análise genómica revelou que estas sequências correspondem a áreas teloméricas e centroméricas dos cromossomas, que são maioritariamente populadas por elementos repetitivos e cópias de transposões e, por esta razão, foram denominadas de clusters de piRNA. Estes clusters podem ser transcritos e produzir piRNAs de ambas as cadeias genómicas, que são subsequentemente acoplados a uma proteína Piwi. Os complexos Piwi-piRNA formados podem agora procurar sequências complementares de transposões e provocar a sua degradação, protegendo assim a integridade do genoma. Esta descoberta revelou uma nova função para zonas genómicas que até agora eram consideradas “lixo genético”. Assim, estas sequências funcionam como um sistema imunitário genético, impedindo que elementos móveis como os transposões provoquem alterações na informação que será transmitida à descendência. Considerando o estado de conservação das proteínas Piwi e o seu largo espectro de acção, parece improvável que a sua função seja dispensável em ovários de mamíferos. Recentemente foi identificado um quarto gene Piwi-like (PIWIL3) em muitos mamíferos (incluindo humanos e bovinos) que se encontra ausente do genoma do ratinho, razão pela qual ainda não foi estudado. Adicionalmente, o perfil de pequenos RNAs em oócitos de bovino revelou a existência de uma população acentuada de piRNAs, situação que não se observa em ratinho. Estas observações indicam que até este e os outros genes da família Piwi não forem examinados em outros animais, não se pode excluir por completo a sua participação no desenvolvimento da linha germinal feminina. Por esta razão, este projecto foi desenvolvido utilizando a vaca como organismo modelo. Apesar das limitações óbvias de manipulação, a investigação em bovino tem algumas vantagens. Grandes quantidades de ovários podem ser obtidos como material descartado de matadouros locais, a partir dos quais óvulos saudáveis podem ser extraídos. Adicionalmente, estes óvulos podem ser maturados in vitro, fertilizados, e podem manter-se em cultura até ao momento de implantação, isto é, até a fase de blastocisto. Neste trabalho demonstramos que a proteína Piwi-like 3 é expressa em oócitos de bovino, ao contrário da situação observada em ratinho. Em primeiro lugar, determinámos através de RT-PCR que a expressão de PIWIL3 encontra-se restrita aos tecidos germinais de bovino, sendo apenas detectável significativamente em ovários e testículos. De seguida verificou-se a presença de mRNA de PIWIL3 dentro desse contexto, focando a nossa atenção nos ovários. Para isso, foram recolhidas amostras em diferentes pontos do desenvolvimento de oócitos e embriões: vesícula germinal, metafase I, metafase II, zigoto, 2 células, 4 células, 8 células, morula e blastocisto. Utilizando novamente PCR, observamos que PIWIL3 encontra-se presente ao longo de todo o processo de maturação e durante o desenvolvimento embrionário pré-implantação. Para obter uma descrição mais detalhada da expressão deste gene, foi utilizada a técnica de PCR quantitativo, que permite detectar se o gene em estudo se encontra sob regulação. Os nossos resultados não mostraram nenhuma alteração significativa na expressão de PIWIL3 nos pontos temporais testados. Foi apenas detectado um ligeiro aumento de expressão no momento da metafase II, o que sugere um papel para esta proteína durante o processo de maturação. Contudo, a presença de mRNA não implica a existência de proteína nesta altura e, por esta razão, realizaram-se experiências de imunocitoquímica para comprovar a presença e localização da proteína. Em contraste com o padrão observado para mRNA, a proteína Piwi-like 3 foi detectada numa janela temporal menos abrangente, estando apenas presente durante a maturação do oócito até à formação dos pronúcleos. Neste contexto, verificou-se que Piwi-like 3 localiza-se junto dos cromossomas durante o evento da meiose, acompanhando todos os movimentos provocados pelo fuso meiótico. Por fim, com o objectivo de levar a cabo estudos funcionais, foi testada a possibilidade de introduzir RNA fluorescente em oócitos de bovino. Se possível, no futuro poderá introduzir-se RNAs de dupla cadeia que utilizem a maquinaria de RNAi para diminuir os níveis de PIWIL3 em oócitos, simulando uma célula mutante. Os nossos resultados mostram que é possível introduzir RNAs em oócitos sem consequências graves para a célula, abrindo a porta para estudos funcionais em oócitos de bovino. Os nossos resultados apontam para uma possível função para Piwi-like 3 ao longo do processo de maturação, particularmente durante a meiose. Tendo em conta trabalhos relacionados em outros organismos, especulamos também sobre a possibilidade de Piwi-like 3 e piRNAs serem acumulados maternalmente, sobre a participação desta proteína no transporte e localização de mRNA e no silenciamento de transposões. Este trabalho alerta-nos também da importância de conduzir e desenvolver estudos em organismos alternativos, uma vez que a situação observada em animais modelo não representa necessariamente uma classe inteira de animais.
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34

Beyret, Ergin. "Function of the Mouse PIWI Proteins and Biogenesis of Their piRNAs in the Male Germline." Diss., 2009. http://hdl.handle.net/10161/1583.

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PIWI proteins belong to an evolutionary conserved protein family as the sister sub-family of ARGONAUTE (AGO) proteins. While AGO proteins are functionally well-characterized and shown to mediate small-RNA guided gene regulation, the function of PIWI proteins remain elusive. Here we pursued functional characterization of PIWI proteins by studying MILI and MIWI, two PIWI proteins in the mouse.

We first show that both MIWI and MILI co-immunoprecipitate with a novel class of non-coding small RNAs from the post-natal mouse testis extract, which are named Piwi-interacting RNAs (piRNAs). Our cloning efforts identified thousands of different piRNA sequences, mostly derived from intergenic regions. Interestingly, both MILI and MIWI piRNAs correspond to the same regions on the genome and differ primarily in length. We propose piRNAs in the adult testis are produced by the processing of long, single stranded RNA precursors, based on the observation that piRNAs originate in clusters from a number of sites on the genome in a head-to-tail homology. In support, we bioinformatically predicted putative promoters, and yeast one hybrid analysis on two such regions found out that they interact with Krueppel C2H2 type zinc finger transcription factors. We did not observe the features of the "ping-pong" mechanism in their biogenesis: Both MILI and MIWI piRNAs are biased for 5` Uracil without an Adenine bias on the 10th nucleotide position, and do not significantly consist of sequences complementary to each other along their first 10nt. Moreover, MILI piRNAs are not down-regulated in Miwi-/- testis. These results indicate that the post-natal testicular piRNAs are produced independent of the ping-pong mechanism.

Although piRNAs are highly complex, PAGE and in situ analyses showed that piRNAs are germ cell-specific with predominant expression in spermatocytes and round spermatids, suggestive of a meiotic function. Correspondingly, we found that Miwi-/-; Mili-/- mice undergo only male infertility with terminal spermatogenic arrest during meiosis. piRNAs show a nucleo-cytoplasmic distribution, with enrichment in the chromatoid and dense bodies, two male germ cell-specific structures. The dense body has been implicated in synapsis and in the heterochromatinization of the sex chromosomes during male meiosis, a process known as meiotic sex chromosome inactivation (MSCI). Our histological analysis on Miwi-/-; Mili-/- testes showed that, while the overall synapsis is not affected, the sex chromosomes retain the euchromatin marker acetyl-H4K16 and lacks the heterochromatin marker H3K9-dimethyl. These observations indicate that murine PIWI proteins are necessary for MSCI. Moreover, we identified piRNA production from the X chromosome before MSCI, and propose PIWI proteins utilize piRNAs to target and silence unpaired chromosomal regions during meiosis.


Dissertation
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35

"Function of the Mouse PIWI Proteins and Biogenesis of Their piRNAs in the Male Germline." Diss., 2009. http://hdl.handle.net/10161/1583.

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36

Webster, Alexandre. "Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells." Thesis, 2015. https://thesis.library.caltech.edu/8857/1/AWebster_PhDThesis_2015.pdf.

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The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

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Rogers, Alicia Kathryn. "Mechanisms of Transcriptional Silencing by the Nuclear Piwi Protein in Drosophila Germ Cells." Thesis, 2018. https://thesis.library.caltech.edu/10796/41/AKRogers_formatted_thesis_final.pdf.

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An important characteristic for life is the ability to persist – to reproduce and defend oneself against different stresses. The ability of a species to persist from one generation to the next heavily depends on the integrity of the genetic material being passed down, and thus organisms have developed strategies to ensure the integrity of their genomes remain in tact. In Metazoan germlines, piwi proteins and their associated piwi-interacting RNAs (piRNAs) provide a RNA-interference (RNAi) based defense system against the expression of transposable elements (TEs). TE expression is detrimental to an organism’s genome – resulting in disruption of genes, double-stranded DNA breaks, and germ cell death – ultimately leading to the sterility of the organism. In Drosophila melanogaster, the piRNA pathway is composed of two cytoplasmic piwi clade Argonuate proteins, Aubergine (Aub) and Argonaute3 (Ago3), and a single nuclear piwi clade Argonuate protein, Piwi. The piwi clade Argonaute proteins bind piRNAs to form effector complexes that repress TE sequences.

The work presented in this thesis examines the role of the nuclear piwi clade Argonaute – Piwi – and the mechanisms by which Piwi accomplishes its functions. Chapter Two presents how Piwi/piRNA complexes identify genomic loci expressing TEs and direct the establishment of a repressive chromatin state to transcriptionally silence the loci. In Chapter Three, we explore the piRNA-induced transcriptional silencing (piRITS) pathway using a heterologous reporter based tethering system in vivo. We discuss how the recruitment of Piwi alone to a locus is not sufficient to induce repression, and establish a model for the connection bridging the Piwi/piRNA complex and effector silencing complex in the piRITS pathway. In Chapter Four, we employ our heterologous reporter based tethering system to explore the mechanism of piRNA precursor selection in the two cell types that make up Drosophila ovaries. We uncover a common mechanism of piRNA biogenesis in the two cell types and establish a unifying model of piRNA substrate selection. Finally, in Chapter Five, as essential step to understanding how Piwi achieves its nuclear function, we developed a heterologous two-hybrid system to identify factors that directly interact with Piwi. Overall, the work presented in this thesis provides a piece of the groundwork in understanding the mechanisms of transcriptional silencing of TEs in germ cells by Piwi. The work proposes that Piwi has dual functions in the nucleus. First, upon target recognition, Piwi recruits the piRITS complex to target loci to accomplish Piwi- mediated transcriptional silencing by deposition of H3K9me3. Then, Piwi recruits the RDC complex to specifically bind H3K9me3 at target loci to allow piRNA-production from the locus.

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38

Wang, Dafu. "Rubisco activase - a typical AAA+ protein with unique features, and, PIFI - a novel chloroplast protein functioning in chlororespiration /." 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3243018.

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Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006.
Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6238. Adviser: Archie R. Portis, Jr. Includes bibliographical references (leaves 138-154). Available on microfilm from Pro Quest Information and Learning.
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39

Ehlers, Claudia. "Functional analysis of the GlnK1 protein of Methanosarcina mazei strain Gö1: Aspects of nitrogen regulation." Doctoral thesis, 2004. http://hdl.handle.net/11858/00-1735-0000-0006-AC4D-A.

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PII-Proteine, zu denen GlnB und GlnK zählen, sind ubiquitär verbreitete kleine Regulatorproteine, die den internen Stickstoffzustand der Zelle sensieren und weiterleiten und hierdurch maßgeblich an der Regulation des Stickstoffmetabolismus beteiligt sind.Ziel dieser Arbeit war es, das GlnK1-Protein aus dem methanogenen Archeaon Methanosarcina mazei Stamm Gö1 umfassend zu charakterisieren und seine potentielle Rolle in der Regulation des Stickstoffmetabolismus aufzuklären. Das M. mazei GlnK1-Protein weist den typischen Tyrosin51-Rest auf, der in Bakterien stickstoffabhängig posttranslationell modifiziert wird. Sowohl in vitro als auch in vivo Experimente haben jedoch gezeigt, dass GlnK1 in M. mazei nicht stickstoffabhängig modifiziert wird. Weitere strukturelle Unterschiede zu bakteriellen PII-Proteinen haben Experimente zur Bildung von Heterotrimeren aufgezeigt. Trotz dieser deutlichen Unterschiede haben Komplementationsversuche ergeben, dass das archaeelle GlnK-Protein in der Lage ist, E. coli GlnK funktionell zu komplementieren. Dieses läßt darauf schließen, dass das GlnK1-Protein in M. mazei auch in der Regulation des Stickstoffmetabolismus involviert ist. Um dieses zu bestätigen, wurde eine chromosomale M. mazei glnK1-Mutante generiert. Hierfür war es erforderlich, zunächst ein funktionelles System zur Transformation von M. mazei Gö1 zu entwickeln. Es gelang, (i) durch Selektion einer potentiellen spontanen Zellwandmutante von M. mazei, die eine stark verbesserte Plattierungseffizienz aufwies, sowie (ii) durch mehrere Modifizierungen des von W. Metcalf (Urbana) entwickelten Liposomen-vermittelten Transformationsprotokolls für Methanosarcina-Stämme M. mazei Gö1 genetisch zugänglich zu machen. Wachstumsanalysen des konstruierten M. mazei glnK1-Mutantenstamms zeigten einen partiell reduzierten Wachstumsphänotyp unter stickstofflimitierenden Bedingungen. Quantitative Reverse Transkriptions-PCR Analysen ausgewählter Gene ergaben allerdings, dass das GlnK1 keinen Einfluss auf die Transkription stickstoffregulierter Gene ausübt.Sowohl biochemische Experimente mit gereinigtem Enzym als auch in vivo Versuche zeigten jedoch, dass das GlnK1-Protein mit der Glutamin-Synthetase (GlnA) interagiert und hierdurch deren Enzymaktivität inhibiert. Ein aktivierender Effekt auf die GlnA Enzymaktivität wurde hingegen bei Anwesenheit von 2-Oxoglutarat beobachtet, welches den internen Stickstoffstatus wiederspiegelt. Aus der Gesamtheit der Ergebnisse läßt sich folgendes hypothetisches Regulationsmodel ableiten: Unter Stickstofflimitierung wird 2-Oxoglutarat akkumuliert, welches die Glutamine-Synthetase Aktivität stark stimuliert; bei einem Übergang zu Stickstoffüberschuss wird die Glutamine-Synthetase sowohl durch einen reduzierten 2-Oxoglutarat-Spiegel als auch durch direkte Protein-Interaktion mit GlnK1 inaktiviert. Letzteres dient der Feinregulation und ermöglicht schnell auf eine veränderte Stickstoffversorgung reagieren zu können.
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40

Tammam, Stephanie. "Characterization of PilP from the Type IV Pilus System of Pseudomonas aeruginosa." Thesis, 2012. http://hdl.handle.net/1807/43398.

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Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.
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LINHARTOVÁ, Markéta. "Analysis of the role of PilA proteins in the cyanobacterium \kur{Synechocystis} sp. PCC 6803." Master's thesis, 2009. http://www.nusl.cz/ntk/nusl-50359.

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The PilA proteins are major components of pili fibres which are essential for motility in bacteria. This project was focused on the role of the PilA proteins in cyanobacteria, specifically in the alternative functions that are not directly related to motility. The major task of this project was also to develop a method for the purification of the PilA1 protein from Synechocystis under native conditions and to analyze purified PilA1 protein in detail.
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42

Weiß, Martin. "A protein in search of a function: The c-di-AMP-binding protein DarA of Bacillus subtilis." Doctoral thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C177-D.

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43

Abdelmadjid, Imen. "Fonction de l'AmtB dans la régulation de la nitrogénase chez Rhodobacter capsulatus." Thèse, 2010. http://hdl.handle.net/1866/3876.

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La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium.
The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.
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44

Kus, Julianne. "Diversity of Pseudomonas aeruginosa Type IV Pilins and Identification of a Novel D-arabinofuranose Post-translational Modification." Thesis, 2008. http://hdl.handle.net/1807/11222.

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The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.
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