Dissertations / Theses on the topic 'GGDEF'

Parte da produção de fatores de virulência em bactérias do gênero Xanthomonas esta sob controle de um grupo de genes localizados no locus rpf (regulation of pathogenicity factors), que respondem ao aumento da densidade celular num processo chamado quorum sensing. Os genes que codificam as proteínas do sistema Rpf de Xanthomonas axonopodis pv citri (Xac) foram clonados no vetor pOBD por Alegria (2004), e usados como iscas em ensaios de dois híbridos, contra uma biblioteca de Xac clonada no vetor pOAD. Neste ensaio, foram observadas interações entre RpfC-RpfG, RpfC-RpfF, RpfF-RpfF e RpfC-CMF. O gene cmf tem um ortólogo, cuja função esta relacionada com o processo de quorum sensing em Dictyostelium. Para confirmar essas interações, RpfC e seus domínios, RpfG, RpfF e CMF foram expressas e purificadas, produzidos anticorpos, e foram efetuados ensaios de ligação in vitro. Em adição, o domínio HD-GYP de RpfG, que apresenta atividade de fosfodiesterase, também foi usado como isca no ensaio de dois híbridos. Interessantemente, a maioria de suas presas foi derivada de domínios GGDEF (diguanilato ciclase) de um grupo de proteínas de Xac. Em bactérias, muitos fenótipos, como a ativação da virulência, a formação de biofilme e a mobilidade, são controlados pelo processo de quorum sensing e por diGMP cíclico. Neste trabalho demonstramos uma ligação direta entre quorum sensing e diGMP cíclico, representada pela interação entre HD-GYP/GGDEF. Finalmente, estudos com um mutante para o gene cmf interrompido, envolvendo formação de biofilme, produção de goma xantana e patogenicidade, evidenciam que CMF tem uma função relevante no processo de quorum sensing em Xanthomonas.
In Xanthomonas a group of genes named regulators of the pathogenicity factors (rpf) control the synthesis of virulence factors as a function of cellular density in a process termed quorum sensing. Alegria (2002) cloned the rpf genes from Xanthomonas axonopodis pv citri (Xac) in the pOBD vector and used them as baits in two hybrid assays against a Xac prey library cloned in the pOAD vector and showed that RpfC interacts with RpfG, RpfF and CMF. Homologous of the cmf gene are found only in amoebas such as Dictyostelium, where plays a central function in the quorum sensing process. In this work, we expressed and purified RpfC and its domains, RpfG, RpfF and CMF and raised antibodies against these polypeptides. In vitro assays demonstrated the following interactions: RpfC-RpfF, RpfC-RpfG and RpfC-CMF. We show that RpfG and CMF interact with the response regulator domain of RpfC, and interactions RpfG and CMF also interact with the histidine phosphotransfer domain of RpfC. In addition, the recently characterized HD-GYP phosphodiesterase domain of RpfG was used as bait in the two hybrid assays. Interestingly, the majority of its preys were derived from a set of Xac proteins that possess GGDEF domains (diguanilate cyclase). In bacteria, many complex processes such virulence, motility and biofilm production are controlled by quorum sensing process and by levels of the second messenger cyclic diGMP. Our results demonstrate a direct link between quorum sensing and diGMP cyclic signaling pathways in the form of a direct physical interaction between the RpfG HD-GYP domain and GGDEF domains. Finally, studies with a Xac cmf-mutant show that CMF plays an important role in the quorum sensing process in Xanthomonas, including biofilm production, synthesis of xanthan gum and pathogenicity.

Legionella pneumophila est un pathogène intracellulaire, agent de la Légionellose, et dont le réservoir dans l'environnement est constitué d'amibes aquatiques comme Acanthamoeba castellani. Mes objectifs de thèse étaient l'identification de mécanismes moléculaires contrôlant la virulence et la multi-résistance chez L. pneumophila, et en particulier l'exploration du rôle des protéines " GGDEF/EAL ". Les domaines GGDEF et EAL sont retrouvés dans des enzymes permettant respectivement de synthétiser (diguanylate cyclase, DGC) ou dégrader (phosphodiestérase, PDE) le di-GMP cyclique, un second messager spécifique des bactéries, qui participe au contrôle de fonctions clés comme la virulence ou la mobilité. L. pneumophila Lens possède 22 gènes codant des protéines GGDEF/EAL, et dont la plupart sont exprimés lorsqu'elle est dans sa phase virulente. L'activité enzymatique des 22 protéines " GGDEF/EAL " a été analysée in vitro : sur 10 protéines purifiées, 6 sont des DGC, dont 2 présentes une double activité DGC/PDE. L'inactivation de 5 gènes des 22 gènes et la surexpression de 2 autres entrainent une baisse de la virulence vis-à-vis d'A. castellanii. De plus, nous avons montré que l'activité DGC d'au moins 2 de ces protéines est requise lors du cycle infectieux. Enfin, nous avons décrit un système à deux composants original comprenant l'histidine kinase (HK) Lpl0330, capable de s'autophosphoryler sur un nouveau domaine HisKA, retrouvé dans 64 autres HK potentielles, et Lpl0329, le premier régulateur de réponse à double activité enzymatique caractérisé, dont la phosphorylation conduit à moduler le taux de di‐GMPc en favorisant une de ses deux activités (Levet-Paulo et al., 2011).

A formação de biofilmes bacterianos é um fenômeno bem conhecido, caracterizado pela formação de uma comunidade bacteriana estática, embebida em uma matriz exopolimérica, regulada pela molécula sinalizadora c-di-GMP. Os domínios proteicos que catalisam a síntese (GGDEF) e degradação (EAL e HD-GYP) de c-di-GMP estão presentes em grande quantidade em quase todos os genomas bacterianos sequenciados até hoje. Dentre as diversas proteínas envolvidas nas vias de sinalização mediadas por esse nucleotídeo, uma grande parcela são proteínas transmembranares que possuem ambos domínios GGDEF e EAL. Funcionalmente, esses domínios se apresentam em todas combinações: ambos degenerados ou conservados e combinações GGDEF-degenerado/EAL-conservado ou vice-versa. Enquanto que domínios conservados potencialmente apresentam atividade catalítica, os degenerados geralmente convertem-se em domínios estruturais ou receptores de c-di-GMP. Embora recentes estudos estruturais revelaram detalhes de proteínas com ambos domínios degenerados (LapD) ou ativos (MorA), pouco se sabe sobre uma das combinações mais representativas: GGDEF-degenerado/EAL-conservado. Nesse trabalho, realizamos um estudo estrutural e funcional da proteína STM3615 de Salmonella enterica, que apresenta um domínio periplasmático de função desconhecida, seguido pelos domínios citoplasmáticos HAMP, GGDEF-degenerado e EAL-conservado. Através de diferentes construções citoplasmáticas solúveis de STM3615, confirmamos que essa proteína apresenta atividade fosfodiesterase, mesmo quando o domínio EAL encontra-se isolado. Corroborando com sua atividade catalítica, estudos em solução, tais como SAXS e cromatografia de exclusão molecular, mostraram que o EAL isolado de STM3615 é dimérico, um pré-requisito para ser ativo. Utilizando uma construção com os domínios GGDEF-EAL determinamos sua estrutura cristalográfica a uma resolução de 2,5 Å. Comparada com proteínas de arquitetura próxima, como o receptor de c-di-GMP LapD de Pseudomonas fluorescens, ou a enzima bifuncional MorA de Pseudomonas aeruginosa, sua estrutura se assemelha muito mais a essa última. Em particular, a hélice que conecta os domínios GGDEF e EAL possui a mesma extensão que a de MorA, maiores que a encontrada em LapD. Como a hélice pequena de LapD está relacionada com sua plasticidade conformacional interdomínios, a estrutura apresentada nesse trabalho sugere as proteínas dual domain cataliticamente ativas (EAL-mono ou bifuncionais) sejam estruturalmente rígidas. Combinando esses resultados com uma análise computacional feita em outras 150 sequências representativas de proteínas dual domain, propomos mecanismos catalíticos distintos para as enzimas bifuncionais e as EAL-monofuncionais. Enquanto que essas últimas formam dímeros estáveis através do domínio EAL, numa conformação apta para interagir e degradar c-di-GMP, as enzimas bifuncionais apresentam transições oligoméricas mediadas por interação de c-di-GMP com EAL, impondo atividades ciclase (GGDEF) e fosfodiesterase (EAL) excludentes. Por fim, baseados nesses mecanismos e na arquitetura de STM3615, ainda especulamos mecanismos funcionais in vivo compatíveis com o tema emergente de interações proteicas e localização do sinal nas vias de sinalização mediadas por c-di-GMP.
The formation of bacterial biofilms is a well-established phenomenon regulated by the signaling molecule c-di-GMP, characterized by the establishment of a static bacterial community embedded in a exopolymeric matrix. The domains responsible for the synthesis (GGDEF) or degradation (EAL and HD-GYP) of c-di-GMP are present in multiple proteins in nearly all bacterial genomes sequenced to date. Among the multiple and structurally diverse proteins involved in c-di-GMP signaling and biosynthesis, a large class are transmembrane proteins bearing both EAL and GGDEF domains. Functionally, these domains are presented in all combinations: both degenerate or conserved and combinations GGDEF-degenerated/EAL-conserved or vice versa. While the predicted conserved domains exhibit catalytic activity, the degenerate usually converted into structural domains or c-di-GMP receptors. While structural studies have revealed details of proteins with both domains degenerated (LapD) or conserved (MorA), little is known about one of the most representative combinations: GGDEF-degenerated/EAL-conserved. In this work, we conducted a structural and functional study of Salmonella enterica STM3615 protein, which has a periplasmic domain of unknown function, followed by cytoplasmic domains HAMP, GGDEF-degenerated and EAL-conserved. Through different soluble cytoplasmic constructs of STM3615, we confirmed that this protein has phosphodiesterase activity, even with the isolated EAL domain. In agreement with its catalytic activity, solution studies, such as SAXS and size exclusion chromatography, showed that STM3615 isolated EAL is dimeric, a prerequisite for phosphodiesterase activity. Using a construct with the isolated EAL-GGDEF domains, we determine its crystal structure to a resolution of 2.5 Å. Compared to the architectural closed c-di-GMP receptor LapD from Pseudomonas fluorescens and the bifunctional enzyme MorA from Pseudomonas aeruginosa, STM3615 structure is more similar to the latter. In particular, the α-helix connecting the domains GGDEF and EAL has similar extension, longer than the helix found in LapD. Given that this helix in LapD is essential for its inter-domain conformational plasticity, the structure presented in this study suggests the dual domain catalytically active proteins are structurally rigid. Combining these results with a computational analysis with 150 representative sequences containing the tandem GGDEF-EAL domains, we propose distinct catalytic mechanisms for bifunctional and monofunctional EAL enzymes. While the latter form stable dimers through the EAL domain, a conformation prompted to interact and degrade c-di-GMP, the bifunctional enzymes present oligomeric transitions mediated by interaction of c-di-GMP with EAL domain, imposing excluding cyclase (GGDEF) or phosphodiesterase (EAL) activities. Finally, based on these mechanisms and STM3615 architecture, we also speculated about functional mechanisms in vivo consistent with the emerging theme of protein interactions and localized signal involved in signaling pathways mediated by c-di-GMP.

O diguanilato cíclico (c-di-GMP) é uma molécula de sinalização intracelular que atua na regulação de importantes processos bacterianos como motilidade, formação de biofilme e virulência. As diguanilato ciclases (DGCs), contendo um domínio GGDEF ativo, catalisam a formação de c-di-GMP a partir de duas moléculas de GTP. A bactéria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) é o agente causal do cancro cítrico, uma doença que ataca todas as variedades e espécies de citros. O genoma de Xac codifica 31 proteínas contendo domínios GGDEF. Treze destas proteínas possuem também domínios PAS e/ou GAF, que são ubíquos domínios sensores e de sinalização. Para tentar entender melhor o papel na sinalização por c-di-GMP das interações entre domínios GGDEF e domínios PAS e/ou GAF, estudos bioquímicos e funcionais foram realizados com as proteínas XAC0610 e XAC2446. XAC0610 contém um domínio GAF, quatro domínios PAS e um domínio GGDEF conservado. Análises fenotípicas com a linhagem nocaute XacΔ0610 mostraram que XAC0610 atua na regulação da motilidade e sobrevivência de Xac ao tratamento com H2O2. Ensaios de atividade enzimática demonstraram que XAC0610 é uma DGC cataliticamente ativa, e que a mutação sítio-dirigida de um resíduo conservado de lisina (Lys759) provoca uma grande redução na atividade de DGC. Os domínios GAF e PAS de XAC0610 aparentemente não atuam como domínios sensores, entretanto são importantes para a dimerização da proteína, necessária para a obtenção de altos níveis de atividade de DGC. Além disso, várias observações sugerem que XAC0610 não é submetida à inibição alostérica pelo produto, um mecanismo regulatório comumente utilizado para o controle da atividade de DGC. Por outro lado, os dados de cinética enzimática de XAC0610HIS-35-880 revelaram um efeito de cooperatividade positiva para a ligação dos substratos, com uma constante de dissociação para a ligação da primeira molécula de GTP (K1) cerca de 3-5 vezes maior que a constante de dissociação para a ligação da segunda molécula de GTP (K2). A partir deste estudo, nós apresentamos um esquema cinético geral mais apropriado para as análises dos dados cinéticos de enzimas DGCs e propomos que a ligação cooperativa do substrato talvez possa desempenhar um importante papel na regulação in vivo da atividade de algumas DGCs, aumentando sua sensibilidade a pequenas variações nos níveis celulares de GTP. Outra proteína caracterizada neste trabalho, XAC2446 possui um domínio GAF e um domínio GGDEF que, ao contrário do domínio GGDEF de XAC0610, não deve apresentar atividade de DGC. Mesmo assim, análises funcionais mostraram que XAC2446 regula negativamente a formação de biofilme e positivamente a motilidade de Xac. Ensaios de duplo híbrido em leveduras identificaram que XAC2446 interage com XAC2897, contendo um domínio GGDEF potencialmente ativo, e XAC1185, contendo um domínio HD fosfohidrolase de (p)ppGpp. Alguns estudos indicam que altos níveis celulares de c-di-GMP e baixos níveis de (p)ppGpp podem ser necessários durante a formação de biofilme. XAC2446 talvez possa atuar como um inibidor da atividade enzimática de XAC2897 e XAC1185 e influenciar, indiretamente e antagonicamente, tanto os níveis celulares de c-di-GMP quanto de (p)ppGpp.
Cyclic di-GMP is a bacterial second messenger that regulates a range of functions, including cellular motility, biofilm formation and virulence. This molecule is produced from two GTP substrates by the activity of diguanylate cyclases (DGCs) containing a GGDEF domain. The phytopathogenic bacteria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) causes citrus canker in a wide variety of citrus species. The Xac genome codes for 31 proteins with GGDEF domains. Thirteen of the 31 Xac GGDEF domain-containing proteins also possess PAS (Per-Arnt-Sim) or GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domains that are ubiquitous signaling and sensory domains. In order to better understand the relationship between these commonly associated domains, biochemical and functional studies were carried out with the XAC0610 and XAC2446 proteins. XAC0610 is a large multi-domain protein containing one GAF domain, four PAS domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. XAC0610 DGC activity was also impaired by removal of the N-terminal GAF and PAS domains, which are probably needed for proper protein dimerization. Furthermore, experimental and in silico analysis suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately three to five times greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. The other characterized protein, XAC2446, has a GAF domain and a degenerated GGDEF domain. Unlike XAC0610, XAC2446 should not present DGC activity. Nevertheless, functional analysis of XAC2446 demonstrated that it plays a role in the regulation of Xac motility and biofilm formation. A yeast two-hybrid screen identifies XAC2897 (a potentially active GGDEF domain-containing protein) and XAC1185 (a (p)ppGpp hydrolase) as specific binding partners of the XAC2446 protein. As indicated by studies in other bacteria, high cellular levels of c-di-GMP and low levels of (p)ppGpp may be both required for biofilm formation. It is possible that XAC2446 might have a role in the antagonistic regulation of c-di-GMP and (p)ppGpp cellular levels by acting as an inhibitor of both XAC2897 and XAC1185 enzymatic activities.

Die Entwicklung des grampositiven Bodenbakteriums Streptomyces ist in einem komplexen Lebenszyklus koordiniert, bestehend aus drei Stufen: vegetativem Hyphenwachstum, Luftmycelbildung und Sporulation. C-di-GMP kontrolliert die Enwicklung über zwei Effektorproteine: dem Masterregulator BldD und dem Anti-Sigmafaktor RsiG. In dieser Arbeit konnte gezeigt werden, dass das membranständige GGDEF-EAL Protein CdgC eine wichtige aktive Diguanylatzyklase (DGC) in S. venezuelae ist. Chromosomale Deletion von cdgC führte zu einer flachen, gräulichen Koloniemorphologie mit radialen Stegen und hydrophiler Oberfläche sowie zu frühzeitiger Sporulation ohne Lufthyphenbildung. Phänotypische Analysen zeigten, dass die DGC-Aktivität von CdgC essentiell ist für dessen biologische Rolle und deuten auf einen zusätzlichen Protein-spezifischen morphologischen Effekt von CdgC hin. CdgC-FLAG akkumuliert im Laufe des Lebenszyklus und scheint BldD-abhängig über eine c-di-GMP vermittelte Feedbackschleife reguliert zu werden. Frühere RNA-seq Daten, verifiziert für repräsentative Gene mittels qRT-PCR, deuten eine differentielle Expression der Bestandteile des hydrophoben Mantels als Ursache der Lufthyphendefizienz an. Konfokalmikroskopische Aufnahmen des bakteriellen Tubulin-Homologons FtsZ deuten einen c-di-GMP-sensitiven Einfluss von CdgC auf die Koordination der Zellteilung an. Zudem konnte nachgewiesen werden, dass CdgC mit sich selbst sowie drei potentiellen Membranproteinen interagiert. Demnach trägt CdgC zur Koordination von Zellteilungs- und hydrophoben Zelloberflächenproteinen bei und beeinflusst damit c-di-GMP-abhängig den Zeitpunkt der Sporenbildung. Insgesamt führt diese Studie CdgC als GGDEF-EAL-Tandemprotein mit spezifischem Knockout- Phänotyp ein, der von seiner DGC-Aktivität sowie seinem Membrananker bestimmt wird. Zudem ist CdgC, als Reaktion auf eine noch unbekannte Signalübertragungskaskade, an der Koordinierung von Zeitpunkt und Verlauf der Sporulation ausschlaggebend beteiligt.
The proliferation of Gram-positive soil bacteria Streptomyces is temporally and genetically coordinated with a complex developmental life cycle, including three main stages of differentiation: vegetative hyphal growth, formation of aerial mycelium and sporulation. The key factor of Streptomyces developmental control is c-di-GMP with to-date two identified effector proteins: the master regulator BldD and the anti-sigma factor RsiG. In this thesis, the membrane-associated GGDEF-EAL protein CdgC, was identified as a major active diguanylate cyclase (DGC) in S. venezuelae. Deletion of cdgC results in the unique flat gray colony morphology with radial wrinkles and a hydrophilic surface, that shows enhanced sporulation without forming aerial hyphae. Phenotypic analyses suggest, that the DGC activity is essential for its biological role, but hint to an additional protein specific role. The protein levels of CdgC-FLAG were found to accumulate during the life cycle of S. venezuelae. Further investigation of CdgC-FLAG in a strain carrying a DNA-binding deficient BldD_D116A allele indicated, that BldD represses the expression of CdgC in a regulatory feedback loop along with the DGCs CdgA, CdgB and CdgE. RNA‐sequencing data indicated that reduced expression levels of the major compounds of the hydrophobic sheath result in the initiation of sporulation out of the vegetative mycelium and were verified for representative examples via qRT-PCR. Confocal microscopic imaging of the bacterial tubulin homolog FtsZ indicated a contribution of CdgC via its DGC activity in coordination of the cell division. In addition, BTH screenings revealed self-interaction and identified three membrane associated interaction partners. In conclusion, this study introduces the GGDEF-EAL tandem protein CdgC, whose specific knockout phenotype is governed by its DGC activity and membrane association. CdgC seems to drive timing and mode of sporulation in response to an unknown signal to a major extend.

Segundos mensageiros nucleotídicos são amplamente utilizados por bactérias para se adaptar às mudanças ambientais e fisiológicas. Neste cenário destaca-se o c-di-GMP, um segundo mensageiro praticamente universal em bactérias responsável por controlar a transição do estilo de vida bacteriano. Em geral, altos níveis celulares de c-di-GMP promovem um estado séssil, de formação de biofilme, enquanto baixos níveis induzem a motilidade. Xanthomonas citri subsp. citri (Xac), um fitopatógeno de grande importância econômica no Brasil, possui uma complexa regulação da sinalização de c-di-GMP, possuindo mais de 30 proteínas envolvidas na síntese, na degradação e na detecção deste segundo mensageiro. Dentre essas proteínas, destacam-se as que possuem os domínios de síntese e degradação presentes na mesma cadeia polipetídica, os domínios GGDEF e EAL respectivamente. A análise das estruturas primárias das 11 proteínas GGDEF-EAL codificadas pelo genoma de Xac revelou que a maior parte delas (6) provavelmente possui o domínio GGDEF inativo, enquanto o EAL é ativo. Três possivelmente possuem ambos os domínios ativos enquanto outras duas possuem ambos os domínios inativos. O nocaute do gene xac2382 que codifica uma dessas proteínas (que possui um domínio periplasmático seguido dos domínios citoplasmáticos HAMP-GGDEF-EAL), demonstra um aumento de motilidade e uma diminuição na formação de biofilme. Construções de fragmentos da proteína revelaram que XAC2382 necessita pelo menos dos domínios HAMP-GGDEF para complementar a cepa nocaute e que a atividade de diguanilato ciclase é essencial para isto. O domínio periplasmático de XAC2382 se mostrou interagir com XAC2383, uma proteína codificada por um gene presente no mesmo cluster do gene de XAC2382, e essa interação parece importante para o controle da motilidade de Xac. A estrutura de XAC2383 foi resolvida por cristalografia de raios X na qual foi revelada uma topologia típica de proteínas da família das periplasmic binding proteins (PBPs) possuindo ainda uma cavidade carregada positivamente contendo um motivo Ser-Thr-Ser (amnioácidos 152-154) importante para a ligação de compostos com grupos fosfatos ou fosfonatos. A mutação sítio dirigida nesse motivo aboliu os efeitos na motilidade dependentes dessa proteína. Esses resultados sugerem que XAC2383 é um sensor periplasmático de um composto eletronegativo e esta proteína interage com XAC2382 regulando a motilidade bacteriana. XAC0495, uma proteína com ambos os domínios GGDEF-EAL provavelmente inativos, pode fazer parte de um sistema de dois componentes com a histidina quinase XAC0494. XAC0495 se comporta como um monômero em solução e possui um formato alongado, como revelado por experimentos de SAXS.
Nucleotide based second messengers are widely used by bacteria in signaling pathways that mediate adaptations to environmental and physiological changes. c-di-GMP is a nucleotide second messenger ubiquitous in Gram-negative bacteria, where it plays a role in many important behaviors that define bacterial lifestyle. In general, high cellular levels of c-di-GMP promote biofilm formation, while low levels induce bacterial motility. Xanthomonas citri subsp. Citri (Xac), a pathogen of great economic importance in Brazil, has a complex repertoire of c-di-GMP signaling molecules, with more than 30 genes coding for proteins involved in the synthesis, degradation and detection of this second messenger. Among these proteins, many have both GGDEF and EAL domains (often associated with c-di-GMP synthesis and degradation, respectively) present in the same polypeptide chain. Analysis of the primary structure of 11 GGDEF-EAL proteins coded by the Xac genome revealed that six most likely possess an inactive GGDEF domain plus an active EALdomain. Another three proteins have both domains active while the other two have both domains inactive. The knockout of the xac2382 gene, coding for a protein which contains a periplasmic domain followed by cytoplasmic HAMP, GGDEF (active) and EAL (active) domains, shows an increase in motility and a decrease in biofilm formation. Constructions containing fragments of this protein revealed that constructs containing at least the HAMP and GGDEF domains are able to complement the knockout strain and that diguanilate cyclase activity is essential for this. The XAC2382 periplasmic domain was shown to interact with a protein encoded by a gene situated in the same cluster, XAC2383, and that this interaction seems crucial for the control of Xac motility. The structure of XAC2383 was solved by X-ray crystallography and was shown to adopt a topology typical of the periplasmic binding proteins (PBP) family. The protein possesses a positively charged groove that contains a Ser-Thr-Ser motif (152STS154) important for the binding of compounds with phosphate or phosphonate groups. Site-directed mutagenesis of this motif abolished the effects on motility caused by this protein. These results suggest that XAC2383 is a periplasmic protein responsible for sensing a compound with electronegative characteristics and which interacts with XAC2382, thereby regulating the bacterial motility. Another protein, XAC0495 (with both GGDEF-EAL domains probably inactive) may be part of a two-component system with the histidine kinase XAC0494. Small-angle X-ray scattering (SAXS) experiments reveal that XAC0495 exists as an elongated monomer in solution.

Streptomyceten weisen einen komplexen Lebenszyklus auf, dessen Verlauf durch den sekundären Botenstoff Bis-(3´- 5´)-zyklisches dimeres Guanosinmonophosphat (c-di-GMP) und die c-di-GMP-Effektorproteine BldD und RsiG reguliert wird. Der Auf- bzw. Abbau von c-di-GMP wird von Diguanylatzyklasen (DGC) mit GGDEF-Domänen bzw. Phosphodiesterasen (PDE) mit EAL- oder HD-GYP-Domänen katalysiert. In S. venezuelae, einem Modellorganismus der Streptomyceten, konnten zehn potenziell c-di-GMP metabolisierende Enzyme identifiziert werden, von welchen mit RmdA und RmdB zwei GGDEF-EAL-Tandem-Proteine im Fokus dieser Arbeit stehen. Die chromosomale Deletion der für RmdA und RmdB kodierenden Gene führt zu einer ausgeprägten Verzögerung der Entwicklung in S. venezuelae. Mit Hilfe chromosomaler Mutationen konnten die EAL-Motive der EAL-Domänen als essenziell für die in vivo Funktion beider Proteine identifiziert werden. Beide Proteine zeigen in vitro PDE-Aktivität und RmdA konnte als bifunktionales Enzym charakterisiert werden, da es in vitro auch DGC-Aktivität aufweist. Mittels Nukleotidextraktionen konnte RmdB als Haupt-PDE in S. venezuelae identifiziert werden, welche über den gesamten Entwicklungsverlauf für den Abbau von c-di-GMP verantwortlich ist. Aber auch RmdA hat während des Übergangs von der vegetativen zur reproduktiven Wachstumsphase Einfluss auf die globale zelluläre c-di-GMP Konzentration. Durchgeführte Transkriptomanalysen und qRT-PCR-Experimente ergaben, dass in den Deletionsmutanten die Expression einiger wichtiger entwicklungsspezifischer Gene im Vergleich zum Wildtyp herunterreguliert ist. Dies ist vermutlich auf die erhöhten c-di-GMP Konzentrationen in den Deletionsmutanten zurückzuführen, wodurch die Aktivität der c-di-GMP-Effektorproteine BldD und RsiG beeinflusst wird und die verzögerte Entwicklung der Deletionsmutanten erklärt werden kann. Weiterhin konnte gezeigt werden, dass RmdB mit dem Sigmafaktor der Sporulation, WhiG, interagieren kann.
Streptomycetes show a complex life cycle. The transition between the different developmental stages is regulated by the secondary messenger bis- (3´- 5´) -cyclic dimeric guanosine monophosphate (c-di-GMP) and the c-di-GMP effector proteins BldD and RsiG. c-di-GMP is synthesized by diguanylate cyclases (DGCs) with GGDEF domains, and its degradation is catalyzed by phosphodiesterases (PDE) with EAL or HD-GYP domains. In S. venezuelae, the Streptomyces strain which was used as a model organism in this work, there are ten potentially c-di-GMP metabolizing enzymes, of which two GGDEF-EAL tandem proteins, RmdA and RmdB, are the focus of this work. The deletion of the genes coding for RmdA and RmdB leads to a pronounced developmental delay in S. venezuelae. With the help of chromosomal mutations, the EAL motif was identified as essential for the in vivo function of RmdA and RmdB. Furthermore, both proteins were characterized in vitro as active PDEs and RmdA as a bifunctional enzyme, which also showed DGC activity. RmdB was identified as the master PDE in S. venezuelae by means of nucleotide extraction and is responsible for the hydrolysis of c-di-GMP over the course of development investigated. Also RmdA has an influence on the global cellular c-di-GMP concentration during the transition from the vegetative to the reproductive growth phase. A transcriptome analysis, qRT-PCR experiments and related follow-up experiments showed that the deletion of rmdA and rmdB leads to a differential expression of genes which code for important development-specific factors and regulators. This is presumably due to the increased c-di-GMP concentrations in the deletion mutants, with the c-di-GMP effector proteins BldD and RsiG delaying the transition to the next growth phase. Furthermore, it could be shown that RmdB can interact with the sigma factor of sporulation, WhiG.

Lyme disease is the most common tick-borne disease in North America, with approximately 35,000 cases reported to the Centers for Disease Control in 2008. The genome of its causative agent, Borrelia burgdorferi, encodes for a set of genes involved in the metabolism and regulatory activities of the second messenger nucleotide, cyclic-di-GMP (c-di-GMP). Rrp1 is a response regulatory-diguanylate cyclase, and its regulatory capability is likely mediated via production of c-di-GMP, as it lacks a DNA-binding domain. One known class of c-di-GMP effector/binding proteins are those that harbor a PIlZ domain. The genome of B. burgdorferi strain 5A4 encodes for one chromosomally-carried PilZ domain, which we have designated PlzA. Additionally, certain B. burgdorferi strains encode for a second PilZ domain-containing protein (PlzB) which is plasmid-carried. Both PlzA and PlzB were found to bind specifically to c-di-GMP, and c-di-GMP binding by PlzA was found to be dependant upon arginine residues in the c-di-GMP binding region. Additionally, expression of PlzA was found to be upregulated by tick feeding and was constitutive in the mammalian host. We next constructed two deletion/allelic exchange mutants – one with the targeted deletion of PlzA, and on ethat replaced PlzA with PlzB in a strain lacking the plzB gene. Our studies demonstrated that ΔplzA was deficient in motility and was also non-infectious in the mouse model of B. burgdorferi infection. Additionally, this strain remained viable in larval Ixodes ticks. Also, B31-plzB KI was deficient in motility, as well as infectivity, demonstrating that PlzB is unable to complement for functions fo PlzA in vitro and in vivo and that it may play other roles in the biology of B. burgdorferi strains carrying the plzB gene. These studies represent the first identification of a c-di-GMP binding protein in any spirochete, but also represent the first demonstration of the importance of PilZ domain proteins in a spirochetal system. We additionally examined the effects of c-di-GMP synthesis and breakdown in the related bacterium, B. hermsii, a causative agent of tick-borne relapsing fever (TBRF). Deletion mutants in Rrp1 (B. hermsii’s sole diguanylate cyclase) and PdeA (B. hermsii’s only EAL domain-containing phosphodiesterase) were created. These strains were analyzed in order to determine: 1) the effect(s) of the losse of Rrp1/PdeA on intracellular spirochete c-di-GMP levels, and 2) the effects of Rrp1/PdeA on the establishment of murine infection and on gross motility/chemotaxis. It was demonstrated that c-di-GMP accumulates intracellularly in the cells lacking PdeA. Additionally, spirochetes were shown to chemotax towards N-acetyl-glucosamine (NAG) and they did not form soft agar swarms. In contrast, cells lacking Rrp1 did not accumulate detectable levels of c-di-GMP, demonstrated a reduced ability to chemotax towards NAG, and swarmed on soft agar in a fashion indistinguishable from wild type. Despite these differences in phenotype, both mutant strains display an attenuated murine infectivity. These results indicate that c-di-GMP is indeed important in the TBRF spirochete, B. hermsii and this vital second messenger plays key roles in virulence, motility, and chemotaxis. These studies also pave the way for future investigation of B. hermsii through use of targeted genetic manipulation.

c-di-GMP stellt einen wichtigen Regulator bei der Kontrolle von Motilität, Virulenz und der Biofilmbildung in Bakterien dar. Aufgrund der Vielfalt c-di-GMP-synthetisierender (Diguanylatzyklasen, DGC) bzw. abbauender (Phosphodiesterasen, PDE) Enzyme, c-di-GMP-bindender Effektoren und zellulärer Antworten etablierte sich die Theorie paralleler Regulationsmodule, die sich aus DGC, PDE, Effektor und Zielmolekül zusammensetzen und spezifische Prozesse wie die Cellulose-Synthese kontrollieren. Während die Aktivierung dieser durch c-di-GMP bereits verstanden und damit Effektor und Zielmolekül bekannt sind, konnten dem Modul bisher keine spezifisch wirkenden DGCs und PDEs zugeordnet werden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die Proteine DgcC und PdeK in E. coli spezifisch auf die Cellulose-Synthese in Makrokolonie-Biofilmen wirken und ein solches c-di-GMP-Modul bilden. Beide sind aktiv an der Umsetzung von c-di-GMP beteiligt und mittels Interaktionen Teil eines Multi-Protein-Komplexes, der auch die Cellulose-Synthase-Einheiten BcsA und BcsB umfasst. Aufgrund dieser Co-Lokalisation können DgcC und PdeK die c-di-GMP-Konzentration in unmittelbarer Umgebung zum c-di-GMP-bindenden Effektor BcsA kontrollieren. DgcC-PdeK somit stellen das erste Regulationsmodul dar, welches durch lokalisierte Synthese und Abbau von c-di-GMP wirkt und dessen Spezifität aufgrund multipler Protein-Interaktionen gewährleistet wird. 2011 kam es in Mitteleuropa zu einem schweren Ausbruch von Shiga-Toxin-produzierenden E. coli O104:H4, in dessen Verlauf fast 4000 Menschen erkrankten und etwa 20% ein Hämolytisch-urämisches Syndrom entwickelten. Die in dieser Arbeit erlangten Ergebnisse legen nahe, dass die einzigartige Kombination aus Toxin-Produktion und Biofilm-assoziierter Eigenschaften – das Potential zur c-di-GMP-Akkumulation (TL Povolotsky), verstärkte CsgD-Synthese und vor allem keine Cellulose-Produktion– möglicherweise zur erhöhten Virulenz dieses E. coli O104:H4 beitragen.
c-di-GMP represents an important regulator in the control of motility, virulence and biofilm formation. Due to the multiplicity of c-di-GMP-forming (diguanylate cyclases, DGCc) and -degrading (phosphodiesterases, PDEs) enzymes, c-di-GMP-binding effectors and cellular outputs, the theory of parallel existing c-di-GMP-regulation modules was established. Such a module consists of a DGC, a PDE, an effector and a target controlling a specific cellular output such as cellulose synthesis. Whereas the activation of cellulose synthesis is well understood, and therefore effector and target molecule are known, so far no DGC or PDE has been associated with the cellulose-specific c-di-GMP-module. Within the framework of this work it was shown that DgcC and PdeK act specifically on the regulation of cellulose synthesis in macrocolony biofilms, thus forming a c-di-GMP module. Both proteins are enzymatically active concerning c-di-GMP metabolism and through protein-interactions part of a multi-protein-complex, which includes the cellulose synthase-subunits BcsA and BcsB, too. Due to this co-localisation DgcC and PdeK can control the c-di-GMP-concentration in close proximity to the c-di-GMP-binding cellulose-synthase BcsA. Therefore, DgcC-PdeK represents the first signalling module, which acts through local c-di-GMP-synthesis and -degradation and controls specifically cellulose because of multiple protein-interactions with the synthase-complex. In 2011 a serious outbreak of shiga toxin producing E. coli O104:H4 occurred in Middle Europe with nearly 4000 patients of whom approximately 20% developing haemolytic uraemic syndrome. The results obtained in this study suggest that a unique combination of shiga toxin production and biofilm-associated properties – the potential of c-di-GMP accumulation (see PhD Thesis T. L. Povolotsky), enhanced CsgD-synthesis at 37°C and especially no cellulose production – potentially contribute to the enhanced virulence of E. coli O104:H4.

Legionella pneumophila est une bactérie aquatique qui prolifère en se répliquant à l’intérieur de cellules amibiennes. Elle peut persister dans ces environnements en vivant en communauté sous forme de biofilms. L’inhalation par l’Homme d’eau contaminée, vaporisée par les réseaux d’eau chaude ou les tours aéro‐réfrigérantes, peut mener à l’infection des macrophages pulmonaires qui se traduit par une grave pneumonie appelée légionellose. Le di‐GMP cyclique (diGMPc) est impliqué, chez diverses espèces bactériennes, dans la transition entre les modes de vie mobiles et sessiles, et chez certains pathogènes, dans la régulation de la virulence. Mon travail de thèse vise à démontrer l’implication des voies de signalisation à diGMPc dans le contrôle de la virulence et de la formation de biofilms par L. pneumophila. Cette implication a été étudiée grâce à l’inactivation systématique de chacun des gènes codant les protéines du métabolisme du diGMPc chez la souche L. pneumophila Lens. Notre étude a révélé que trois de ces protéines, Lpl0780, Lpl0922 et Lpl1118, sont spécifiquement requises pour le contrôle de la virulence et, plus particulièrement, pour la survie précoce lors de l’infection de cellules‐hôtes via l’orchestration de la sécrétion de facteurs de virulence dans la cellule‐hôte. Lpl1118 participerait également à la biogénèse de la vacuole de réplication. Cinq autres de ces protéines sont impliquées dans la régulation de la formation et de l’architecture des biofilms. L’une d’elles est, plus particulièrement, requise pour la formation de biofilms en présence d’oxyde nitrique. Ces résultats contribuent à une meilleure compréhension de l’organisation complexe et spécifique des voies de signalisation à diGMPc chez L. pneumophila et pourraient permettre d’envisager une lutte plus efficace contre ce pathogène
Legionella pneumophila is a bacterium that proliferates in fresh water environments through the replication within amoebas. These bacteria can persist in these environments through biofilm formation. The inhalation of aerosolized contaminated water through hot water systems or cooling towers can induce the infection of human lungs, leading to a severe pneumonia called legionellosis. Cyclic di‐GMP (c‐di‐GMP) in involved, in various bacterial species, in the motility‐to‐sessility transition, and in some pathogens, in virulence control. My work aims to demonstrate the involvement of signaling pathways that use c‐di‐GMP in virulence control and biofilm formation of L. pneumophila. This involvement was investigated by systematically inactivating each gene encoding a c‐di‐GMP‐metabolizing enzyme in L. pneumophila Lens strain. Our work revealed that 3 of these proteins, Lpl0780, Lpl0922 and Lpl1118 are specifically involved in virulence control and, particularly, in the early survival during host cell infection through the orchestration of virulence factors secretion within host cell. Lpl1118 is particularly required for replicative vacuole biogenesis. Five other proteins, participate in the formation and architecture of biofilms. One of them is more specifically involved in biofilm formation in the presence of nitric oxide. These results help to better understand the complexity and the specificity of c‐di‐GMP signaling pathways in L. pneumophila and should allow the exploration of more effective ways to fight this pathogen

碩士
中臺科技大學
醫學生物科技研究所
98
Abstract Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger with a role in the regulation of a range of cellular function. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases, the enzymes characterized by the presence of a GGDEF domain, whereas the hydrolysis of c-di-GMP is catalyzed by phosphodiesterases, the enzymes noted for possessing an EAL or a HD-GYP domain. Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The bacterial genome encodes 37 proteins with GGDEF, EAL or HD-GYP domain. In this study, we showed that over-expression of GGDEF domain protein genes Xcc1294 and Xcc2731 or their deletion mutants caused different phenotypic changes when compared with wild type strain. When Xcc1294 was over-expressed or deleted, no significant differences were found including extracellular enzymes, exopolysaccharides, motility, and adhesion. While over-expression of Xcc2731 in wild-type Xcc caused (i) decrease in production of virulence factor extracellular enzymes and exopolysaccharides, (ii) reduction in motility, and (iii) reduction in cell attachment.

博士
國立中興大學
生物化學研究所
100
c-di-GMP is a key signalling molecule involved in regulating many important biological functions in bacteria. The synthesis of c-di-GMP is catalyzed by the GGDEF-domain-containing diguanylate cyclase (DGC), the activity of which is regulated by the binding of product at the allosteric inhibitory (I) site. However, a significant number of GGDEF domains lack the RxxD motif characteristic of the allosteric I site. Here, the structure of XCC4471GGDEF, the GGDEF domain of a DGC from Xanthomonas campestris, in complex with c-di-GMP has been solved. Unexpectedly, the structure of the complex revealed a GGDEF-domain dimer cross-linked by two molecules of c-di-GMP at the strongly conserved active sites. In the complex (c-di-GMP)2 adopts a novel partially intercalated form, with the peripheral guanine bases bound to the guanine-binding pockets and the two central bases stacked upon each other. Alteration of the residues involved in specific binding to c-di-GMP led to dramatically reduced KD values between XCC4471GGDEF and c-di-GMP. In addition, these key residues are strongly conserved among the many thousands of GGDEF-domain sequences identified to date. These results indicate a new product-bound form for GGDEF-domain containing proteins obtained via (c-di-GMP)2 binding at the active site. This novel XCC4471GGDEF–c-di-GMP complex structure may serve as a general model for the design of lead compounds to block the DGC activity of GGDEF-domain containing proteins in X. campestris or other microorganisms that contain multiple GGDEF-domain proteins.

Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to manipulate genetically. An increased knowledge about the physiology of this model organism will facilitate the development of engineered E.coli strains for applications such as production of biofuels and biofilm control. The aims of this work were the application of protein engineering to increase E. coli hydrogen production, the identification of the proteins regulating extracellular DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal 3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation. The Escherichia coli hydrogen production rate was increased 9 fold through random mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) enhances hydrogen production by increasing transcription of the four transcriptional units regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen production 6 fold and 4 fold, respectively. The complete E. coli genome was screened to identify proteins that affect eDNA production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E. coli is related to direct secretion. Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN increased swimming motility and eDNA as expected for low c-di-GMP levels. However, contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30 fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP levels should be reduced for initial biofilm formation because motility is important for initial attachment to a surface.

碩士
國立中興大學
生物化學研究所
100
Cyclic nucleotide second messengers such as cAMP, cGMP, c-di-GMP and c-di-AMP which represent a important cornerstone in signal transduction mechanism of microorganisms.For several decades, bacteria have been known to use cAMP to control a variety of processes, from utilization of alternative sugars to motility and virulence. The bacteria also use cGMP to control the cycst formation to survive in a unfavorable environment. The cyclic dimeric GMP (c-di-GMP) as ubiquitous second messengers that is used by most bacteria to regulate a diverse range of important cellular functions including biofilm formation, motility, and virulence factor production. Cellular levels of c-di-GMP are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), DGCs with the catalytically active part: GGDEF domain which can convert two molecules of guanosine-triphosphate (GTP) into c-di-GMP and, by contrast, PDEs with the EAL and HD-GYP domain which can hydrolytically cleave the c-di-GMP into pGpG or guanosine-monophosphate (GMP). The purpose of this study is to explore the biological functions and structure of the SM0233 protein. The predicted function of SM0233 is the putative cyclic nucleotide-binding and GGDEF domain regulator.We have determined the structure of GGDEF domain of SM0233 from S. maltophilia to a resolution of 1.9 Å. In order to understand the regulatory mechanism of SM0233 between cAMP and cGMP. We use the isothermal titration calorimetry（ITC）to measure of the binding constant between the SM0233﹑cAMP﹑cGMP and c-di-GMP. We found that the KD value of SM0233 bound cGMP or cAMP is 0.42 and 2.8μM. Because the GGDEF domain of SM0233 lack the RxxD motif characteristic of the allosteric inhibit site﹐and therefore can not be combined with c-di-GMP. The pyrophosphate assay data also shows that the requirement for DGC activity of SM0233 proetein must by containing the coiled-coil motif or cNMP binding domain. And we also found in the presence of cAMP and cGMP，the DGC activity of SM0233 is enhanced. Based on the experiment results of isothermal titration calorimetry and pyrophosphate assay﹐SM0233 is a GGDEF protein with the DGC activity. When the intracellular concentration of cAMP or cGMP is enhanced, the second messengers can bind with the cNMP binding domain of SM0233 and increase the DGC activity. According to previous studies, the XC0249 protein can interact with XcRpfG in Xanthomonas campestris. The SM0233 was homologous protein of XC0249 in S. maltophilia through a bioinformatic study. The gel filtration data shows that SmRpfGD81E interacted with the GGDEF domain of SM0233, but how such binding changes the activity of SM0233 remains unknown.

Biofilm is a microbial community whose formation is regulated by the dinucleotide cyclic-di GMP. The GGDEF diguanylate cyclases and EAL or HD-GYP phosphodiesterases control the balance of c-di-GMP. Most of these proteins have N-terminal sensing domains indicating that their activities are modulated by cellular and environmental stimuli. Moreover there are proteins bearing both the GGDEF and EAL or HD-GYP domains named hybrid proteins. The cross-talk between these two domains and with other regulatory domains is crucial to understand the output and the regulation of hybrid proteins. Given that nutrients are among the major driving forces guiding the change of c-diGMP levels, the characterization of the mechanistic details linking nutrients sensing to c-di-GMP homeostasis is very interesting and up to now poorly characterized. The aim of study was to identify a putative protein in P. aeruginosa able to link the nutrient state to c-di-GMP signalling. In particular my project has been focused on the characterization of the Hybrid Protein RmcA (Redox modulator of c-di-GMP A) from Pseudomonas aeruginosa. RmcA is a multidomain transmembrane protein carrying a periplasmic Venus Fly Trap (VFT) sensory domain, a transmembrane helix, three Per-Arnt-Sim (PAS) domain and one light, oxygen, or voltage domain (LOV) and finally the GGDEF-EAL domains.Few structural data are available on Hybrid Protein family of enzymes and these data indicate that the output of these proteins is regulated by a reciprocal allosteric control between the two GGDEF and EAL domains occuring via inter-domain interactions and ligand-induced conformationals changes. In addition, the final output is difficult to predict since it depends also on the interaction between the two domains and the upstream sensing/regulatory ones. During the enzymatic characterization of RmcA, we observed alternative and novel reactivity of the GGDEF domain which has been analysed more deeply, also in comparison with other GGDEFcontaining enzymes. We found that DUAL protein showed an unusual catalytic activity converting GTP to GMP. We therefore investigated whether this unexpected feature is a common feature of different GGDEF containing proteins, including both DGCs and hybrid proteins. This side project has been useful for the overall study presented in this PhD thesis, since it allowed us to establish the best experimental conditions to study this domain.