E. coli and Salmonella enterica have multiple c-di-GMP cyclases and phosphodiesterases. Absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di- GMP-binding protein, for motility inhibition. This study demonstrates that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, with the primary interaction site located within FliM. Interaction of YcgR with these proteins induces a CCW motor bias and reduces the efficiency of torque generation, thus inhibiting both chemotaxis and the speed of movement. In collaboration with David Blair’s group at the University of Utah, we propose a "backstop brake" model showing how both effects of YcgR on the motor can result from an initial disruption of the FliM/FliG interface, followed by destabilization and disorganization of the FliG C-terminal domain, which interacts with the stator protein MotA. Support for this order of events i.e. induction of a CCW bias followed by reduction of torque, is provided for S. enterica motors. Data from single motor analysis show that E. coli and S. enterica motors have inherently different properties, but that YcgR is solely responsible for disruption of motor function in both bacteria. This study also finds that E. coli and S. enterica employ c-di-GMP in additional and different pathways to impede motility. Inhibition of motility and chemotaxis may represent a bacterial strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed. / text

Xylella fastidiosa is an important bacterial plant pathogen that colonizes the xylem of hundreds of plant species. X. fastidiosa cause Pierce's disease in grapevine by occlusion of the xylem by extensive bacterial colonization, extracellular polysaccharides and the formation of a biofilm. These traits are mediated in a cell-density manner by a cell-to-cell signaling system that transduces a diffusible signaling factor (DSF). This dissertation demonstrates that PD1994, PD1617 and RpfG regulate important traits for bacterial virulence such as cell-cell signaling, biofilm formation and cell aggregation. X. fastidiosa strains harboring mutations in pd1994 (which encodes for a defective GGDEF- EAL-domain protein) and in pd1617 (which encodes for a EAL-domain protein) have increased growth rate, increased biofilm formation, increased plant colonization and decreased cell aggregation. Gene expression analysis of the pd1994 mutant strain showed overexpression of rpfF, which is a DSF synthase, suggesting that PD1994 regulates DSF signaling by repressing rpfF expression. Additionally, the pd1994mutant showed overexpression of pd1617 and rpfG (with EAL and HD-GYP domains respectively, that may be responsible for c-di-GMP turnover), which suggested that this mutant may have low c-di-GMP levels and that PD1994 regulates c-di-GMP turnover by repression of RpfG activity and PD1617 gene expression. X. fastidiosa harboring a mutation on rpfG exhibited decreased biofilm formation while it had no effect in growth or cell aggregation. Together, these results suggest that PD1994, PD1617 and RpfG regulate the DSF regulatory network by controlling the turnover of the second messenger c-di-GMP.

Acinetobacter baumannii is an increasingly multidrug-resistant Gram-negative bacterial pathogen and contributes to many hospital-acquired infections. Discovering new treatments against Acinetobacter baumannii infections is necessary as the pathogen adapts to the antimicrobials prescribed by physicians. Cyclic di-GMP (c-di-GMP), a bacterial second messenger, can regulate various phenotypes including biofilm formation, desiccation tolerance, motility, etc.; many of these phenotypes may help A. baumannii better survive a hospital environment, such as dryness on hospital surfaces. Up to twelve c-di-GMP modulating enzymes (CMEs) and two c-di-GMP binding proteins are predicted to be encoded by this pathogen. Diguanylate cyclases (DGCs) produce c-di-GMP, whereas phosphodiesterases (PDEs) degrade c-di-GMP. More c-di-GMP that can bind to its binding proteins means more biofilm formation and less motility. Of the eleven CMEs, 7 are DGCs, 2 are PDEs, and 3 encode both domains (DGCs/PDEs). I hypothesized that biofilm formation, desiccation tolerance, and motility were controlled by c-di-GMP and that we could target these parts of the c-di-GMP signaling network for new treatments. If we disrupt these genes, then we should see a reduction in the regulatory effects of these phenotypes. In this investigation, we generated mutants with a single gene knockout or transposon mutagenesis in two different A. baumannii strains: 17978, a historical laboratory strain that exhibits swarming motility and AB5075, a recent clinical isolate that exhibits twitching motility. To test biofilm formation, we let the mutants grow to their maximum concentration in 96-well plates, stained the plates with crystal violet, and quantified the crystal violet that stained the biofilm. To test for motility, a LB agar plate was stabbed to the plastic surface or dropped on the agar surface with diluted culture to determine the presence of twitching or swarming motility, respectively. To test for desiccation tolerance, we washed the cultures in distilled water to rid the sample of any salt, serially diluted the samples in solution, and plated them out onto LB agar plates. Bacterial counts were quantified before and after desiccation to determine survival of each mutant. From these experiments, 6 DGCs, 1 PDE, and 2 DGCs/PDEs were shown to regulate biofilm formation in AB5075. Furthermore, a PDE and a DGC/PDE were shown to regulate twitching motility in AB5075, while a single DGC was required for tolerating dryness. In strain 17978, we have found a PDE and 4 DGCs that are necessary for swarming motility and are currently conducting biofilm and desiccation tolerance assays. So far, we’ve identified a role for c-di-GMP in A. baumannii biofilm formation, motility, and desiccation survival. Inhibiting the regulation of these pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

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.

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.

Des conditions de carence entrainent une mort cellulaire développementale chez le protiste Dictyostelium discoideum. Dans un système in vitro, des cellules de Dictyostelium sont mises en conditions de carence, puis l'addition des inducteurs DIF-1 ou c-di-GMP conduit à une mort cellulaire vacuolaire. DIF-1 est un polyketide produit par Dictyostelium et induisant la différenciation des cellules pré-tiges. Le dinucléotide cyclique c-di-GMP était connu comme un second messager chez les procaryotes, et comme un déclencheur de l'immunité innée dans des cellules de mammifères. Il a été montré par d'autres que des cellules de Dictyostelium peurent produire et détecter c-di-GMP.Pour analyser la signalisation par c-di-GMP chez Dictyostelium, nous avons utilisé la mutagénèse aléatoire et la mutagénèse ciblée. En utilisant des mutants inactivant stlB ou dmtA, nous avons démontré que DIF-1 endogène ou exogène est nécessaire pour la signalisation par c-di-GMP dans Dictyostelium. En conséquence, nous avons amélioré l'étape de sélection dans une mutagenèse aléatoire en utilisant c-di-GMP et un peu de DIF-1 comme inducteurs, ce qui a produit plusieurs mutants. Par ailleurs j’ai testé par mutagenèse ciblée des hypothèses basées sur les informations connues dans Dictyostelium ou d'autres types de mort cellulaire. Trois molécules ont été essayées, DDX41 comme récepteur putatif de c-di-GMP, l' uniport mitochondrial pour le Ca2+(MCU) et la Na+/K+ATPase (IonA).En résumé, au cours de ma thèse, nous avons démontré une relation entre la signalisation c-di-GMP et a signalisation DIF-1 dans Dictyostelium et identifié plusieurs nouvelles molécules de la mort cellulaire par mutagenèse aléatoire. / The protist Dictyostelium discoideum undergoes development cell death when under starvation. To investigate the molecular mechanism of Dictyostelium cell death, an in vitro system has been used. Dictyostelium cells were starved and then cell death was induced by DIF-1 or c-di-GMP. About 40h after induction, cells underwent vacuolar cell death. DIF-1 is a polyketide, produced by Dictyostelium prespore cells, which induces prestalk cell differentiation. c-di-GMP was well known not only as a second messenger produced and sensed by bacteria but also as a trigger of innate immunity in mammalian cells. Dictyostelium was recently found by another laboratory to produce and sense c-di-GMP. To analyze c-di-GMP signaling in Dictyostelium cell death, we used random mutagenesis and targeted mutagenesis. By using the knockout mutants stlB- and dmtA-, we demonstrated that endogenous or exogenous DIF-1 is required for c-di-GMP signaling in Dictyostelium. In contrast, endogenous c-di-GMP is not necessary for exogenous DIF-1 signaling. As a consequence, we improved the selection step in random mutagenesis by using c-di-GMP and a little DIF-1 as inducers, which produced several mutants. Another part of my project was to test by targeted mutagenesis some hypotheses, based on known information in Dictyostelium or other similar cell death types. Three molecules have been tested, the c-di-GMP putative receptor DDX41, the mitochondrial Ca2+ uniporter (MCU) and the Na+/K+-ATPase (IonA).In summary, during my thesis, we have demonstrated a relation between c-di-GMP signaling and DIF-1 signaling in Dictyostelium and identified several new cell death molecules by random mutagenesis.

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.

Bei den meisten Bakterien wird die Biofilmbildung durch das Botenmolekül c-di-GMP stimuliert. Durch die enzymatische Aktivität von c-di-GMP-synthetisierenden Diguanylatzyklasen/DGC und c-di-GMP-abbauenden Phosphodiesterasen/PDE wird der c-di-GMP-Gehalt als eine Antwort auf diverse Stress- und suboptimale Umweltbedingungen reguliert. Vor allem Gram-negative Bakterien haben multiple DGC/PDE. So besitzt Escherichia coli K-12 29 solche Proteine, darunter 12 DGC, 13 PDE sowie 4 degenerierte Proteine ohne enzymatische Funktion. Dieses komplexe c-di-GMP-Kontrollsystem reguliert die Produktion der extrazellulären Biofilmmatrix, die in E. coli während des Übergangs in die stationäre Wachstumsphase stattfindet. Das Ziel dieser Arbeit war es zu untersuchen, ob die 29 DGC/PDE von E. coli ein spezifisches Interaktom bilden, das DGC/PDE-Pärchen enthält. Durch umfangreiche Two-Hybrid-Untersuchungen konnte gezeigt werden, dass es ein solches Interaktom in der Biofilmregulationskaskade tatsächlich gibt, das allerdings nicht in Pärchen organisiert ist. Vielmehr wird die Biofilmbildung von einer Kerngruppe von Enzymen, welche multiple Interaktionen untereinander und mit anderen DGC/PDE aufweisen, kontrolliert. Die Funktionsweise der Kerngruppe von Enzymen könnte jedoch möglicherweise unter bestimmten Wachstumsbedingungen durch Interaktionen mit weiteren Proteinen moduliert werden. Die Dynamik des Interaktionsnetzwerks ermöglicht vermutlich eine rationelle Ressourcenverwaltung in den verschiedenen Zonen des Biofilms, was zum Aufbau der komplexen Matrixarchitektur beitragen könnte, und eine hohe Anpassungsfähigkeit der Bakterien und der von ihnen aufgebauten Biofilmstrukturen gewährleisten könnte. Insgesamt führt diese Arbeit aus einer systemischen Perspektive zu einem neuen Modell der lokalen Biofilmbildungsregulation durch den Botenstoff c-di-GMP und legt die Basis für weitere Untersuchungen der daran beteiligten Mechanismen einzelner GGDEF/EAL-Domäne-haltiger Proteine in E. coli. / The messenger molecule c-di-GMP stimulates the formation biofilms in most bacteria species. The enzymatic activities of the diguanylate cyclases/DGC and the phosphodiesterases/PDE adjust the c-di-GMP content in response to diverse stress and suboptimal environmental conditions. Above all, Gram-negative bacteria have multiple GGDEF/EAL domain proteins. Escherichia coli K-12 possesses 29 of such proteins: 12 DGCs, 13 PDEs and 4 so called degenerate proteins without any enzymatical function. Mainly, this complex c-di-GMP control system regulates the production of the extracellular biofilm matrix, which in E. coli takes place during the switch into the stationary growth phase. The main compounds of the matrix are amyloid curli-fibers and exoplysaccaride cellulose. The goal of this work was to investigate, whether the 29 DGC/PDE from E. coli develop a specific interactome containing additional DGC/PDE pairs. In a comprehensive two-hybrid study, it could be demonstrated that there is indeed a specific interactome in the biofilm formation cascade. However, this interactome does not contain additional DGC/PDE pairs. Mainly, the core group of enzymes, which have multiple interactions among each other and with other DGC/PDE, controls biofilm formation. Under certain growth conditions the mode of action of the core enzymes might be adjusted through the interaction with other proteins. Presumably, the dynamics of the interaction network allows managing the resources in the different biofilm zones efficiently, which could conribute to the complex organisation of the matrix architecture. Therefore, the rapid adaptation of bacteria and the formed biofilm structures could be better organized. Altogether, this work provides a new model for the local regulation of the biofilm formation by the secondary messenger c-di-GMP guided from a systemical perspective. Hereby, the basis for further investigations on regulation mechanisms of individual DGC/PDE was set. / Переход от подвижного и планктонообразного образа жизни к формированию биоплёнок является важной и интересной особенностью различных микроорганизмов. Кишечная палочка (Escherichia coli) представляет собой удобный модельный организм для изучения подобных трансформаций. У этой грамотрицательной бактерии образование биоплёнки обусловлено внутриклеточной аккумуляцией циклического дигуанилата (цикло-диГМФ). Известно, что активность ферментов, синтезирующих (дигуанилатциклазы/ДГЦ) и разлагающих (диэстеразы/ДЭ) эти сигнальные молекулы, меняется в ответ на стрессовые и субоптимальные раздражители. Кишечная палочка имеет 12 ДГЦ, 13 ДЭ и четыре дегенерированных протеина. Цель данной работы – изучить специфический, важный при формировании биоплёнки интерактом и выяснить, способны ли другие ДГЦ/ДЭ образовывать дополнительные ДГЦ/ДЕ модули и вносить свой вклад в формирование биоплёнки. В работе были изучены молекулярные взаимодействия, ответственные за формирование биоплёнок у кишечной палочки. Так, было доказано отсутствие в интерактоме дополнительных локальных ДГЦ/ДЕ модулей, участвующих при каскадных процессах регуляции роста биоплёнки. Установлено, что процесс формирования биоплёнки в большей степени контролируется основной группой ферментов, которые имеют множественные взаимодействия между собой и с другими ДГЦ/ДЭ. Вероятнее всего, такие взаимодействия способны модулировать работу основных ферментов при определенных условиях культивирования. Динамика подобной сети взаимодействий позволяет микроорганизмам целесообразно использовать свои клеточные ресурсы при образовании биоплёнок и вносит свой вклад в её сложную архитектуру, повышая тем самым приспособляемость бактерий и созданных ими сложных биоплёночных структур к внешним условиям. В целом, в данной работе предложена новая модель локальной регуляции образования биоплёнки с помощью сигнальной молекулы цикло-диГМФ и заложен фундамент для дальнейших исследований механизмов действия отдельных ДГЦ/ДЭ.

Recentemente, o bis-(3\',5\')-di-guanosina monofosfato cíclico (c-di-GMP) surgiu como uma importante molécula sinalizadora nas bactérias. Essa molécula foi identificada como uma das responsáveis pelo controle do comportamento bacteriano e está relacionada com a patogenicidade e a adaptação de diversas bactérias, coordenando a expressão de genes envolvidos com virulência, motilidade e formação de biofilme. O mecanismo pelo qual c-diGMP atua vem sendo motivo de estudo de vários grupos de pesquisa nos últimos anos. Já foi demonstrado o papel dessa molécula em diferentes etapas do controle da expressão gênica. Acredita-se que a manipulação dos níveis de c-di-GMP pode ser uma nova abordagem terapêutica contra bactérias patogênicas. Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que atua como um patógeno oportunista, causando infecções em pacientes imunocomprometidos, sendo o maior causador de infecções crônicas em pacientes portadores de fibrose cística. O genoma de P. aeruginosa PA14 apresenta vários genes que codificam proteínas envolvidas no metabolismo e/ou ligação de c-di-GMP, o que pode indicar um amplo papel regulatório deste nucleotídeo nessa bactéria. Uma associação infundada entre níveis elevados de c-di-GMP e a resistência aos antibióticos é geralmente assumida, já que altos níveis de c-di-GMP levam à formação de biofilme, que é comprovadamente um modo de crescimento mais resistente. Nesse trabalho, utilizando uma abordagem proteômica, mostramos que Pseudomonas aeruginosa PA14 regula a expressão de cinco porinas em resposta a variações nos níveis de c-di-GMP, independentemente dos níveis de mRNA. Uma dessas porinas, OprD, é responsável pela entrada do antibiótico β-lactâmico imipenem na célula e é menos abundante em condições de alto c-di-GMP. Também demonstramos que linhagens com altos níveis de c-di-GMP apresentam uma vantagem competitiva de crescimento em relação a linhagens com níveis mais baixo de c-di-GMP quando crescidas em meio contendo imipenem. Em contraste, observamos que células planctônicas com elevados níveis c-di-GMP são mais sensíveis a tobramicina. Em conjunto, estes resultados mostram que c-di-GMP pode regular a resistência a antibióticos em sentidos opostos, e independentemente do crescimento em biofilme / Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3\'-5\'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this dinucleotide emerged as an important intracellular signal molecule controlling bacterial behavior. Diverse molecular mechanisms have been described as targets for c-di-GMP, but several questions remain to be addressed. An association between high c-di-GMP levels and antibiotic resistance is largely assumed, since high c-di-GMP upregulates biofilm formation and the biofilm mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Pseudomonas aeruginosa is a versatile gamma-proteobacterium that behaves as an opportunistic pathogen to a broad range of hosts. The ability of P. aeruginosa to form biofilms contributes to its virulence and adaptation to different environments. The P. aeruginosa PA14 genome presents several genes encoding proteins involved in metabolism or binding to c-di-GMP, which may indicate a wide regulatory role of this nucleotide in this bacterium. Here, using a proteomic approach, we show that Pseudomonas aeruginosa PA14 regulates the amount of five porins in response to c-di-GMP levels, irrespective of their mRNA levels. One of these porins is OprD, decreased in high c-di-GMP conditions, which is responsible for the uptake of the β-lactam antibiotic imipenem. We also demonstrate that this difference leads strains with high c-di-GMP to be more resistant to imipenem even when growing as planktonic cells, giving them a competitive advantage over cells with low c-di-GMP. Contrastingly, we found that planktonic cells with high c-di-GMP levels are more sensitive to aminoglycosides antibiotics. Together, these findings show that c-di-GMP levels can regulate the antibiotic resistance to different drugs in opposite ways and irrespective of a biofilm mode of growth.

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.