Academic literature on the topic 'GGDEF'

ABSTRACT Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

ABSTRACT Proteins containing GGDEF domains are encoded in the majority of sequenced bacterial genomes. In several species, these proteins have been implicated in biosynthesis of exopolysaccharides, formation of biofilms, establishment of a sessile lifestyle, surface motility, and regulation of gene expression. However, biochemical activities of only a few GGDEF domain proteins have been tested. These proteins were shown to be involved in either synthesis or hydrolysis of cyclic-bis(3′→5′) dimeric GMP (c-di-GMP) or in hydrolysis of cyclic AMP. To investigate specificity of the GGDEF domains in Bacteria, six GGDEF domain-encoding genes from randomly chosen representatives of diverse branches of the bacterial phylogenetic tree, i.e., Thermotoga, Deinococcus-Thermus, Cyanobacteria, spirochetes, and α and γ divisions of the Proteobacteria, were cloned and overexpressed. All recombinant proteins were purified and found to possess diguanylate cyclase (DGC) activity involved in c-di-GMP synthesis. The individual GGDEF domains from two proteins were overexpressed, purified, and shown to possess a low level of DGC activity. The oligomeric states of full-length proteins and individual GGDEF domains were similar. This suggests that GGDEF domains are sufficient to encode DGC activity; however, enzymatic activity is highly regulated by the adjacent sensory protein domains. It is shown that DGC activity of the GGDEF domain protein Rrp1 from Borrelia burgdorferi is strictly dependent on phosphorylation status of its input receiver domain. This study establishes that majority of GGDEF domain proteins are c-di-GMP specific, that c-di-GMP synthesis is a wide-spread phenomenon in Bacteria, and that it is highly regulated.

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

ABSTRACT GGDEF and EAL domain proteins are involved in turnover of the novel secondary messenger cyclic di(3′→5′)-guanylic acid (c-di-GMP) in many bacteria. The rdar morphotype, a multicellular behavior of Salmonella enterica serovar Typhimurium characterized by the expression of the extracellular matrix components cellulose and curli fimbriae is controlled by c-di-GMP. In this work the roles of the EAL and GGDEF-EAL domain proteins on rdar morphotype development were investigated. Knockout of four of 15 EAL and GGDEF-EAL domain proteins upregulated rdar morphotype expression and expression of CsgD, the central regulator of the rdar morphotype, and partially downregulated c-di-GMP concentrations. More-detailed analysis showed that the EAL domain protein STM4264 and the GGDEF-EAL domain protein STM1703, which highly downregulated the rdar morphotype, have overlapping yet distinct functions. Another subset of EAL and GGDEF-EAL domain proteins influenced multicellular behavior in liquid culture and flagellum-mediated motility. Consequently, this work has shown that several EAL and GGDEF-EAL domain proteins, which act as phosphodiesterases, play a determinative role in the expression level of multicellular behavior of Salmonella enterica serovar Typhimurium.

Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3′-5′)-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria–Bertani medium, with 15 being under σ S control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 °C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5′-phosphoguanylyl-(3′-5′)-guanosine in these strains revealed that overexpression of most genes promoted modest 1–10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

ABSTRACT In this work, we describe a new gene controlling lateral flagellar gene expression. The gene encodes ScrG, a protein containing GGDEF and EAL domains. This is the second GGDEF-EAL-encoding locus determined to be involved in the regulation of swarming: the first was previously characterized and named scrABC (for “swarming and capsular polysaccharide regulation”). GGDEF and EAL domain-containing proteins participate in the synthesis and degradation of the nucleotide signal cyclic di-GMP (c-di-GMP) in many bacteria. Overexpression of scrG was sufficient to induce lateral flagellar gene expression in liquid, decrease biofilm formation, decrease cps gene expression, and suppress the ΔscrABC phenotype. Removal of its EAL domain reversed ScrG activity, converting ScrG to an inhibitor of swarming and activator of cps expression. Overexpression of scrG decreased the intensity of a 32P-labeled nucleotide spot comigrating with c-di-GMP standard, whereas overexpression of scrG Δ EAL enhanced the intensity of the spot. Mutants with defects in scrG showed altered swarming and lateral flagellin production and colony morphology (but not swimming motility); furthermore, mutation of two GGDEF-EAL-encoding loci (scrG and scrABC) produced cumulative effects on swarming, lateral flagellar gene expression, lateral flagellin production and colony morphology. Mutant analysis supports the assignment of the primary in vivo activity of ScrG to acting as a phosphodiesterase. The data are consistent with a model in which multiple GGDEF-EAL proteins can influence the cellular nucleotide pool: a low concentration of c-di-GMP favors surface mobility, whereas high levels of this nucleotide promote a more adhesive Vibrio parahaemolyticus cell type.

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.