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‣ Contribution to the functional analysis of genes involved in cyclic GMP signaling in bacteria of the Burkholderia cepacia complex

Alves, Sofia Patrão Neves, 1989-
Fonte: Universidade de Lisboa Publicador: Universidade de Lisboa
Tipo: Dissertação de Mestrado
Publicado em //2013 Português
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Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2013; O complexo Burkholderia cepacia (Bcc) é constituído actualmente por 18 espécies de bactérias patogénicas oportunistas, capazes de causar infecções respiratórias graves e por vezes letais doentes com fibrose quística (FQ). A FQ é uma doença genética autossómica recessiva mais comum em caucasianos e é causada por mutações no gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), que está localizado no cromossoma 7 e codifica uma proteína de 1480 aminoácidos que funciona como canal transportador de iões cloreto (Cl-) nas membranas epiteliais. Vários estudos têm sido realizados com o objectivo de entender quais os mecanismos moleculares associados aos processos de colonização e persistência destas bactérias nos doentes com FQ. Normalmente, os organismos pertencentes ao Bcc começam por colonizar o tracto respiratório dos doentes com FQ, podendo em alguns casos invadir o epitélio e sobreviver dentro de células epiteliais e de macrófagos. As infecções causadas pelo Bcc em doentes com FQ têm um desenvolvimento clinico imprevisível, variando desde assintomático até ao “síndroma cepacia”...

‣ Control of Formation and Cellular Detachment from Shewanella oneidensis MR-1 Biofilms by Cyclic di-GMP

Thormann, Kai M.; Duttler, Stefanie; Saville, Renee M.; Hyodo, Mamoru; Shukla, Soni; Hayakawa, Yoshihiro; Spormann, Alfred M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2006 Português
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Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3′,5′)guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a ΔmxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

‣ DgrA is a member of a new family of cyclic diguanosine monophosphate receptors and controls flagellar motor function in Caulobacter crescentus

Christen, Matthias; Christen, Beat; Allan, Martin G.; Folcher, Marc; Jenö, Paul; Grzesiek, Stephan; Jenal, Urs
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Bacteria are able to switch between two mutually exclusive lifestyles, motile single cells and sedentary multicellular communities that colonize surfaces. These behavioral changes contribute to an increased fitness in structured environments and are controlled by the ubiquitous bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP). In response to changing environments, fluctuating levels of c-di-GMP inversely regulate cell motility and cell surface adhesins. Although the synthesis and breakdown of c-di-GMP has been studied in detail, little is known about the downstream effector mechanisms. Using affinity chromatography, we have isolated several c-di-GMP-binding proteins from Caulobacter crescentus. One of these proteins, DgrA, is a PilZ homolog involved in mediating c-di-GMP-dependent control of C. crescentus cell motility. Biochemical and structural analysis of DgrA and homologs from C. crescentus, Salmonella typhimurium, and Pseudomonas aeruginosa demonstrated that this protein family represents a class of specific diguanylate receptors and suggested a general mechanism for c-di-GMP binding and signal transduction. Increased concentrations of c-di-GMP or DgrA blocked motility in C. crescentus by interfering with motor function rather than flagellar assembly. We present preliminary evidence implicating the flagellar motor protein FliL in DgrA-dependent cell motility control.

‣ Cyclic Di-GMP Stimulates Protective Innate Immunity in Bacterial Pneumonia▿

Karaolis, David K. R.; Newstead, Michael W.; Zeng, Xianying; Hyodo, Mamoru; Hayakawa, Yoshihiro; Bhan, Urvhashi; Liang, Hallie; Standiford, Theodore J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
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Innate immunity is the primary mechanism by which extracellular bacterial pathogens are effectively cleared from the lung. We have previously shown that cyclic di-GMP (c-di-GMP [c-diguanylate]) is a novel small molecule immunomodulator and immunostimulatory agent that triggers protective host innate immune responses. Using a murine model of bacterial pneumonia, we show that local intranasal (i.n.) or systemic subcutaneous (s.c.) administration of c-di-GMP prior to intratracheal (i.t.) challenge with Klebsiella pneumoniae stimulates protective immunity against infection. Specifically, i.n. or s.c. administration of c-di-GMP 48 and 24 h prior to i.t. K. pneumoniae challenge resulted in significantly increased survival. Pretreatment with c-di-GMP resulted in a 5-fold reduction in bacterial CFU in the lung (P < 0.05) and an impressive >1,000-fold decrease in CFU in the blood (P < 0.01). c-di-GMP administration stimulated a robust innate response to bacterial challenge, characterized by enhanced accumulation of neutrophils and αβ T cells, as well as activated NK and αβ T lymphocytes, which was associated with earlier and more vigorous expression of chemokines and type I cytokines. Moreover, lung macrophages recovered from Klebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase and nitric oxide ex vivo than did macrophages isolated from infected mice pretreated with the control...

‣ A cyclic-di-GMP receptor required for bacterial exopolysaccharide production

Lee, Vincent T; Matewish, Jody M; Kessler, Jennifer L; Hyodo, Mamoru; Hayakawa, Yoshihiro; Lory, Stephen
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Publicado em /09/2007 Português
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Bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been shown to be a global regulatory molecule that modulates the reciprocal responses of bacteria to activate either virulence pathways or biofilm formation. The mechanism of c-di-GMP signal transduction, including recognition of c-di-GMP and subsequent phenotypic regulation, remain largely uncharacterized. The key components of these regulatory pathways are the various adaptor proteins (c-di-GMP receptors). There is compelling evidence suggesting that, in addition to PilZ domains, there are other unidentified c-di-GMP receptors. Here we show that the PelD protein of Pseudomonas aeruginosa is a novel c-di-GMP receptor that mediates c-di-GMP regulation of PEL polysaccharide biosynthesis. Analysis of PelD orthologues identified a number of conserved residues that are required for c-di-GMP binding as well as synthesis of the PEL polysaccharide. Secondary structure similarities of PelD to the inhibitory site of diguanylate cyclase suggest that a common fold can act as a platform to bind c-di-GMP. The combination of a c-di-GMP binding site with a variety of output signalling motifs within one protein domain provides an explanation for the specificity for different cellular responses to this regulatory dinucleotide.

‣ Vibrio parahaemolyticus ScrC Modulates Cyclic Dimeric GMP Regulation of Gene Expression Relevant to Growth on Surfaces▿

Ferreira, Rosana B. R.; Antunes, Luis Caetano M.; Greenberg, E. Peter; McCarter, Linda L.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type. scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities, and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.

‣ Role of Cyclic Di-GMP during El Tor Biotype Vibrio cholerae Infection: Characterization of the In Vivo-Induced Cyclic Di-GMP Phosphodiesterase CdpA▿

Tamayo, Rita; Schild, Stefan; Pratt, Jason T.; Camilli, Andrew
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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In Vibrio cholerae, the second messenger cyclic di-GMP (c-di-GMP) positively regulates biofilm formation and negatively regulates virulence and is proposed to play an important role in the transition from persistence in the environment to survival in the host. Herein we describe a characterization of the infection-induced gene cdpA, which encodes both GGDEF and EAL domains, which are known to mediate diguanylate cyclase and c-di-GMP phosphodiesterase (PDE) activities, respectively. CdpA is shown to possess PDE activity, and this activity is regulated by its inactive degenerate GGDEF domain. CdpA inhibits biofilm formation but has no effect on colonization of the infant mouse small intestine. Consistent with these observations, cdpA is expressed during in vitro growth in a biofilm but is not expressed in vivo until the late stage of infection, after colonization has occurred. To test for a role of c-di-GMP in the early stages of infection, we artificially increased c-di-GMP and observed reduced colonization. This was attributed to a significant reduction in toxT transcription during infection. Cumulatively, these results support a model of the V. cholerae life cycle in which c-di-GMP must be down-regulated early after entering the small intestine and maintained at a low level to allow virulence gene expression...

‣ Cyclic-di-GMP Regulates Extracellular Polysaccharide Production, Biofilm Formation, and Rugose Colony Development by Vibrio vulnificus▿

Nakhamchik, Alina; Wilde, Caroline; Rowe-Magnus, Dean A.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3′-5′)-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However...

‣ The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase ▿ †

Rao, Feng; Qi, Yaning; Chong, Hui Shan; Kotaka, Masayo; Li, Bin; Li, Jinming; Lescar, Julien; Tang, Kai; Liang, Zhao-Xun
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (β/α)8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (Km) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in Km correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains...

‣ YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity*

Rao, Feng; See, Rui Yin; Zhang, Dongwei; Toh, Delon Chengxu; Ji, Qiang; Liang, Zhao-Xun
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5′-pApA and 5′-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (Km) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ΔyybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.

‣ YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

Malone, Jacob G.; Jaeger, Tina; Spangler, Christian; Ritz, Daniel; Spang, Anne; Arrieumerlou, Cécile; Kaever, Volkhard; Landmann, Regine; Jenal, Urs
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong...

‣ CdpA Is a Burkholderia pseudomallei Cyclic di-GMP Phosphodiesterase Involved in Autoaggregation, Flagellum Synthesis, Motility, Biofilm Formation, Cell Invasion, and Cytotoxicity ▿

Lee, Hwee Siang; Gu, Feiyu; Ching, Shi Min; Lam, Yulin; Chua, Kim Lee
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Cyclic diguanylic acid (c-di-GMP) is an intracellular signaling molecule involved in regulation of cellular functions such as motility, biofilm formation and virulence. Intracellular level of c-di-GMP is controlled through opposing diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities of GGDEF and EAL domain proteins, respectively. We report the identification and characterization of cdpA, a gene encoding a protein containing an EAL domain in the Gram-negative soil bacillus and human pathogen Burkholderia pseudomallei KHW. Purified recombinant CdpA protein exhibited PDE activity in vitro. Evidence that CdpA is a major c-di-GMP-specific PDE in B. pseudomallei KHW was shown by an 8-fold-higher c-di-GMP level in the cdpA-null mutant as compared to the wild type and the complemented cdpA mutant. The presence of higher intracellular c-di-GMP levels in the cdpA-null mutant was associated with increased production of exopolysaccharides, increased cell-to-cell aggregation, absence of flagella and swimming motility, and increased biofilm formation. The relevance of CdpA in B. pseudomallei virulence was demonstrated by a 3-fold reduction in invasion of human lung epithelial cells and a 6-fold reduction in cytotoxicity on human macrophage cells infected with the cdpA mutant.

‣ Identification and molecular characterization of a cyclic-di-GMP effector protein, PlzA (BB0733): additional evidence for the existence of a functional cyclic-di-GMP regulatory network in the Lyme disease spirochete, Borrelia burgdorferi

Freedman, John C.; Rogers, Elizabeth A.; Kostick, Jessica L.; Zhang, Hongming; Iyer, Radha; Schwartz, Ira; Marconi, Richard T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The Borrelia burgdorferi Rrp1 protein is a diguanylate cyclase that controls a regulon consisting of ~10% of the total genome. Because Rrp1 lacks a DNA-binding domain, its regulatory capability is most likely mediated through the production of bis-(3′–5′)-cyclic dimeric GMP (c-di-GMP). C-di-GMP binds to and activates the regulatory activity of proteins that harbor a PilZ domain. The occurrence of a PilZ domain within a protein is not in and of itself sufficient to convey c-di-GMP binding, as other structural aspects of the protein are important in the interaction. In this study, we have assessed the expression and c-di-GMP binding ability of the sole PilZ domain-containing protein of B. burgdorferi B31, PlzA. PlzA was determined to be upregulated by tick feeding and to be expressed during mammalian infection. The gene is highly conserved and present in all Borrelia species. Analyses of recombinant PlzA demonstrated its ability to bind c-di-GMP and site-directed mutagenesis revealed that this interaction is highly specific and dependent on Arg residues contained within the PilZ domain. In summary, this study is the first to identify a c-di-GMP effector molecule in a spirochete and provides additional evidence for the existence of a complete c-di-GMP regulatory network in the Lyme disease spirochete...

‣ Cyclic di-GMP Signaling Regulates Invasion by Ehrlichia chaffeensis of Human Monocytes ▿ †

Kumagai, Yumi; Matsuo, Junji; Hayakawa, Yoshihiro; Rikihisa, Yasuko
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Cyclic di-GMP (c-di-GMP) is a bacterial second messenger produced by GGDEF domain-containing proteins. The genome of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, encodes a single protein that contains a GGDEF domain, called PleD. In this study, we investigated the effects of c-di-GMP signaling on E. chaffeensis infection of the human monocytic cell line THP-1. Recombinant E. chaffeensis PleD showed diguanylate cyclase activity as it generated c-di-GMP in vitro. Because c-di-GMP is not cell permeable, the c-di-GMP hydrophobic analog 2′-O-di(tert-butyldimethylsilyl)-c-di-GMP (CDGA) was used to examine intracellular c-di-GMP signaling. CDGA activity was first tested with Salmonella enterica serovar Typhimurium. CDGA inhibited well-defined c-di-GMP-regulated phenomena, including cellulose synthesis, clumping, and upregulation of csgD and adrA mRNA, indicating that CDGA acts as an antagonist in c-di-GMP signaling. [32P]c-di-GMP bound several E. chaffeensis native proteins and two E. chaffeensis recombinant I-site proteins, and this binding was blocked by CDGA. Although pretreatment of E. chaffeensis with CDGA did not reduce bacterial binding to THP-1 cells, bacterial internalization was reduced. CDGA facilitated protease-dependent degradation of particular...

‣ Systematic analysis of cyclic di-GMP signaling enzymes and their role in biofilm formation and virulence in Yersinia pestis

Bobrov, Alexander G.; Kirillina, Olga; Ryjenkov, Dmitri A.; Waters, Christopher M.; Price, Paul A.; Fetherston, Jacqueline D.; Mack, Dietrich; Goldman, William E.; Gomelsky, Mark; Perry, Robert D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Cyclic di-GMP (c-di-GMP) is a signaling molecule that governs the transition between planktonic and biofilm states. Previously we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-β-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-β-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signaling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signaling network likely resulting from the different disease cycles of these human pathogens.

‣ Tyrosine Phosphatase TpbA of Pseudomonas aeruginosa Controls Extracellular DNA via Cyclic Diguanylic Acid Concentrations

Ueda, Akihiro; Wood, Thomas K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/2010 Português
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Inactivating the tyrosine phosphatase TpbA of Pseudomonas aeruginosa PA14 induces biofilm formation by 150-fold via increased production of the second messenger cyclic diguanylic acid (c-di-GMP). Here, we show the tpbA mutation reduces extracellular DNA (eDNA) and that increased expression of tpbA increases eDNA; hence, eDNA is inversely proportional to c-di-GMP concentrations. Mutations in diguanylate cyclases PA0169, PA4959, and PA5487 and phosphodiesterase PA4781 corroborate this trend. The tpbA mutation also decreases cell lysis while overexpression of tpbA increases cell lysis. To further link c-di-GMP concentrations and eDNA, the gene encoding phosphodiesterase PA2133 was overexpressed which increased eDNA and decreased biofilm formation by decreasing c-di-GMP. Furthermore, the effect of the tpbB mutation along with the tpbA mutation was examined because loss of TpbB restored the phenotypes controlled by enhanced c-di-GMP in the tpbA mutant. The tpbA tpbB double mutations restored eDNA to that of the PA14 wild-type level. These findings suggest that c-di-GMP, rather than TpbA, controls eDNA. Hence, TpbA acts as a positive regulator of eDNA and cell lysis by reducing c-di-GMP concentrations.

‣ The Structure of an Unconventional HD-GYP Protein from Bdellovibrio Reveals the Roles of Conserved Residues in this Class of Cyclic-di-GMP Phosphodiesterases

Lovering, Andrew L.; Capeness, Michael J.; Lambert, Carey; Hobley, Laura; Sockett, R. Elizabeth
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 11/10/2011 Português
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Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins...

‣ Exploring Environmental Control of Cyclic di-GMP Signaling in Vibrio cholerae by Using the Ex Vivo Lysate Cyclic di-GMP Assay (TELCA)

Koestler, Benjamin J.; Waters, Christopher M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2013 Português
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Vibrio cholerae senses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. V. cholerae encodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system in V. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named the ex vivo lysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show that V. cholerae grown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP...

‣ Identification of a cyclic-di-GMP-modulating response regulator that impacts biofilm formation in a model sulfate reducing bacterium

Rajeev, Lara; Luning, Eric G.; Altenburg, Sara; Zane, Grant M.; Baidoo, Edward E. K.; Catena, Michela; Keasling, Jay D.; Wall, Judy D.; Fields, Matthew W.; Mukhopadhyay, Aindrila
Fonte: Frontiers Media S.A. Publicador: Frontiers Media S.A.
Tipo: Artigo de Revista Científica
Publicado em 29/07/2014 Português
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We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn2+-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However...

‣ Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

Roelofs, Kevin G.; Jones, Christopher J.; Helman, Sarah R.; Shang, Xiaoran; Orr, Mona W.; Goodson, Jonathan R.; Galperin, Michael Y.; Yildiz, Fitnat H.; Lee, Vincent T.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 27/10/2015 Português
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Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele...