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‣ c-di-GMP (3′-5′-Cyclic Diguanylic Acid) Inhibits Staphylococcus aureus Cell-Cell Interactions and Biofilm Formation

Karaolis, David K. R.; Rashid, Mohammed H.; Chythanya, Rajanna; Luo, Wensheng; Hyodo, Mamoru; Hayakawa, Yoshihiro
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2005 Português
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Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3′,5′-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillin-resistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms...

‣ The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains†

Schmidt, Andrew J.; Ryjenkov, Dmitri A.; Gomelsky, Mark
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2005 Português
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The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg2+ or Mn2+, strongly inhibited by Ca2+, and independent of protein oligomerization. Linear dimeric GMP was shown to be 5′pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5′pGpG into GMP...

‣ The Cyclic-di-GMP Phosphodiesterase BinA Negatively Regulates Cellulose-Containing Biofilms in Vibrio fischeri▿

Bassis, Christine M.; Visick, Karen L.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
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Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the ΔbinA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together...

‣ Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions

Rogers, Elizabeth A.; Terekhova, Darya; Zhang, Hong-Ming; Hovis, Kelley M.; Schwartz, Ira; Marconi, Richard T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Two-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi, a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase–response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1–Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1, constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome...

‣ Structural insight into the mechanism of cyclic di-GMP hydrolysis by EAL domain phosphodiesterases

Tchigvintsev, Anatoli; Xu, Xiaohui; Singer, Alexander; Chang, Changsoo; Brown, Greg; Proudfoot, Michael; Cui, Hong; Flick, Robert; Anderson, Wayne F.; Joachimiak, Andrzej; Galperin, Michael Y.; Savchenko, Alexei; Yakunin, Alexander F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

‣ Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

Merritt, Judith H.; Ha, Dae-Gon; Cowles, Kimberly N.; Lu, Wenyun; Morales, Diana K.; Rabinowitz, Joshua; Gitai, Zemer; O’Toole, George A.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 19/10/2010 Português
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The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa.

‣ STING is a direct innate immune sensor of cyclic-di-GMP

Burdette, Dara L.; Monroe, Kathryn M.; Sotelo-Troha, Katia; Iwig, Jeff S.; Eckert, Barbara; Hyodo, Mamoru; Hayakawa, Yoshihiro; Vance, Russell E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/09/2011 Português
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The innate immune system detects infection by employing germline-encoded receptors specific for conserved microbial molecules. Recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFN), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of IFN induction1,2, and requires signaling via Tank Binding Kinase 1 (TBK1), and its downstream transcription factor, Interferon Regulatory Factor 3 (IRF3). In addition, a transmembrane protein called STING (STimulator of INterferon Genes; also called MITA, ERIS, MPYS, TMEM173) functions as an essential signaling adaptor linking cytosolic detection of DNA to the TBK1/IRF3 signaling axis3–7. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signaling molecules in bacteria8, were also shown to induce a STING-dependent type I interferon response9–12. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP) and that this binding is competed by unlabelled cyclic dinucleotides but not by other nucleotides or nucleic acids. Furthermore...

‣ Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease▿†

Wang, Hongxia; Wu, Jian-He; Ayala, Julio C.; Benitez, Jorge A.; Silva, Anisia J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2011 Português
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Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings...

‣ Quantification of high-specificity cyclic diguanylate signaling

Massie, Jonathan P.; Reynolds, Evan L.; Koestler, Benjamin J.; Cong, Jian-Ping; Agostoni, Marco; Waters, Christopher M.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Cyclic di-GMP (c-di-GMP) is a second messenger molecule that regulates the transition between sessile and motile lifestyles in bacteria. Bacteria often encode multiple diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that produce and degrade c-di-GMP, respectively. Because of multiple inputs into the c-di-GMP–signaling network, it is unclear whether this system functions via high or low specificity. High-specificity signaling is characterized by individual DGCs or PDEs that are specifically associated with downstream c-di-GMP–mediated responses. In contrast, low-specificity signaling is characterized by DGCs or PDEs that modulate a general signal pool, which, in turn, controls a global c-di-GMP–mediated response. To determine whether c-di-GMP functions via high or low specificity in Vibrio cholerae, we correlated the in vivo c-di-GMP concentration generated by seven DGCs, each expressed at eight different levels, to the c-di-GMP–mediated induction of biofilm formation and transcription. There was no correlation between total intracellular c-di-GMP levels and biofilm formation or gene expression when considering all states. However, individual DGCs showed a significant correlation between c-di-GMP production and c-di-GMP–mediated responses. Moreover...

‣ Structures of the PelD Cyclic Diguanylate Effector Involved in Pellicle Formation in Pseudomonas aeruginosa PAO1

Li, Zhi; Chen, Jui-Hui; Hao, Yue; Nair, Satish K.
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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Background: Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to PelD is required for pellicle formation by Pseudomonas aeruginosa.

‣ Enzymatically Active and Inactive Phosphodiesterases and Diguanylate Cyclases Are Involved in Regulation of Motility or Sessility in Escherichia coli CFT073

Spurbeck, Rachel R.; Tarrien, Rebecca J.; Mobley, Harry L. T.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 09/10/2012 Português
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Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC...

‣ Detection of Cyclic Diguanylate G-Octaplex Assembly and Interaction with Proteins

Lieberman, Ori J.; DeStefano, Jeffery J.; Lee, Vincent T.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 07/01/2013 Português
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Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of Gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated.

‣ Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 05/06/2013 Português
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Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32.

‣ Cyclic di-GMP Sensing via the Innate Immune Signaling Protein STING

Yin, Qian; Tian, Yuan; Kabaleeswaran, Venkataraman; Jiang, Xiaomo; Tu, Daqi; Eck, Michael J.; Chen, Zhijian J.; Wu, Hao
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.

‣ ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa

Jones, Christopher J.; Newsom, David; Kelly, Benjamin; Irie, Yasuhiko; Jennings, Laura K.; Xu, Binjie; Limoli, Dominique H.; Harrison, Joe J.; Parsek, Matthew R.; White, Peter; Wozniak, Daniel J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 06/03/2014 Português
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The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon...

‣ Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

Martínez-Gil, Marta; Ramos-González, María Isabel; Espinosa-Urgel, Manuel
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2014 Português
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LapA and LapF are large extracellular proteins that play a relevant role in biofilm formation by Pseudomonas putida. Current evidence favors a sequential model in which LapA is first required for the initial adhesion of individual bacteria to a surface, while LapF participates in later stages of biofilm development. In agreement with this model, lapF transcription was previously shown to take place at late times of growth and to respond to the stationary-phase sigma factor RpoS. We have now analyzed the transcription pattern of lapA and other regulatory elements that influence expression of both genes. The lapA promoter shows a transient peak of activation early during growth, with a second increase in stationary phase that is independent of RpoS. The same pattern is observed in biofilms although expression is not uniform in the population. Both lapA and lapF are under the control of the two-component regulatory system GacS/GacA, and their transcription also responds to the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP), although in surprisingly reverse ways. Whereas expression from the lapA promoter increases with high levels of c-di-GMP, the opposite is true for lapF. The transcriptional regulator FleQ is required for the modulation of lapA expression by c-di-GMP but has a minor influence on lapF. This work represents a further step in our understanding of the regulatory interactions controlling biofilm formation in P. putida.

‣ A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

Feirer, Nathan; Xu, Jing; Allen, Kylie D.; Koestler, Benjamin J.; Bruger, Eric L.; Waters, Christopher M.; White, Robert H.; Fuqua, Clay
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Artigo de Revista Científica
Publicado em 30/06/2015 Português
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The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR...

‣ C-di-GMP is an effective immunomodulator and vaccine adjuvant against pneumococcal infection

Ogunniyi, A.; Paton, J.; Kirby, A.; McCullers, J.; Cook, J.; Hyodo, M.; Hayakawa, Y.; Karaolis, D.
Fonte: Elsevier Sci Ltd Publicador: Elsevier Sci Ltd
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
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Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or pneumococcal surface protein A (PspA) before pneumococcal challenge, were investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of Streptococcus pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in a significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and a significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases.; http://www.elsevier.com/wps/find/journaldescription.cws_home/30521/description#description; Abiodun D. Ogunniyi...

‣ Expression, crystallization and preliminary crystallographic analysis of PilZ(XAC1133) from Xanthomonas axonopodis pv. citri

GUZZO, Cristiane R.; FARAH, Chuck S.
Fonte: WILEY-BLACKWELL PUBLISHING, INC Publicador: WILEY-BLACKWELL PUBLISHING, INC
Tipo: Artigo de Revista Científica
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Proteins containing PilZ domains are widespread in Gram-negative bacteria and have recently been shown to be involved in the control of biofilm formation, adherence, aggregation, virulence-factor production and motility. Furthermore, some PilZ domains have recently been shown to bind the second messenger bis(3'-> 5') cyclic diGMP. Here, the cloning, expression, purification and crystallization of PilZ(XAC1133), a protein consisting of a single PilZ domain from Xanthomonas axonopodis pv. citri, is reported. The closest PilZ(XAC1133) homologues in Pseudomonas aeruginosa and Neisseria meningitidis control type IV pilus function. Recombinant PilZ(XAC1133) containing selenomethionine was crystallized in space group P6(1). The unit-cell parameters were a = 62.125, b = 62.125, c = 83.543 angstrom. These crystals diffracted to 1.85 angstrom resolution and a MAD data set was collected at a synchrotron source. The calculated Matthews coefficient suggested the presence of two PilZ(XAC1133) molecules in the asymmetric unit.

‣ Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector

Dahlstrom, Kurt M.; Giglio, Krista M.; Collins, Alan J.; Sondermann, Holger; O’Toole, George A.
Fonte: American Society of Microbiology Publicador: American Society of Microbiology
Tipo: Text
Publicado em 15/12/2015 Português
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Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network.