π-Helix controls activity of oxygen-sensing diguanylate cyclases

Abstract The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

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Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Applied and Environmental Microbiology ◽

10.1128/aem.01638-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6167-6174 ◽

Cited By ~ 20

Author(s):

Xiaohui Gao ◽

Xiao Dong ◽

Sundharraman Subramanian ◽

Paige M. Matthews ◽

Caleb A. Cooper ◽

...

Keyword(s):

Bacillus Subtilis ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Threshold Level ◽

Metabolic Enzymes ◽

Guanosine Monophosphate ◽

Content Type ◽

Diguanylate Cyclases ◽

Rapid Characterization ◽

Green Fluorescent

ABSTRACTMicrobial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite ofBacillus subtilisstrains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs inClostridium difficileusing parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putativeC. difficile630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility inBacillus subtilis.

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Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

Microbiology ◽

10.1099/mic.0.076828-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1427-1439 ◽

Cited By ~ 18

Author(s):

H. Tan ◽

J. A. West ◽

J. P. Ramsay ◽

R. E. Monson ◽

J. L. Griffin ◽

...

Keyword(s):

Biofilm Formation ◽

Cellular Localization ◽

Bacterial Species ◽

Guanosine Monophosphate ◽

Domain Family ◽

Pectobacterium Atrosepticum ◽

Diguanylate Cyclases ◽

Signalling Molecule ◽

Apparent Correlation ◽

Ggdef Domain

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.

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Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507245112 ◽

2015 ◽

Vol 112 (36) ◽

pp. E5048-E5057 ◽

Cited By ~ 75

Author(s):

Mona W. Orr ◽

Gregory P. Donaldson ◽

Geoffrey B. Severin ◽

Jingxin Wang ◽

Herman O. Sintim ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Active Site ◽

Half Life ◽

Biofilm Formation ◽

Degradation Pathway ◽

Dependent Manner ◽

Capillary Action ◽

Diguanylate Cyclases ◽

Active Site Mutants ◽

Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

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Putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00297-21 ◽

2021 ◽

Author(s):

Zhexian Liu ◽

Sarzana S. Hossain ◽

Zayda Morales Moreira ◽

Cara H. Haney

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune Responses ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Pathogen ◽

Guanosine Monophosphate ◽

Genetic Approach ◽

Chronic Infections ◽

Host Immune Responses ◽

Primary Mechanism

Pseudomonas aeruginosa , an opportunistic bacterial pathogen can synthesize and catabolize a number of small cationic molecules known as polyamines. In several clades of bacteria polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, L-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or through a metabolic derivative. Here we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa . Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that L-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation, but via a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and L-arginine induced a significant increase in the intracellular level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor arginine promote biofilm and c-di-GMP synthesis in P. aeruginosa . Importance: Biofilm formation allows bacteria to physically attach to a surface, confers tolerance to antimicrobial agents, and promotes resistance to host immune responses. As a result, regulation of biofilm is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switching that favors chronic infection.

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Expression of Candidate Cold Stress and Metabolic Related Genes in Acidithiobacillus ferrivorans PQ33 Strain Using Ferrous Iron as Electron Donor

Solid State Phenomena ◽

10.4028/www.scientific.net/ssp.262.368 ◽

2017 ◽

Vol 262 ◽

pp. 368-371

Author(s):

Gregory Guerra-Bieberach ◽

Robert Ccorahua-Santo ◽

Anika Eca ◽

Jordan Bernaldo ◽

Tito Sánchez ◽

...

Keyword(s):

Candidate Genes ◽

Low Temperatures ◽

Biofilm Formation ◽

Iron Oxidation ◽

Trehalose Synthase ◽

Adaptation Mechanisms ◽

Diguanylate Cyclases ◽

Cold Adaptations ◽

Trehalose Synthesis ◽

Andean Mining

The identification of genes involved in cold adaptations of psychrotolerant bacteria Acidithiobacillus ferrivorans is important for biomining processes that take place at low temperatures like Andean mining installations in Peru. We have performed relative quantification RT-qPCR on candidate genes to have a role in adaptations at low temperature (5°C). The candidate genes analyzed were six: Two trehalose synthesis pathway genes, trehalose synthase (treS) and malto-oligosiltrehalose trehalohydrolase (treZ) showing no overexpression at 5°C. Two diguanylate cyclases genes related to exopolymer synthesis and biofilm formation (designated as dgc-I and dgc-II in this paper) were overexpressed at 21°C. The rusA and rusB genes involved in iron oxidation showed no significant change for rusA and no expression for rusB gene in any of both conditions. Genes rpoC, gyrB and alaS were validated as reference genes. These results show congruency with trancriptomics studies about gene expression of A. ferrivorans. Furthermore, the trehalose synthesis genes show no overexpression at low temperatures suggesting that other cold adaptation mechanisms are involved.

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More than Enzymes That Make or Break Cyclic Di-GMP—Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli

mBio ◽

10.1128/mbio.01639-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 60

Author(s):

Olga Sarenko ◽

Gisela Klauck ◽

Franziska M. Wilke ◽

Vanessa Pfiffer ◽

Anja M. Richter ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Second Messenger ◽

Bacterial Biofilm ◽

Interaction Patterns ◽

Systematic Analysis ◽

E Coli ◽

Diguanylate Cyclases ◽

Hub Proteins ◽

Effector Target

ABSTRACT The bacterial second messenger bis-(3′-5′)-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is “local” c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli . Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or “supermodule” of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli . Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems. IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli , together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as “lonely players” without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of “local” c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.

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A Surrogate Matrix-Based Approach Toward Multiplexed Quantitation of an sGC Stimulator and cGMP in Ocular Tissue and Plasma

Toxicologic Pathology ◽

10.1177/0192623320948836 ◽

2020 ◽

pp. 019262332094883

Author(s):

Kenneth Lin ◽

Pablo Cabral ◽

Oscar Ekpenyong ◽

Suzy El Bader ◽

Joana Galvao ◽

...

Keyword(s):

Soluble Guanylate Cyclase ◽

Cyclic Guanosine Monophosphate ◽

Ocular Blood Flow ◽

Guanosine Monophosphate ◽

Ocular Tissue ◽

Target Engagement ◽

Aqueous Humor Outflow ◽

Discovery Research ◽

Liquid Chromatography Tandem Mass ◽

Surrogate Matrix

A liquid chromatography–tandem mass spectrometry assay was developed and qualified for the multiplexed quantitation of a small molecule stimulator of soluble guanylate cyclase (sGC) and its target engagement biomarker, 3′,5′-cyclic guanosine monophosphate (cGMP), in ocular tissues and plasma from a single surrogate matrix calibration curve. A surrogate matrix approach was used in this assay due to the limited quantities of blank ocular matrices in a discovery research setting. After optimization, the assay showed high accuracy, precision, and recovery as well as parallelism between the surrogate matrix and the biological matrices (rabbit plasma, vitreous, and retina–choroid). This assay provided pharmacokinetic and target engagement data after intravitreal administration of the sGC stimulator. The nitric oxide-sGC-cGMP pathway is a potential target to address glaucoma. Increasing sGC-mediated production of cGMP could improve aqueous humor outflow and ocular blood flow. The sGC stimulator showed dose-dependent exposure in rabbit vitreous, retina–choroid, and plasma. The cGMP exhibited a delayed yet sustained increase in vitreous humor but not retina–choroid. Multiplexed measurement of both pharmacokinetic and target engagement analytes reduced animal usage and provided improved context for interpreting PK and PD relationships.

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Proteins Associated with the Myxococcus xanthus Extracellular Matrix

Journal of Bacteriology ◽

10.1128/jb.01007-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7634-7642 ◽

Cited By ~ 26

Author(s):

Patrick D. Curtis ◽

James Atwood ◽

Ron Orlando ◽

Lawrence J. Shimkets

Keyword(s):

Extracellular Matrix ◽

Biofilm Formation ◽

Myxococcus Xanthus ◽

Sodium Dodecyl ◽

Wild Type ◽

Fruiting Body Formation ◽

Ecm Proteins ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

In The Wild

ABSTRACT Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

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Interactions of the c-di-GMP riboswitch with its second messenger ligand

Biochemical Society Transactions ◽

10.1042/bst0390647 ◽

2011 ◽

Vol 39 (2) ◽

pp. 647-651 ◽

Cited By ~ 23

Author(s):

Kathryn D. Smith ◽

Scott A. Strobel

Keyword(s):

Biofilm Formation ◽

Second Messenger ◽

Signalling Pathway ◽

Guanosine Monophosphate ◽

Binding Kinetics ◽

Bacterial Motility ◽

Kinetic Properties ◽

Related Molecule ◽

Kinetically Controlled ◽

Phosphate Backbone

The c-di-GMP [bis-(3′–5′)-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.

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Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

10.1101/402909 ◽

2018 ◽

Author(s):

Matthias Christen ◽

Cassandra Kamischke ◽

Hemantha D. Kulasekara ◽

Kathleen C. Olivas ◽

Bridget R. Kulasekara ◽

...

Keyword(s):

Small Molecules ◽

Small Molecule ◽

High Throughput ◽

Biofilm Formation ◽

High Throughput Screening ◽

Diguanylate Cyclase ◽

Chronic Infections ◽

Structure Activity ◽

Diguanylate Cyclases ◽

Small Molecule Modulators

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low µM range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

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