Recombinant Diguanylate cyclase yddV (yddV)

What is YddV and what is its primary function?

YddV from Escherichia coli (Ec) is a globin-coupled heme-based oxygen sensor protein that displays diguanylate cyclase activity in response to oxygen availability . As a diguanylate cyclase, YddV catalyzes the synthesis of bis-(3′,5′)-cyclic di-guanylic acid (c-di-GMP), an important bacterial signaling molecule. The protein contains a heme domain that acts as an oxygen sensor and regulates the enzymatic activity of the protein based on oxygen concentrations. YddV plays a critical role in bacterial signaling pathways that control various cellular processes, including biofilm formation, by influencing the production of adhesion factors and extracellular polysaccharides (EPS) .

How does YddV function as an oxygen sensor?

YddV functions as an oxygen sensor through its globin-coupled heme domain. The oxygen sensing mechanism involves the binding of O₂ to the Fe(II) heme complex, which triggers conformational changes that activate the diguanylate cyclase domain. Research has shown that the Fe(II)-O₂ complex represents an active form of YddV with a turnover number of 0.022 min⁻¹, whereas the reduced Fe(II) form without oxygen binding shows minimal activity (<0.01 min⁻¹) . This oxygen-dependent activation mechanism allows bacteria to modulate c-di-GMP production in response to environmental oxygen levels, thereby regulating various physiological processes including biofilm formation.

What is the relationship between YddV and cyclic di-GMP homeostasis?

YddV functions in tandem with phosphodiesterase Ec DOS to regulate c-di-GMP homeostasis in bacteria. Studies have demonstrated that YddV catalyzes the synthesis of c-di-GMP from GTP, while Ec DOS degrades c-di-GMP to pGpG . The turnover number of YddV (0.066 min⁻¹ for Fe(III) and 0.022 min⁻¹ for Fe(II)-O₂) is considerably lower than that of Ec DOS-Fe(II)-O₂ (61 min⁻¹), indicating that the YddV reaction is the rate-determining step in this regulatory pathway . This coordinated system allows precise control over c-di-GMP levels in response to oxygen availability, demonstrating that both O₂ binding and the heme redox state of YddV are critical factors in c-di-GMP regulation.

What methods are recommended for studying YddV oxygen sensing mechanisms?

To study YddV oxygen sensing mechanisms, researchers should employ a combination of spectroscopic and biochemical approaches:

• UV-Visible Spectroscopy: To characterize different heme states (Fe(II), Fe(III), Fe(II)-O₂, Fe(II)-CO, Fe(II)-NO) and monitor autoxidation rates.

• Resonance Raman Spectroscopy: To analyze the vibrational characteristics of heme-ligand interactions, particularly for identifying hydrogen bonding networks with bound O₂ or CO. Studies have identified specific Raman frequencies for Fe-O₂ (565 cm⁻¹) and Fe-CO (495 cm⁻¹) in wild-type YddV .

• Time-Resolved Step-Scan FTIR Studies: To determine site-specific protein dynamics following ligand photodissociation and investigate the communication pathway between the distal residues and the heme .

• Electrochemical Analysis: To determine redox potentials of the heme domain, as previously measured at -17 mV versus the standard hydrogen electrode for YddV-heme .

• Enzyme Activity Assays: To quantify turnover numbers for different heme states using radioisotope-labeled GTP substrate or HPLC-based methods.

These methodological approaches should be combined with site-directed mutagenesis to investigate the roles of specific amino acid residues in oxygen sensing and catalytic activity.

How can researchers effectively express and purify recombinant YddV?

The successful expression and purification of recombinant YddV typically follows this protocol:

• Expression System: Clone the yddV gene into a pET-based expression vector with a His-tag for purification.

• Expression Conditions: Transform the construct into E. coli BL21(DE3) or similar expression strain. Culture cells at 37°C until OD₆₀₀ reaches 0.6-0.8, then induce with 0.5-1.0 mM IPTG.

• Heme Reconstitution: To ensure proper heme incorporation, add δ-aminolevulinic acid (0.5 mM) during induction and/or supplement with hemin during cell lysis.

• Purification Steps:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole

  • Purify using Ni-NTA affinity chromatography with imidazole gradient elution

  • Further purify by size exclusion chromatography using Superdex 200 column

• Quality Control: Verify protein purity by SDS-PAGE and confirm heme incorporation by measuring the Soret band absorption ratio (A₄₁₀/A₂₈₀).

This protocol yields the intact YddV protein. For isolated domain studies, researchers often express the heme domain (YddV-heme) separately from the catalytic domain to investigate their specific properties and interactions.

What are the best approaches for measuring diguanylate cyclase activity of YddV?

Measuring the diguanylate cyclase activity of YddV requires sensitive assays that can detect c-di-GMP production. The following approaches are recommended:

For optimal results, reaction conditions should include:

• Buffer: 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂

• GTP concentration: 100-200 μM

• Enzyme concentration: 0.5-2 μM

• Temperature: 25-30°C

• Time points: Multiple measurements over 60 minutes

To control the redox and ligation state of the heme, researchers should prepare samples under defined oxygen conditions or add specific ligands (CO, NO) to investigate their regulatory effects on enzyme activity.

How do specific amino acid residues in the heme pocket affect oxygen sensing and enzymatic activity?

Studies on YddV have identified critical residues in the heme pocket that significantly influence oxygen binding and enzymatic regulation:

• Tyrosine 43 (Tyr43): Located at the heme distal side, Tyr43 forms hydrogen bonds with both O₂ and CO molecules bound to the Fe(II) complex. Resonance Raman spectroscopy has shown that the Y43F mutation alters the frequencies representing ν(Fe-O₂) from 565 cm⁻¹ in wild-type to 559 cm⁻¹ in the mutant, and ν(Fe-CO) from 495 cm⁻¹ to 505 cm⁻¹ . This indicates that the hydroxyl group of Tyr43 electrostatically interacts with bound gaseous ligands, stabilizing the Fe(II)-O₂ complex.

• Effects of Tyr43 Mutations:

  • Y43A and Y43L mutants exhibit very low O₂ affinities, preventing detection of their Fe(II)-O₂ complexes

  • Y43W mutant shows a significantly increased O₂ dissociation rate constant (>150 s⁻¹) compared to wild-type (22 s⁻¹)

  • Y43F and Y43W mutants demonstrate higher autoxidation rate constants (0.069 and 0.12 min⁻¹, respectively) than wild-type (0.0076 min⁻¹)

• Leucine 65 (Leu65): The L65M and L65T mutations affect ligand dynamics, suggesting this residue plays a role in controlling ligand access to the heme pocket .

These findings demonstrate that the distal pocket architecture, particularly the presence of hydrogen bond donors like Tyr43, is essential for stabilizing the oxygen-bound form of YddV and consequently regulating its enzymatic activity.

What is the molecular mechanism of signal transduction between the heme domain and the catalytic domain of YddV?

The signal transduction mechanism between the oxygen-sensing heme domain and the catalytic domain of YddV involves a sophisticated allosteric regulation pathway:

• Heme State-Dependent Conformational Changes: Time-resolved step-scan FTIR studies have revealed that ligand binding to the heme iron induces protein backbone relaxations that propagate from the heme pocket to distant regions of the protein. The rate of decay of amide I vibrations coincides with the rate of CO rebinding, suggesting coupling between ligation dynamics and protein backbone relaxation .

• Role of Heme Propionates: The heme propionates exist in both protonated and deprotonated states and appear to play a critical role in transmitting the signal. Changes in the heme ligation state alter the environment sensed by the heme propionates, potentially triggering conformational changes that affect the catalytic domain .

• Proposed Signaling Pathway:

  • O₂ binding to Fe(II) heme → Stabilization by distal pocket residues

  • Conformational changes in the globin domain → Altered interactions of heme propionates

  • Signal propagation through connecting helices → Repositioning of catalytic residues

  • Enhanced substrate binding and catalysis in the GGDEF domain

This model explains why different heme ligation states (Fe(II)-O₂, Fe(II)-CO, Fe(III), etc.) exhibit varying levels of enzymatic activity, with the oxygen-bound and ferric forms showing the highest activity levels.

How do the kinetic parameters of YddV compare with other diguanylate cyclases?

YddV displays distinctive kinetic characteristics when compared to other diguanylate cyclases (DGCs):

The relatively low turnover number of YddV compared to the associated phosphodiesterase Ec DOS (61 min⁻¹) suggests that YddV activity is the rate-limiting step in c-di-GMP turnover . This kinetic arrangement allows for precise control of c-di-GMP levels, where small changes in YddV activity can significantly impact signaling outcomes.

The specificity of YddV in stimulating poly-N-acetylglucosamine (PNAG) production distinguishes it from other DGCs like AdrA and YcdT, which do not induce PNAG production when overexpressed . This functional specificity suggests that different DGCs may regulate distinct downstream targets despite catalyzing the same biochemical reaction.

How does YddV regulate biofilm formation in bacteria?

YddV plays a critical role in bacterial biofilm formation through a specific regulatory pathway:

• PNAG Production Pathway: Research has demonstrated that YddV specifically stimulates the expression of the pgaABCD operon, which encodes the biosynthetic machinery for poly-N-acetylglucosamine (PNAG), a major extracellular polysaccharide (EPS) component of bacterial biofilms .

• Enzymatic Activity Requirement: The stimulation of PNAG production requires an intact GGDEF motif in YddV, which is responsible for its diguanylate cyclase activity. Mutations that inactivate this motif abolish both the stimulation of PNAG production and activation of pgaABCD expression .

• Specific Regulatory Role: Notably, not all diguanylate cyclases share this function. Overexpression of other DGCs such as AdrA and YcdT does not induce PNAG production, suggesting that YddV has a specialized role in this particular regulatory pathway .

• Oxygen-Dependent Regulation: Since YddV activity is modulated by oxygen levels through its heme sensor domain, this creates a mechanism for oxygen-dependent regulation of biofilm formation. Under conditions where the Fe(II)-O₂ complex forms (aerobic conditions), YddV shows enhanced activity, potentially promoting biofilm formation in oxygen-rich environments.

This regulatory pathway illustrates how bacteria can integrate environmental oxygen sensing with biofilm formation through the specialized activity of YddV, demonstrating a sophisticated mechanism for adapting to changing environmental conditions.

What experimental approaches can be used to study YddV-regulated gene expression?

To study YddV-regulated gene expression, particularly focusing on the pgaABCD operon, researchers should employ the following experimental approaches:

• Transcriptional Reporter Assays:

  • Generate pgaABCD promoter-lacZ or pgaABCD promoter-GFP fusions

  • Measure β-galactosidase activity or fluorescence in wild-type vs. yddV knockout strains

  • Compare expression levels under conditions of YddV overexpression with varying oxygen tensions

• Quantitative RT-PCR:

  • Extract RNA from bacterial cultures with different yddV expression levels

  • Perform reverse transcription and quantitative PCR targeting pgaABCD transcripts

  • Normalize results to appropriate housekeeping genes (e.g., rpoD or gyrB)

• RNA-Seq Analysis:

  • Conduct genome-wide transcriptome analysis of wild-type, yddV knockout, and YddV-overexpressing strains

  • Identify differentially expressed genes as potential YddV-regulated targets

  • Perform pathway and gene ontology analyses to identify biological processes affected by YddV activity

• Chromatin Immunoprecipitation (ChIP) Assays:

  • To investigate potential binding of c-di-GMP-responsive transcription factors to the pgaABCD promoter

  • Use antibodies against candidate transcription factors or epitope-tagged versions

  • Analyze ChIP data to identify direct regulatory interactions

• Phenotypic Assessment:

  • Quantify PNAG production using congo red binding assays or immunoblotting with PNAG-specific antibodies

  • Measure biofilm formation using crystal violet staining or confocal microscopy

  • Correlate phenotypic measurements with YddV activity and gene expression data

These complementary approaches provide a comprehensive framework for understanding the regulatory networks controlled by YddV and the mechanisms by which c-di-GMP signaling influences gene expression in bacteria.

How can insights from YddV research be applied to developing anti-biofilm strategies?

Research on YddV provides several promising avenues for developing anti-biofilm strategies:

• Targeting Heme-Oxygen Interactions:

  • Small molecules that compete with O₂ for binding to the YddV heme domain could inhibit its activation

  • Compounds that mimic the Fe(II) or Fe(II)-NO states of YddV would maintain the enzyme in its low-activity form

  • Potential targets include the critical Tyr43 residue, which stabilizes oxygen binding through hydrogen bonding

• Disrupting Signal Transduction:

  • Molecules that interfere with the conformational changes coupling oxygen binding to catalytic activation

  • Compounds targeting the protein dynamics or heme propionate interactions that mediate signal propagation

• Direct Enzyme Inhibition:

  • Competitive inhibitors that bind to the GTP binding site in the GGDEF domain

  • Allosteric inhibitors that stabilize inactive conformations of the catalytic domain

  • Substrate analogs that occupy the active site without being converted to c-di-GMP

• Downstream Intervention:

  • Targeting the pgaABCD operon expression or PNAG biosynthesis machinery

  • Developing enzymes that degrade PNAG or interfere with its incorporation into the biofilm matrix

  • Engineering phages or delivery systems to express PNAG-degrading enzymes within biofilms

• Combined Approaches:

  • Multi-target strategies addressing both YddV activity and other c-di-GMP signaling components

  • Coupling YddV inhibitors with conventional antibiotics to enhance penetration into biofilms

  • Environmental manipulation of oxygen levels to modulate YddV activity in clinical settings

The oxygen-sensing capability of YddV makes it particularly interesting as a target in environments with variable oxygen tensions, such as wounds, respiratory infections, or industrial biofilms. Inhibitors specifically designed to function under these conditions could provide selective anti-biofilm activity without disrupting beneficial commensal bacteria in other niches.

What are the most promising unexplored aspects of YddV function and regulation?

Several promising research directions for YddV remain unexplored or underdeveloped:

• Structural Studies: While functional data on YddV is available, high-resolution structural information of the full-length protein in different ligation and activation states would provide crucial insights into the signal transduction mechanism. Techniques like cryo-EM could reveal conformational changes associated with oxygen binding and activation.

• Post-translational Modifications: Investigation of potential post-translational modifications that might regulate YddV activity independently of oxygen sensing. Phosphorylation, acetylation, or other modifications could integrate additional cellular signals into c-di-GMP production.

• Interaction Partners: Identification of proteins that directly interact with YddV could reveal additional regulatory mechanisms and integration points with other signaling pathways. Proteomic approaches such as co-immunoprecipitation coupled with mass spectrometry would be valuable.

• Temporal Dynamics: Most studies have examined steady-state conditions, but the temporal dynamics of YddV activation in response to changing oxygen levels remain poorly understood. Real-time measurements of c-di-GMP production following oxygen transitions would provide insights into signaling kinetics.

• Environmental Regulation: Beyond oxygen, other environmental factors may influence YddV activity directly or indirectly. Investigating the effects of pH, temperature, redox state, and nutrient availability on YddV function could reveal additional regulatory mechanisms.

These research directions would significantly enhance our understanding of YddV function and potentially reveal new approaches for modulating bacterial biofilm formation in various contexts.

How might advanced spectroscopic techniques further elucidate YddV mechanisms?

Advanced spectroscopic techniques offer powerful approaches to resolve remaining questions about YddV function:

• Time-Resolved Crystallography: This technique could capture structural changes in real-time following ligand photolysis, providing direct visualization of the conformational changes that propagate from the heme domain to the catalytic domain.

• Nuclear Magnetic Resonance (NMR): Solution NMR studies of isotopically labeled YddV domains could map residue-specific changes in chemical shifts upon oxygen binding, identifying the communication pathway between domains.

• Electron Paramagnetic Resonance (EPR): EPR spectroscopy could provide detailed information about the electronic structure of the heme iron in different ligation states and how mutations affect the properties of the iron center.

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): This technique could identify regions of YddV that undergo conformational changes upon oxygen binding by measuring changes in solvent accessibility.

• Fluorescence Resonance Energy Transfer (FRET): By introducing fluorescent probes at strategic positions, FRET measurements could monitor domain movements and conformational changes in real-time under varying oxygen conditions.

• Advanced Raman Techniques: Techniques like ultrafast coherent anti-Stokes Raman spectroscopy could provide unprecedented temporal resolution of vibrational changes following oxygen binding, revealing the earliest events in signal transduction.

These spectroscopic approaches, particularly when combined with site-directed mutagenesis and computational modeling, would provide comprehensive insights into the molecular mechanisms underlying YddV function.