Recombinant Pseudomonas syringae pv. tomato Protein CrcB homolog (crcB)

What is the CrcB homolog in Pseudomonas syringae pv. tomato and what is its primary function?

The CrcB motif is a conserved domain found in the 5' untranslated regions (UTRs) of genes encoding DNA repair mechanisms, ion transporters (K+, Cl-), and formate hydrogen lyase. In bacterial systems, CrcB expression is critically important for reducing fluoride concentration in cells and mitigating fluoride toxicity . In Pseudomonas syringae pv. tomato, the CrcB homolog likely performs similar functions while potentially having species-specific adaptations related to plant pathogenicity and environmental stress responses.

The primary function of CrcB appears to be linked to fluoride resistance. Highly conserved nucleotides of the CrcB motif undergo significant conformational changes in the presence of sodium fluoride (NaF), as demonstrated through in-line probing methods . The CrcB motif functions within a fluoride riboswitch mechanism, where fluoride binding to the aptamer domain leads to the formation of an anti-terminator stem, allowing RNA polymerase access for transcription activation. Conversely, in the absence of fluoride ions, the riboswitch forms a terminator stem that halts transcription .

How is CrcB expression regulated in Pseudomonas syringae pv. tomato?

CrcB expression regulation in P. syringae pv. tomato appears to be complex and may involve multiple regulatory mechanisms. While specific information on CrcB regulation is limited in the search results, we can extrapolate from related regulatory systems. The CrcZ and CrcX small non-coding RNAs (formerly designated psr1 and psr2) in P. syringae pv. tomato DC3000 are dependent upon RpoN together with the two-component system CbrAB, and their expression is influenced by the carbon source present in the medium .

Similar to other Pseudomonas species like P. aeruginosa and P. putida, where expression of crcZ is activated by the CbrA/CbrB two-component sensor-regulator system, quantitative RT-PCR experiments have shown that inactivation of the cbrB gene significantly reduces expression of both crcZ and crcX in P. syringae pv. tomato DC3000 . This suggests that CrcB might also be part of carbon catabolite repression networks, though direct experimental evidence is needed to confirm this relationship.

What differentiates CrcB from other regulatory proteins in the Pseudomonas syringae complex?

The Pseudomonas syringae complex encompasses a diverse group of bacterial strains organized into at least 13 phylogroups with 23 distinct clades, making it challenging to establish universal characteristics . Unlike many other regulatory proteins that may be specific to certain phylogroups, the CrcB motif appears to be relatively conserved across bacterial species, functioning in fluoride detection and resistance systems.

Unlike type III secretion system (T3SS) effectors and toxins that vary significantly between phylogroups and contribute to host specificity, CrcB is more likely to be involved in fundamental cellular processes related to ion homeostasis. This is consistent with its role in reducing fluoride toxicity, which would be a conserved survival mechanism rather than a host-specific virulence factor .

What are the optimal expression systems for recombinant production of Pseudomonas syringae pv. tomato CrcB homolog?

While the search results don't provide specific expression protocols for P. syringae CrcB, we can recommend methodological approaches based on similar recombinant proteins:

Table 1: Comparison of Expression Systems for Recombinant CrcB Production

What purification strategies yield the highest purity and functional integrity of recombinant CrcB?

A multi-step purification protocol would be most effective:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with imidazole gradient elution (50-250 mM).

• Intermediate Purification: Ion exchange chromatography based on the theoretical isoelectric point of CrcB.

• Polishing Step: Size exclusion chromatography using Superdex 75 or 200 columns depending on the molecular weight.

Table 2: Troubleshooting Common Purification Issues with CrcB

For functional studies, it's crucial to verify protein folding using circular dichroism spectroscopy and maintain the protein in a buffer mimicking physiological conditions of P. syringae cytoplasm.

What experimental methods are most effective for assessing CrcB function in fluoride resistance?

Given CrcB's role in fluoride resistance , several complementary approaches can be employed:

• Growth Inhibition Assays: Compare wild-type, CrcB knockout, and CrcB-overexpressing P. syringae strains in media containing varying fluoride concentrations (0-50 mM NaF). Measure growth curves over 48 hours using spectrophotometry.

• Fluoride Uptake Measurements: Utilize fluoride-selective electrodes or radioactive 18F to quantify intracellular fluoride levels in different strains under standardized conditions.

• Membrane Potential Assays: Since CrcB may function as an ion channel/transporter, measure membrane potential changes using fluorescent dyes like DiSC3(5) upon fluoride exposure.

• In vivo Expression Analysis: Construct transcriptional fusions between the CrcB promoter and reporter genes (e.g., GFP, luciferase) to monitor expression under different fluoride concentrations and environmental conditions.

Table 3: Comparative Analysis of CrcB Functional Assay Methods

When interpreting results, it's essential to include appropriate controls and to consider the multi-factorial nature of fluoride resistance mechanisms.

How can researchers distinguish between direct and indirect effects of CrcB on fluoride riboswitch regulation?

Distinguishing direct from indirect effects requires multiple lines of evidence:

• In vitro Binding Assays: Employ surface plasmon resonance (SPR) or microscale thermophoresis (MST) to measure direct binding between purified recombinant CrcB and synthesized fluoride riboswitch RNA fragments.

• RNA Structure Probing: Utilize selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) to examine structural changes in the riboswitch upon CrcB interaction with and without fluoride ions.

• Protein-RNA Crosslinking: Apply UV crosslinking followed by immunoprecipitation (CLIP) to identify direct interaction sites between CrcB and target RNAs in vivo.

• Mutagenesis Studies: Create point mutations in both CrcB and the riboswitch to identify critical residues/nucleotides for interaction and functional coupling.

• Temporal Analysis: Use time-resolved techniques to determine the sequence of molecular events following fluoride exposure.

When contradictory data emerge, consider that CrcB might have multiple mechanisms of action, including both direct binding to riboswitch elements and indirect effects through other cellular components or signaling pathways.

What structural features of CrcB are critical for its function in the fluoride riboswitch mechanism?

Based on information about fluoride riboswitches, several structural features likely contribute to CrcB function :

• Ion-Binding Domains: CrcB likely contains specific structural motifs that directly interact with fluoride ions or coordinating metals like magnesium.

• RNA-Interaction Interfaces: Regions that specifically recognize and bind to the riboswitch RNA, particularly in the presence of fluoride.

• Conformational Flexibility: The protein likely undergoes significant conformational changes upon fluoride binding, as suggested by the substantial changes observed in riboswitch nucleotides in the presence of NaF .

The study of hydrogen exchange dynamics through NMR spectroscopy has revealed that stabilization of specific base-pairs (like U45·A37 in the B. cereus fluoride riboswitch) due to fluoride ion binding results in transcription regulation by changing dynamics while maintaining structure . Similar mechanisms might be involved in CrcB-mediated processes in P. syringae.

How does magnesium influence CrcB-mediated fluoride recognition and riboswitch function?

Magnesium plays a critical role in fluoride riboswitch function. As demonstrated in the B. cereus fluoride riboswitch, the riboswitch requires magnesium ions to recognize fluoride ions selectively . Upon binding of a magnesium ion, significant conformational changes occur in the riboswitch, resulting in the greatest increase in stability. The fluoride ion then produces further changes in dynamics.

For experimental characterization of magnesium's role:

• Titration Experiments: Conduct fluoride binding assays across a range of magnesium concentrations (0-20 mM) to establish the optimal Mg2+ concentration for CrcB function.

• Metal Chelation Studies: Use chelators like EDTA to selectively remove magnesium and observe effects on CrcB-fluoride interactions.

• Metal Substitution: Replace Mg2+ with other divalent cations (Ca2+, Mn2+, etc.) to determine specificity of the metal requirement.

• Structural Studies: Employ X-ray crystallography or cryo-EM to visualize magnesium-binding sites within the CrcB-riboswitch complex.

When designing experiments, researchers should be aware that results obtained under different magnesium concentrations might not be directly comparable due to the ion's significant impact on riboswitch structure and dynamics.

How can CrcB research inform our understanding of Pseudomonas syringae pathogenicity and environmental adaptations?

Understanding CrcB function can provide insights into several aspects of P. syringae biology:

• Environmental Adaptations: The Pseudomonas syringae complex includes strains from both agricultural and environmental habitats, including those linked to the water cycle . CrcB may contribute to survival in diverse environments containing varying fluoride levels.

• Metabolic Versatility: Connection to carbon source utilization regulation through potential interactions with CrcZ and CrcX regulatory RNAs suggests CrcB might influence the metabolic flexibility that allows P. syringae to thrive in different niches .

• Phylogroup-Specific Variations: The P. syringae complex comprises at least 13 phylogroups with varying pathogenic abilities and environmental adaptations . Comparing CrcB sequences and functions across these groups could reveal evolutionary adaptations.

• Stress Response Networks: CrcB likely participates in broader stress response networks beyond fluoride resistance, potentially including responses to other environmental toxins or plant defense compounds.

Research focusing on these aspects may reveal how CrcB contributes to the remarkable adaptability of P. syringae across diverse environments and its emergence in new plant disease epidemics.

What methodological approaches can resolve contradictory findings regarding CrcB function across different experimental systems?

When faced with contradictory findings, consider these approaches:

• Standardized Experimental Conditions: Develop standardized protocols for CrcB expression, purification, and functional assays to enable direct comparison between laboratories.

• Multi-Strain Validation: Test findings across multiple P. syringae strains representing different phylogroups to distinguish universal CrcB functions from strain-specific adaptations.

• In vivo vs. In vitro Reconciliation: When in vitro and in vivo results conflict, use complementary approaches like:

  • Cell-based validation of in vitro findings

  • Reconstitution of minimal systems to verify complex cellular observations

  • Single-cell analyses to address population heterogeneity effects

• Data Integration Approaches: Apply computational methods to integrate diverse datasets:

Table 4: Data Integration Strategies for Resolving Contradictory CrcB Findings

• Collaboration Networks: Establish multi-laboratory validation studies where key experiments are replicated across different research groups using standardized protocols.

How should researchers design experiments to distinguish the roles of CrcB from other fluoride resistance mechanisms in Pseudomonas syringae?

To isolate CrcB-specific effects from other fluoride resistance mechanisms:

• Genetic Approach:

  • Generate clean deletion mutants (ΔcrcB) using allelic exchange

  • Create complemented strains with wild-type and mutant versions of CrcB

  • Develop CrcB-overexpression strains with inducible promoters

  • Generate double/triple mutants affecting other known fluoride resistance mechanisms

• Biochemical Discrimination:

  • Use fluoride-specific probes to distinguish CrcB-mediated effects from general ion homeostasis

  • Apply specific inhibitors of known fluoride transporters/channels to isolate CrcB contribution

  • Perform metabolic flux analysis with and without CrcB under fluoride stress

• Temporal Resolution:

  • Conduct time-course experiments to determine the sequence of molecular events

  • Use rapid sampling techniques to capture immediate responses to fluoride exposure

  • Apply mathematical modeling to differentiate primary from secondary effects

• Spatial Resolution:

  • Employ fluorescent protein fusions to track CrcB localization during fluoride stress

  • Use subcellular fractionation to determine compartment-specific functions

  • Apply techniques like FRET to detect protein-protein interactions in different cellular regions

When interpreting results, consider that fluoride resistance likely represents a network of interacting mechanisms rather than independent systems, necessitating network analysis approaches for comprehensive understanding.

What are the critical controls and variables to consider when studying CrcB regulation in the context of carbon catabolite repression?

Given the potential connection between CrcB and carbon catabolite repression systems involving CrcZ and CrcX regulatory RNAs , researchers should consider:

Critical Controls:

• Growth media composition standardization (especially carbon sources)

• Growth phase-matched sampling for all experiments

• Parallel analysis of known carbon catabolite repression-regulated genes

• Inclusion of cbrA/cbrB mutants as reference strains

• Complementation controls to verify phenotype specificity

Key Variables to Monitor:

• Carbon source type and concentration

• Growth rate and cellular metabolic state

• Expression levels of crcZ, crcX, and other related regulatory RNAs

• Activity of the CbrA/CbrB two-component system

• Environmental parameters (pH, temperature, osmolarity)

Table 5: Experimental Design for CrcB Carbon Catabolite Repression Studies

When designing these experiments, researchers should be aware that carbon catabolite repression systems can be highly responsive to subtle changes in growth conditions, requiring rigorous standardization and multiple biological replicates.

How can CRISPR-Cas9 genome editing advance functional studies of CrcB in Pseudomonas syringae pv. tomato?

CRISPR-Cas9 technology offers several advantages for CrcB functional studies:

• Precise Genetic Manipulation:

  • Generate scarless deletions, point mutations, or insertions in crcB with minimal off-target effects

  • Create allelic series with graduated functional impacts

  • Introduce epitope tags or fluorescent protein fusions at endogenous loci

• Multiplexed Editing:

  • Simultaneously target crcB and related genes (e.g., components of fluoride response networks)

  • Create combinatorial mutations to dissect genetic interactions

  • Introduce regulatory element modifications alongside coding sequence changes

• High-Throughput Functional Genomics:

  • Develop CRISPR interference (CRISPRi) libraries targeting crcB-related pathways

  • Create saturating mutagenesis libraries of the crcB locus

  • Implement CRISPR activation (CRISPRa) to upregulate crcB under native regulation

• In vivo Dynamics:

  • Apply CRISPR imaging to visualize crcB locus dynamics during fluoride response

  • Use dCas9-based biosensors to detect conformational changes in CrcB or its regulatory elements

  • Implement CRISPR-based proximity labeling to identify interaction partners

For P. syringae specifically, researchers should optimize delivery methods (electroporation of ribonucleoprotein complexes often works well) and consider using alternative Cas proteins if standard SpCas9 shows toxicity or low efficiency.

What insights might structural biology techniques provide about CrcB function that cannot be obtained through genetic and biochemical approaches alone?

Advanced structural biology techniques could reveal:

• Atomic-Level Mechanisms:

  • X-ray crystallography or cryo-EM structures of CrcB alone and in complex with fluoride ions

  • Visualization of conformational changes upon ligand binding

  • Identification of specific fluoride-binding pockets and coordinating residues

• Dynamic Properties:

  • Nuclear magnetic resonance (NMR) to characterize protein dynamics in solution

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational flexibility

  • Single-molecule FRET to observe real-time conformational changes

• Interaction Interfaces:

  • Structural characterization of CrcB-RNA complexes to understand riboswitch regulation

  • Co-crystallization with protein partners to visualize protein-protein interactions

  • Cryoelectron tomography to visualize CrcB in its native membrane environment

• Integration with Computational Approaches:

  • Molecular dynamics simulations based on experimental structures

  • Quantum mechanical calculations of fluoride coordination

  • Machine learning-based prediction of structural changes upon mutations

The combination of multiple structural techniques at different resolutions can overcome limitations of individual methods and provide a comprehensive understanding of CrcB function from atomic to cellular scales.