Recombinant Pectobacterium carotovorum subsp. carotovorum Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Pectobacterium carotovorum subsp. carotovorum and why is it significant in plant pathology research?

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, rod-shaped bacterium belonging to the Enterobacteriaceae family. It is a notorious plant pathogen that causes soft rot disease in various crops, similar to how Pectobacterium atrosepticum causes soft rot and blackleg development in potato plants . The significance of this pathogen stems from its ability to produce and secrete plant cell wall-degrading enzymes, particularly pectinolytic enzymes that macerate plant tissues.

To study this pathogen effectively, researchers typically culture it in standard media such as Luria-Bertani (LB) medium for general growth or specialized media like Pel minimal medium (containing 0.1% yeast extract, 0.1% (NH₄)₂SO₄, 1 mM of MgSO₄, 0.5% glycerol, and 0.5% polygalacturonic acid in 50 mM phosphate buffer, pH 7.0) when studying virulence factors . For experimental infections, infiltration of bacterial suspensions into plant tissues (typically at OD₆₀₀ of 0.8) is commonly performed, with disease progression monitored over time (typically first symptoms appear around 8 hours post-inoculation) .

What techniques are most effective for isolating and purifying recombinant UbiB protein from Pectobacterium carotovorum?

For isolation and purification of recombinant UbiB protein from Pectobacterium carotovorum, a multi-step approach combining molecular cloning and protein purification techniques is most effective:

• Gene Cloning Strategy:

  • PCR amplification of the ubiB gene using designed primers with appropriate restriction sites

  • Insertion into an expression vector (pET system vectors are commonly used)

  • Transformation into a suitable E. coli expression host (BL21(DE3) or derivatives)

• Protein Expression Optimization:

  • Temperature testing (18°C often yields better soluble protein for membrane-associated proteins like UbiB)

  • Induction optimization using varying IPTG concentrations (0.1-1.0 mM)

  • Time-course analysis to determine optimal harvest point

• Protein Purification Protocol:

  • Cell lysis using either sonication or pressure-based disruption

  • Initial clarification by centrifugation (10,000-15,000×g)

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography for further purification

  • Analysis by SDS-PAGE and Western blotting for verification

When analyzing protein purity and characteristics, two-dimensional electrophoresis (2-DE) coupled with mass spectrometry, as demonstrated in studies of other Pcc proteins, can provide detailed information about protein identity and post-translational modifications .

How does UbiB protein expression in Pectobacterium carotovorum differ between in vitro and in vivo conditions?

The expression patterns of proteins in Pectobacterium carotovorum, including potential differences in UbiB expression, show significant variation between in vitro and in vivo conditions. Based on studies of other Pcc proteins, we can outline methodological approaches to investigate these differences:

In comparative studies between in vitro (bacteria grown in culture medium) and in vivo (bacteria isolated from infected plant tissue) conditions, researchers have observed that proteins involved in metabolism, including energy production pathways, often show differential expression. For example, when comparing protein profiles from Pcc grown in Luria-Bertani medium supplemented with plant extracts versus proteins isolated directly from infected plant tissues, researchers identified 53 differentially expressed proteins with expression ratios ≥1.5-fold .

To study UbiB expression differences specifically, a methodological approach would involve:

• Experimental Design:

  • In vitro: Culture Pcc in LB medium (control) and LB supplemented with plant extracts

  • In vivo: Inoculate host plants with Pcc and harvest bacterial cells from infected tissue at specific time points (8-16 hours after inoculation recommended)

• Protein Analysis:

  • Extract total proteins from both conditions using appropriate buffers

  • Analyze using two-dimensional electrophoresis (2-DE)

  • Identify protein spots by mass spectrometry

  • Quantify expression differences using software such as PDQuest

• Confirmation Techniques:

  • qRT-PCR to verify transcriptional changes

  • Western blotting with specific antibodies

  • Functional assays to assess protein activity

From previous studies, proteins isolated from in vivo conditions generally show more pronounced expression changes than those from in vitro conditions with plant extracts, suggesting that the actual plant environment triggers more comprehensive bacterial responses than can be simulated in culture .

What roles do bacterial ubiquinone biosynthesis proteins play in bacterial virulence and pathogenicity?

Ubiquinone biosynthesis proteins, including UbiB, play crucial roles in bacterial pathogenicity through multiple mechanisms that can be methodologically investigated:

• Energy Production and Stress Adaptation:

  • Ubiquinone (Coenzyme Q) functions in the electron transport chain, providing energy required for virulence factor production

  • Under oxygen-limited conditions in plant tissues, efficient energy production systems become critical for bacterial survival and proliferation

  • Methodology: Compare growth rates and ATP production in wild-type versus ubiB mutant strains under aerobic and microaerobic conditions

• Oxidative Stress Response:

  • Ubiquinone acts as an antioxidant that helps bacteria cope with reactive oxygen species produced during plant defense responses

  • Methodology: Challenge bacteria with hydrogen peroxide or superoxide generators and measure survival rates and lipid peroxidation levels

• Connection to Virulence Factor Regulation:

  • Metabolic status affects global regulators of virulence

  • Similar to how FlhDC regulates both motility and production of plant cell wall-degrading enzymes in Pectobacterium , metabolic regulators connected to ubiquinone biosynthesis may influence multiple virulence pathways

  • Methodology: Perform transcriptomic analysis (RNA-Seq) of ubiB mutants compared to wild-type to identify affected virulence pathways

• Experimental Approach to Verify Role in Pathogenicity:

  • Generate ubiB knockout mutants using CRISPR-Cas9 gene editing (similar to the flhDC knockout approach described in the literature)

  • Assess virulence through plant inoculation assays measuring disease progression

  • Quantify extracellular enzyme production using specialized media with appropriate substrates

  • Evaluate motility through swimming or swarming assays on semi-solid media

These investigative approaches provide a framework for understanding how ubiquinone biosynthesis connects to virulence mechanisms in plant pathogenic bacteria.

What genetic approaches are available for studying the function of ubiB gene in Pectobacterium carotovorum?

Several genetic approaches can be employed to investigate ubiB function in Pectobacterium carotovorum:

• CRISPR-Cas9 Gene Editing:

  • This precise gene editing system has been successfully applied in Pectobacterium species to generate knockout mutants

  • Methodology:

    • Design 20 bp spacer oligonucleotides targeting ubiB using sgRNAcas9 software

    • Insert phosphorylated oligonucleotides into a suitable vector (e.g., pSGAb-km) using Golden Gate assembly

    • Transform cells with the vector and ssDNA donor DNA for ubiB gene

    • Confirm successful transformation by colony PCR

    • Cure cells on medium containing sucrose via sacB-counter selection to obtain ΔubiB strain

• Complementation Studies:

  • Essential for verifying phenotypes caused by gene deletion

  • Methodology:

    • Insert the complete ubiB gene into an expression vector (e.g., pBBR1MCS2) with a constitutive promoter

    • Transform the plasmid into the ΔubiB mutant to create a complemented strain

    • Assess restoration of wild-type phenotypes in the complemented strain

• Transcriptional Fusions:

  • For studying gene expression patterns

  • Methodology:

    • Create promoter-reporter fusions (e.g., ubiB promoter fused to GFP or luciferase)

    • Analyze expression under different conditions (nutrients, plant extracts, in planta)

    • Quantify fluorescence or luminescence to determine expression levels

• Site-Directed Mutagenesis:

  • For studying specific functional domains

  • Methodology:

    • Identify conserved amino acid residues in UbiB through sequence alignment

    • Create point mutations in these residues using inverse PCR or overlap extension PCR

    • Express mutated versions and assess functionality through complementation assays

These genetic approaches can be combined with phenotypic assays to comprehensively characterize ubiB function in bacterial metabolism and pathogenicity.

How can differential proteomics approaches be optimized to study UbiB protein interactions in Pectobacterium carotovorum?

Advanced differential proteomics approaches can be optimized for studying UbiB protein interactions through the following methodological framework:

• Protein Complex Purification Strategies:

  • Tandem Affinity Purification (TAP):

    • Create a TAP-tagged UbiB construct by fusing the ubiB gene with sequential affinity tags

    • Express in Pcc and purify protein complexes through sequential affinity steps

    • Identify interaction partners by mass spectrometry

  • Co-immunoprecipitation with stable isotope labeling:

    • Implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) by growing Pcc in media with heavy or light isotope-labeled amino acids

    • Perform immunoprecipitation with anti-UbiB antibodies

    • Quantify enriched proteins through mass spectrometry

• In situ Crosslinking Approaches:

  • Formaldehyde crosslinking to capture transient interactions

  • Photo-activatable crosslinkers for capturing spatial-specific interactions

  • Chemical crosslinkers with varying spacer lengths to accommodate different interaction distances

• Advanced Mass Spectrometry Analysis:

  • Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS):

    • Create a spectral library using data-dependent acquisition

    • Perform SWATH-MS for comprehensive, reproducible quantification

    • Apply to samples from different growth conditions to identify condition-specific interactions

  • Crosslinking MS (XL-MS):

    • Use bifunctional crosslinkers to capture protein-protein interactions

    • Enzymatically digest crosslinked proteins

    • Identify crosslinked peptides through specialized search algorithms

    • Map interaction surfaces at amino acid resolution

• Validation and Structural Analysis:

  • Bimolecular Fluorescence Complementation (BiFC):

    • Fuse UbiB and potential interaction partners to split fluorescent protein fragments

    • Visualize interaction through reconstituted fluorescence

  • Hydrogen-Deuterium Exchange MS (HDX-MS):

    • Identify regions of UbiB protected from exchange in the presence of interaction partners

    • Map binding interfaces at peptide resolution

This comprehensive proteomics workflow can be adapted from approaches used in studying other bacterial proteins, similar to the 2-DE coupled with MS methods that successfully identified 53 differentially expressed proteins in Pcc under various conditions .

What is the relationship between UbiB expression and stress response in Pectobacterium carotovorum during plant infection?

The relationship between UbiB expression and stress response during plant infection requires sophisticated experimental approaches to unravel:

• Time-Course Transcriptomics and Proteomics:

  • Extract RNA and protein from bacteria at various infection stages (early: 0-8h, middle: 8-16h, late: >16h after inoculation)

  • Analyze ubiB transcription by qRT-PCR and RNA-Seq

  • Quantify UbiB protein levels using targeted proteomics (Multiple Reaction Monitoring)

  • Correlate expression patterns with specific infection stages and host defense responses

• Stress-Specific Response Profiling:

  • Challenge bacteria with specific stressors encountered during infection:

    • Oxidative stress: H₂O₂, superoxide generators

    • pH stress: Acidic and alkaline conditions

    • Antimicrobial peptides: Plant defensins

    • Nutrient limitation: Iron restriction, carbon source variation

  • Monitor ubiB expression changes under each condition

  • Create a stress response map to identify which stressors most significantly affect ubiB expression

• In planta Expression Analysis:

  • Create a ubiB promoter-reporter fusion (e.g., GFP, luciferase)

  • Track expression directly in infected plant tissues using confocal microscopy or luminescence imaging

  • Correlate expression with localized plant defense responses

  • Compare expression in susceptible versus resistant host varieties

• Metabolomic Integration:

  • Analyze ubiquinone levels in wild-type and ubiB mutant strains during infection

  • Measure redox balance indicators (NAD⁺/NADH ratio, glutathione levels)

  • Correlate metabolic changes with stress response activation

  • Apply metabolic flux analysis to trace carbon flow through central metabolism

• Comparative Analysis Across Stress Response Regulons:

  • Examine overlap between ubiB regulation and known stress response regulons

  • Create a regulatory network model integrating transcription factors, small RNAs, and post-translational modifications

  • Identify key nodes connecting ubiquinone biosynthesis to virulence factor production

This multi-faceted approach can reveal how UbiB and ubiquinone biosynthesis are integrated into the complex stress response network that Pectobacterium carotovorum employs during plant colonization and disease progression.

How can CRISPR-Cas9 technology be optimized for targeting ubiB gene function in Pectobacterium carotovorum?

Optimizing CRISPR-Cas9 technology for targeting ubiB in Pectobacterium carotovorum requires careful consideration of several technical aspects:

• sgRNA Design Optimization:

  • Computational prediction of effective target sites:

    • Use multiple prediction algorithms (sgRNAcas9, CHOPCHOP, CRISPRscan) to identify optimal target sequences

    • Select targets with minimal off-target potential throughout the Pcc genome

    • Analyze GC content (40-60% ideal) and avoid homopolymer runs

  • Target functional domains based on protein structure prediction:

    • Identify conserved catalytic or binding domains in UbiB

    • Design sgRNAs targeting these critical regions for functional studies

• Delivery System Refinement:

  • Plasmid-based systems:

    • Optimize vector backbone for stability in Pcc (use pSGAb-km or similar validated vectors)

    • Test different selectable markers and origins of replication for compatibility

  • Ribonucleoprotein (RNP) delivery:

    • Purify Cas9 protein and combine with in vitro transcribed sgRNA

    • Optimize electroporation parameters (voltage, resistance, capacitance) specifically for Pcc

    • Test membrane-permeabilizing agents to enhance RNP uptake

• Repair Template Optimization:

  • Single-stranded DNA donors:

    • Optimize length (typically 80-120 nucleotides) and symmetry around cut site

    • Introduce silent mutations in the PAM or seed region to prevent re-cutting

  • Double-stranded DNA donors:

    • Include homology arms of optimized length (500-1000 bp)

    • Consider adding selection markers flanked by FRT sites for subsequent removal

• Validation and Screening Strategies:

  • High-throughput screening methods:

    • Design PCR primers spanning the edited region

    • Implement restriction fragment length polymorphism (RFLP) analysis if edit introduces or removes restriction sites

    • Develop high-resolution melt analysis protocols for detecting successful edits

  • Whole-genome sequencing:

    • Verify absence of off-target modifications

    • Confirm genetic stability of edited regions

• Multiplex Editing Approaches:

  • Simultaneous targeting of ubiB and related genes:

    • Design compatible sgRNAs targeting multiple genes in the ubiquinone biosynthesis pathway

    • Optimize scaffold sequences to enhance expression and processing of multiple sgRNAs

    • Develop efficient transformation protocols for delivering multiple editing components

This methodological framework, adapted from successful CRISPR-Cas9 applications in Pectobacterium species for targeting genes like flhDC , provides a comprehensive approach for precise genetic manipulation of ubiB.

What are the protein-protein interaction networks involving UbiB and how do they influence bacterial metabolism and virulence?

Investigating protein-protein interaction (PPI) networks involving UbiB requires a multi-layered experimental approach:

• High-Throughput Interaction Screening:

  • Bacterial Two-Hybrid (B2H) System:

    • Create fusion constructs of ubiB with the T18 fragment of adenylate cyclase

    • Screen against a genomic library fused to the T25 fragment

    • Select positive interactions on selective media containing X-gal

    • Validate interactions through reciprocal testing

  • Pull-Down Assays with Quantitative Proteomics:

    • Express His-tagged UbiB protein

    • Perform pull-down experiments under various physiological conditions

    • Identify binding partners through LC-MS/MS

    • Quantify interaction strengths using label-free quantification

• Functional Interaction Mapping:

  • Synthetic Genetic Array Analysis:

    • Generate ubiB conditional mutant (depletion strain)

    • Cross with genome-wide deletion library

    • Identify synthetic lethal or synthetic sick interactions

    • Map functional relationships between ubiB and other cellular pathways

  • Protein Complementation Assays:

    • Use split reporter proteins (luciferase or fluorescent proteins)

    • Systematically test protein pairs for interaction-dependent reporter reconstitution

    • Quantify signal strength as a measure of interaction intensity

• Dynamic Interaction Profiling:

  • Temporal Interaction Analysis:

    • Sample bacteria at different growth phases and during infection

    • Perform immunoprecipitation coupled with mass spectrometry

    • Identify condition-specific interaction partners

  • Proximity-Dependent Labeling:

    • Fuse UbiB to BioID or APEX2 enzymes

    • Allow in vivo biotinylation of proximity partners

    • Identify labeled proteins through streptavidin purification and MS

• Network Analysis and Visualization:

  • Integrate PPI data with transcriptomics and metabolomics:

    • Construct comprehensive network models using Cytoscape

    • Identify network motifs and hubs connecting UbiB to virulence systems

    • Apply machine learning algorithms to predict functional modules

  • Network Perturbation Analysis:

    • Systematically delete genes for interaction partners

    • Measure effects on ubiquinone biosynthesis, metabolism, and virulence

    • Identify critical nodes in the network

This comprehensive PPI analysis would reveal how UbiB connects to cellular processes beyond its primary role in ubiquinone biosynthesis, potentially explaining the pleiotrophic effects often observed when disrupting metabolic proteins in bacterial pathogens.

How do post-translational modifications affect UbiB function in Pectobacterium carotovorum and how can these be systematically studied?

Post-translational modifications (PTMs) of UbiB can significantly impact its function, requiring sophisticated methodological approaches for comprehensive characterization:

• Global PTM Profiling:

  • Enrichment Strategies:

    • Phosphorylation: Titanium dioxide or immobilized metal affinity chromatography

    • Acetylation: Anti-acetyllysine antibody enrichment

    • Oxidation: Biotin-switch technique for cysteine modifications

    • Methylation: Anti-methyllysine/arginine antibody enrichment

  • Mass Spectrometry Analysis:

    • Use electron transfer dissociation (ETD) fragmentation to preserve labile modifications

    • Implement parallel reaction monitoring (PRM) for targeted quantification of modified peptides

    • Apply data-independent acquisition for comprehensive modification mapping

• Site-Specific Mutagenesis of Modified Residues:

  • Identify modification sites through MS analysis

  • Create point mutations replacing modifiable residues:

    • Phosphorylation: Ser/Thr/Tyr → Ala (phospho-null) or Asp/Glu (phospho-mimetic)

    • Acetylation: Lys → Arg (acetylation-resistant) or Gln (acetylation-mimetic)

    • Oxidation: Cys → Ser (redox-insensitive)

  • Assess functional consequences through activity assays and in vivo virulence tests

• Dynamic PTM Analysis:

  • Time-Course Experiments:

    • Subject bacteria to relevant stresses (oxidative, pH, temperature)

    • Sample at multiple time points post-stress

    • Quantify changes in modification levels

    • Correlate with ubiquinone production and bacterial fitness

  • In Planta PTM Profiling:

    • Isolate bacteria from infected plant tissue at different infection stages

    • Apply targeted proteomics to measure specific modifications

    • Compare modification patterns between in vitro and in planta conditions

• PTM Crosstalk Analysis:

  • Investigate interdependence between different modifications:

    • Determine if one modification influences the occurrence of others

    • Map modification "codes" that may regulate UbiB activity or interactions

    • Create modification-specific antibodies for tracking modified forms in situ

• Enzyme Identification:

  • Discover the enzymes responsible for UbiB modifications:

    • Perform systematic screening of kinase, acetyltransferase, or other modifier enzyme mutants

    • Implement in vitro modification assays with purified enzymes

    • Use proximity labeling to identify enzymes that physically associate with UbiB

These systematic approaches would provide unprecedented insights into the regulatory mechanisms controlling UbiB function and how they relate to bacterial adaptation and virulence, similar to the protein regulation mechanisms observed in other Pectobacterium proteins studied using 2-DE coupled with MS .

Comparative Analysis of Protein Expression Methods for Studying UbiB in Pectobacterium

Differentially Expressed Proteins in Pectobacterium Under Various Conditions

Data adapted from differential protein expression analysis in Pectobacterium carotovorum

Recommended Experimental Workflow for UbiB Function Analysis

• Gene Characterization Phase:

  • Sequence analysis and structural prediction

  • Protein expression and purification

  • Biochemical activity assays

  • Time: 2-3 months

• Genetic Manipulation Phase:

  • CRISPR-Cas9 knockout generation

  • Complementation strain creation

  • Phenotypic characterization

  • Time: 3-4 months

• Functional Analysis Phase:

  • Proteomics and interactome studies

  • Metabolic profiling of ubiquinone pathway

  • Stress response characterization

  • Time: 3-4 months

• Pathogenicity Assessment Phase:

  • Plant infection assays

  • Virulence factor quantification

  • Comparative analysis with related enzymes

  • Time: 2-3 months

• Integration and Modeling Phase:

  • Data integration and network construction

  • Systems biology modeling

  • Validation experiments

  • Time: 3-4 months

Total estimated project timeline: 13-18 months for comprehensive characterization