Recombinant Xanthomonas axonopodis pv. citri Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the role of UbiB in ubiquinone biosynthesis in Xanthomonas?

UbiB plays a crucial role in the biosynthesis of Coenzyme Q8 (CoQ8, ubiquinone) in Xanthomonas species. Research in X. campestris pv. campestris demonstrates that UbiB is involved in early steps of the ubiquinone biosynthetic pathway. While not directly catalytic, it is essential for efficient CoQ8 biosynthesis.

Methodological approach to determine function:

• Generate targeted deletion mutants of ubiB in Xanthomonas

• Quantify CoQ8 levels using HPLC or LC-MS/MS in wild-type vs. mutant strains

• Analyze accumulating intermediates to pinpoint the affected step

• Perform complementation assays with plasmid-borne ubiB to confirm phenotype specificity

Deletion of ubiB in X. campestris pv. campestris significantly decreases CoQ8 biosynthesis and leads to accumulation of octaprenyl hydroxyl benzoate (OHB), suggesting UbiB facilitates the conversion of OHB to later intermediates in the pathway . This differs from E. coli, where ubiB deletion results in octaprenylphenol pyrophosphate (OPP) accumulation, indicating divergent biosynthetic mechanisms between species .

How does UbiB function within protein complexes in Xanthomonas?

UbiB appears to function as part of a multi-protein complex in Xanthomonas. Experimental evidence reveals key interactions that affect its functionality.

Protein interaction analysis methods:

• Yeast two-hybrid (Y2H) analysis demonstrates strong binding between UbiK and UbiB in X. campestris pv. campestris

• UbiK also interacts with UbiJ, suggesting a potential three-protein regulatory complex

• Complementation studies show that overexpression of UbiB alone has limited effect in ubiJ mutants

• Complete restoration of CoQ8 levels requires the entire UbiJ-UbiB gene cluster

This evidence supports a model where UbiB, UbiJ, and UbiK form a complex involved in regulating CoQ8 biosynthesis in Xanthomonas . Notably, these interaction patterns show species specificity - UbiK from X. campestris interacts with UbiB and UbiJ from X. campestris but has no binding affinity to E. coli homologs .

What phenotypic changes occur when ubiB is deleted in Xanthomonas?

Deletion of ubiB in Xanthomonas produces multiple phenotypic alterations that can be assessed through various experimental approaches.

Multi-dimensional phenotypic analysis:

• Quantify CoQ8 levels: Wild-type X. campestris contains 145.9 pmol/mg wet weight, while ΔubiB shows significantly reduced levels

• Analyze metabolite accumulation: ΔubiB accumulates OHB, a precursor in the CoQ8 pathway

• Measure virulence: Lesion length assays on host plants reveal diminished pathogenicity

• Assess cellular functions: Evaluate motility, adhesion, biofilm formation, and stress response

Results show that ubiB deletion significantly impairs bacterial virulence in plant infection models. When inoculated on Chinese radish, the ΔubiB mutant produced significantly smaller lesions compared to wild-type X. campestris . These virulence defects can be reversed through genetic complementation with a plasmid expressing the functional ubiB gene.

How can researchers differentiate between direct and indirect effects of ubiB deletion?

Distinguishing direct from indirect effects of ubiB deletion requires careful experimental design and controls.

Methodological approaches:

• Construct polar and non-polar deletion mutants to discriminate ubiB-specific effects from operon disruption

• Perform complementation with ubiB alone versus the complete ubiJ-ubiB gene cluster

• Create point mutations in conserved domains rather than complete gene deletions

• Use inducible expression systems to observe acute versus chronic effects of UbiB absence

A critical consideration is the genomic context - in X. campestris pv. campestris, ubiB is located downstream of ubiJ . When analyzing a ubiJ mutant, researchers confirmed that reduced CoQ8 biosynthesis was not due to polar effects on ubiB expression by demonstrating that overexpression of ubiB alone had minimal effect on CoQ8 levels in the ΔubiJ strain .

How does UbiB contribute to bacterial virulence in plant hosts?

UbiB significantly impacts Xanthomonas virulence through multiple potential mechanisms that can be experimentally evaluated.

Methodological approach to virulence assessment:

• Perform scissor clipping inoculation method on host plants

• Measure and compare lesion length between wild-type and mutant strains

• Quantify bacterial populations in planta over time

• Assess expression of virulence-associated genes in the ubiB mutant

In X. campestris pv. campestris, the ΔubiB mutant shows significantly reduced lesion lengths on Chinese radish compared to the wild-type strain . This virulence reduction is comparable to that observed in other CoQ8 biosynthesis mutants (Δcoq7, ΔubiJ, and ΔubiK) . The correlation between CoQ8 biosynthesis and virulence suggests that proper respiratory function is essential for successful host colonization and infection.

What are the optimal expression and purification conditions for recombinant X. axonopodis pv. citri UbiB?

Optimizing the expression and purification of recombinant UbiB presents unique challenges due to its biochemical properties and tendency to form complexes.

Recommended optimization strategy:

• Expression system selection:

  • Use E. coli BL21(DE3) with codon optimization to address potential codon bias between Xanthomonas and E. coli

  • Test both T7 and tightly controlled systems (e.g., pET, pBAD) to minimize toxicity

  • Consider co-expression with UbiJ and UbiK for proper complex formation and folding

• Construct design and conditions:

  • Test both N-terminal and C-terminal His-tags (6-10×His) to identify optimal configuration

  • Compare solubility with different fusion partners (MBP, SUMO, GST)

  • Optimize expression at reduced temperatures (16-20°C) to enhance folding

  • Use low IPTG concentrations (0.1-0.5 mM) and extended expression times (16-24 hours)

• Purification strategy:

  • Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Initial capture using IMAC (Ni-NTA) with gradient elution (20-250 mM imidazole)

  • Secondary purification via ion exchange chromatography

  • Final polishing using size exclusion chromatography in buffer containing 6% trehalose

Commercial preparations of similar proteins are typically stored as lyophilized powders and reconstituted in deionized water to 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C .

What experimental approaches can resolve the specific enzymatic function of UbiB?

Despite extensive research, the precise enzymatic function of UbiB remains unresolved. A multi-faceted approach is necessary to elucidate its biochemical role.

Comprehensive functional characterization strategy:

• In vitro biochemical assays:

  • Express and purify UbiB alone and in complex with UbiJ and UbiK

  • Test activity with potential substrates including OHB (which accumulates in ΔubiB mutants)

  • Screen various cofactors (NAD+, NADH, NADP+, NADPH, ATP, etc.)

  • Analyze reaction products using LC-MS/MS with multiple reaction monitoring

• Structural and computational studies:

  • Obtain high-resolution protein structures through X-ray crystallography or cryo-EM

  • Perform molecular docking with putative substrates

  • Identify conserved motifs through bioinformatic analysis of UbiB homologs

  • Compare with predicted protein kinase family members

• Site-directed mutagenesis:

  • Target conserved residues in predicted catalytic or binding domains

  • Assess impact of mutations on complementation efficiency in ΔubiB strains

  • Evaluate effects on protein-protein interactions, particularly with UbiK

  • Compare mutations in Xanthomonas UbiB with equivalent positions in E. coli UbiB

Current evidence suggests UbiB may function in the conversion of OHB to downstream intermediates in Xanthomonas, potentially through regulatory rather than direct catalytic action .

How do environmental conditions influence UbiB expression and function?

Understanding how environmental factors affect UbiB expression and function provides insight into its role during infection and adaptation processes.

Comprehensive environmental analysis approach:

• Transcriptional regulation:

  • Construct reporter fusions with the ubiB promoter (e.g., ubiBp-gfp, ubiBp-lux)

  • Monitor expression under various conditions:

    • Plant environment-mimicking media

    • Different temperatures (22°C, 28°C, 37°C)

    • Oxidative stress (H₂O₂, paraquat)

    • Nutrient limitation

    • pH variation (5.0-8.0)

• In planta expression analysis:

  • Isolate bacteria from infected plant tissue at different infection stages

  • Perform RT-qPCR targeting ubiB and related genes

  • Use RNA-seq to capture global transcriptional changes

  • Compare expression in compatible vs. incompatible plant interactions

• Protein stability and complex formation:

  • Analyze UbiB-UbiJ-UbiK complex formation under different environmental conditions

  • Assess protein stability and turnover rates using pulse-chase experiments

  • Evaluate post-translational modifications in response to environmental stresses

Previous research has shown that genes involved in LPS biosynthesis in Xanthomonas show altered expression in media that mimic plant environments , suggesting similar regulation may occur for ubiquinone biosynthesis genes like ubiB.

How do UbiB-mediated metabolic changes impact broader bacterial physiology?

UbiB's role in ubiquinone biosynthesis has far-reaching implications for bacterial metabolism and adaptation. Understanding these broader impacts requires integrative approaches.

Systems biology strategy:

• Metabolomic analysis:

  • Compare wild-type and ΔubiB metabolic profiles using untargeted LC-MS

  • Quantify cellular energetics (ATP/ADP ratio, NADH/NAD+ ratio)

  • Measure membrane potential using fluorescent probes

  • Assess redox status using glutathione/oxidized glutathione measurements

• Stress response evaluation:

  • Challenge wild-type and ΔubiB strains with various stressors:

    • Oxidative stress (H₂O₂, superoxide)

    • Membrane stress (detergents, antimicrobial peptides)

    • pH extremes

    • Temperature shifts

  • Monitor survival, growth rates, and morphological changes

  • Assess expression of stress response genes

• Motility and biofilm analysis:

  • Quantify swimming, swarming, and twitching motility

  • Measure biofilm formation using crystal violet staining and confocal microscopy

  • Analyze extracellular polysaccharide production

  • Evaluate cell-to-cell communication and quorum sensing

Research on Xanthomonas LPS mutants has shown increased sensitivity to external stresses and differences in bacterial motilities, in vivo and in vitro adhesion, and biofilm formation . Similar phenotypes may be observed in UbiB mutants due to the essential role of ubiquinone in cellular energetics.

How can comparative genomic approaches inform UbiB functional evolution across Xanthomonas species?

Comparative genomics provides insights into the evolution of UbiB function and its relationship to host specificity and virulence adaptation.

Comparative genomic workflow:

• Sequence analysis across Xanthomonas pathovars:

  • Collect ubiB sequences from diverse Xanthomonas species and pathovars

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Identify conserved domains and pathovar-specific variations

  • Calculate selection pressures (dN/dS ratios) on different protein regions

• Genomic context comparison:

  • Analyze the organization of the ubi gene cluster across species

  • Identify syntenic regions and gene rearrangements

  • Compare promoter sequences to identify regulatory differences

  • Assess horizontal gene transfer signatures

• Structure-function correlation:

  • Map sequence variations onto structural models

  • Identify positions likely to affect substrate specificity or protein interactions

  • Design chimeric proteins to test functional determinants

  • Perform cross-species complementation experiments

This table summarizes key Xanthomonas pathovars for comparative analysis:

Understanding the evolutionary patterns of UbiB across Xanthomonas species may reveal adaptations specific to different plant hosts and infection strategies .

What are the molecular mechanisms by which UbiB influences bacterial virulence?

The link between UbiB function and virulence requires detailed investigation of multiple potential mechanisms.

Multi-level virulence analysis approach:

• Type III secretion system (T3SS) interaction:

  • Quantify expression and function of T3SS components in ΔubiB mutants

  • Measure secretion efficiency of known effector proteins

  • Assess assembly of the T3SS needle complex

  • Evaluate translocation of effectors into plant cells

• Cellular energetics and virulence:

  • Measure ATP production in wild-type vs. ΔubiB strains during infection

  • Analyze energy-dependent processes (motility, secretion, replication)

  • Assess membrane potential using fluorescent dyes

  • Correlate energy status with virulence gene expression

• Oxidative stress response:

  • Challenge bacteria with plant-derived reactive oxygen species (ROS)

  • Compare survival of wild-type and ΔubiB strains

  • Measure expression of ROS detoxification enzymes

  • Assess membrane integrity under oxidative stress

• Host recognition evasion:

  • Analyze pathogen-associated molecular pattern (PAMP) exposure in ΔubiB mutants

  • Measure plant defense responses to wild-type vs. mutant bacteria

  • Assess flagellin and LPS modifications that might affect recognition

  • Evaluate effector-triggered immunity responses

Research on other Xanthomonas virulence determinants has shown that LPS modifications affect basal defense responses in both host and non-host plants . Similar mechanisms may connect UbiB function to virulence through modulation of bacterial surface properties or metabolic adaptation during infection.

How can genomic-phenotypic correlations guide the development of UbiB-targeted antimicrobials?

Leveraging genomic-phenotypic correlations can accelerate the development of targeted antimicrobials against Xanthomonas pathogens.

Integrated drug discovery framework:

• Target validation:

  • Confirm essentiality of UbiB across multiple Xanthomonas pathovars

  • Quantify growth inhibition and virulence reduction in ΔubiB mutants

  • Assess potential for resistance development

  • Compare with homologs in beneficial bacteria to evaluate selectivity potential

• Structure-based drug design:

  • Determine crystal structure of UbiB alone and in complex with UbiJ/UbiK

  • Identify druggable pockets through computational analysis

  • Perform virtual screening against compound libraries

  • Design and synthesize compounds targeting specific structural features

• Phenotypic screening:

  • Develop high-throughput assays for UbiB function

  • Screen compound libraries for CoQ8 biosynthesis inhibition

  • Evaluate effects on bacterial growth and virulence

  • Assess specificity against different bacterial species

• Mode of action studies:

  • Confirm target engagement using thermal shift assays or cellular thermal shift assays

  • Analyze metabolite accumulation patterns to verify on-target effects

  • Generate and characterize resistant mutants

  • Perform transcriptomic analysis to identify compensatory mechanisms

Targeting UbiB offers a promising approach for controlling Xanthomonas infections as it plays a dual role in normal metabolism and virulence . The high conservation of UbiB across Xanthomonas species suggests broad-spectrum potential, while structural differences from plant homologs may enable selective targeting.

What are the challenges in studying UbiB interactions with host plant defenses during infection?

Understanding how UbiB-dependent processes interact with plant immune responses presents unique experimental challenges that require specialized approaches.

Advanced in planta research strategy:

• Tissue-specific infection dynamics:

  • Engineer Xanthomonas strains expressing fluorescently-tagged UbiB

  • Use confocal microscopy to track bacterial localization in plant tissues

  • Correlate UbiB expression levels with infection stages

  • Compare wild-type and modified UbiB variants for plant defense triggering

• Dual RNA-seq analysis:

  • Simultaneously capture bacterial and plant transcriptomes during infection

  • Compare wild-type and ΔubiB infections to identify differentially expressed plant defense genes

  • Track temporal changes in gene expression patterns

  • Validate key findings using RT-qPCR and reporter gene assays

• Metabolic crosstalk investigation:

  • Analyze metabolite exchange between bacteria and host using isotope labeling

  • Identify plant-derived molecules affecting UbiB expression or function

  • Measure bacterial metabolic adaptations in different plant tissues

  • Assess impact of plant defense compounds on UbiB-dependent processes

• Microscale analytical techniques:

  • Use laser capture microdissection to isolate bacteria from specific infection sites

  • Apply single-cell approaches to capture bacterial heterogeneity during infection

  • Develop microfluidic devices to mimic plant microenvironments

  • Combine with imaging mass spectrometry for spatial metabolomics

Previous research shows that Xanthomonas LPS mutants produce a lower increase in the expression of host plant defense-related genes compared to wild-type strains . Similarly, UbiB may influence host recognition patterns, potentially through effects on bacterial viability, surface structures, or secreted molecules.