Recombinant Xanthomonas campestris pv. vesicatoria Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is known about UbiB's involvement in bacterial pathogenicity?

While UbiB itself has not been directly linked to pathogenicity, the ubiquinone biosynthesis pathway it participates in has significant implications for bacterial virulence. Research indicates that proper ubiquinone production is essential for aerobic respiration in bacteria, which directly impacts their metabolic capacity and ability to proliferate in host environments. Studies with related bacteria have shown that mutations in ubiquinone biosynthesis genes can attenuate virulence. For instance, a Salmonella ubiK mutant (UbiK being a protein that interacts with UbiB) showed defects in proliferation in macrophages and mouse infection models . This suggests that the ubiquinone biosynthesis pathway, including UbiB, may be indirectly involved in pathogenicity by supporting the metabolic requirements for bacterial survival during infection.

What are the optimal conditions for expressing and purifying recombinant UbiB?

For optimal expression and purification of recombinant UbiB from X. campestris pv. vesicatoria, researchers should consider the following protocol based on successful approaches with related proteins:

• Expression system: E. coli BL21(DE3) has been successfully used for expression of Ubi proteins. Similar systems can be adapted for UbiB expression.

• Vector selection: pACYCDuet-1 or similar expression vectors have yielded high protein expression levels for related Ubi proteins .

• Induction conditions:

  • Grow cultures to OD600 of 0.6-0.8

  • Induce with 0.5-1.0 mM IPTG

  • Continue expression at 18-25°C for 16-20 hours to enhance protein solubility

• Purification strategy:

  • Harvest cells and lyse in Tris-based buffer (typically 50 mM Tris-HCl, pH 8.0, 300 mM NaCl)

  • Include protease inhibitors to prevent degradation

  • Consider affinity tags (His6 or S-tag) for initial purification

  • Follow with size exclusion chromatography for higher purity

• Storage conditions: Based on product information, the purified protein should be stored in Tris-based buffer with 50% glycerol at -20°C for short-term storage or -80°C for long-term storage . Working aliquots can be maintained at 4°C for up to one week.

Note: Repeated freeze-thaw cycles should be avoided to maintain protein activity and stability .

How can researchers effectively study UbiB-protein interactions?

Several complementary approaches have proven effective for studying UbiB and other Ubi protein interactions:

• Bacterial Two-Hybrid (BACTH) System: This approach has successfully identified interactions between UbiB and other Ubi proteins. The system is based on functional complementation between adenylate cyclase fragments (T18 and T25) expressed from compatible plasmids. When testing UbiB interactions:

  • Create fusion constructs with UbiB fused to both T18 and T25 domains

  • Test interactions in both orientations, as directionality matters (UbiB interactions with UbiK were detected only when UbiK was fused to T18)

  • Include positive controls (known interacting partners) and negative controls

• Co-expression and Co-purification Assays:

  • Express UbiB with a different tag (e.g., S-tag) than potential partners (e.g., His6-tag)

  • Purify the complex using sequential affinity chromatography

  • Verify co-elution by SDS-PAGE and western blotting

• Pull-down Assays:

  • Express UbiB as a fusion with MBP (maltose-binding protein)

  • Purify the fusion protein using amylose resin

  • Detect interacting partners by co-elution followed by immunoblotting

• Yeast Two-Hybrid System:

  • Useful for mapping specific interaction domains

  • Can identify direct protein-protein interactions

  • Has been successfully used to map interaction domains in similar Ubi proteins

What analytical techniques should be used to characterize UbiB structure and oligomeric state?

Based on successful approaches with related proteins, researchers should consider these analytical techniques:

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS):

  • Determines absolute molar mass and oligomeric state

  • Provides information on shape through hydrodynamic radius measurements

  • Can detect heterogeneity in the sample

  • Has been successfully used with related Ubi proteins, revealing trimeric structures with elongated hydrodynamic shapes

• Circular Dichroism (CD) Spectroscopy:

  • Analyzes secondary structure elements

  • Identify the prevalence of α-helices (minima at 208 and 222 nm) or β-sheets

  • Monitors thermal stability and conformational changes

• X-ray Crystallography:

  • Provides high-resolution structural information

  • Requires highly pure, homogeneous protein samples

  • May need extensive screening for crystallization conditions

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

  • Maps solvent-accessible regions and protein dynamics

  • Identifies potential ligand binding sites

  • Particularly useful when crystallography is challenging

What is known about UbiB's interactions with other proteins in the ubiquinone biosynthesis pathway?

Research using bacterial two-hybrid (BACTH) screening has revealed that UbiB interacts with several proteins in the ubiquinone biosynthesis pathway, though these interactions show directional preference. Specifically:

• UbiB-UbiK Interaction: UbiB was found to interact with UbiK, but only when UbiK was fused to the T18 moiety in the bacterial two-hybrid system . This directionality suggests a specific structural constraint in their interaction.

• Network of Interactions: While the direct interaction partners of UbiB have not been fully characterized, the ubiquinone biosynthesis pathway involves multiple protein-protein interactions. UbiK, which interacts with UbiB, has been shown to interact with several other Ubi proteins including UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX .

• UbiK-UbiJ Complex: A significant finding is that UbiK forms a stable complex with UbiJ in a 2:1 stoichiometry (UbiK₂-UbiJ₁), and this interaction involves the C-terminal 50 amino acids of UbiJ . This complex formation might have implications for UbiB function, potentially suggesting the existence of a larger multiprotein complex involved in ubiquinone biosynthesis.

• Functional Significance: The interaction network among Ubi proteins suggests that ubiquinone biosynthesis may involve a multienzyme complex or "metabolon" rather than isolated enzymatic reactions. This organization could enhance the efficiency of the pathway by facilitating substrate channeling between sequential enzymatic steps.

How does the absence of UbiB affect ubiquinone biosynthesis and bacterial growth?

The specific effects of UbiB deficiency have not been directly reported in the provided sources, but insights can be drawn from studies on related proteins in the ubiquinone biosynthesis pathway:

• Impact on Ubiquinone Levels: Mutations in ubiK, a gene encoding a protein that interacts with UbiB, resulted in decreased ubiquinone (UQ₈) content to approximately 18% of wild-type levels in E. coli . Given the interaction between UbiB and UbiK, UbiB deficiency might cause similar reductions in ubiquinone production.

• Accumulation of Intermediates: The ubiK mutant accumulated octaprenylphenol, an early intermediate in the UQ biosynthetic pathway . This suggests that disruption of components in this pathway, potentially including UbiB, may lead to the buildup of specific intermediates, providing clues about the step(s) affected.

• Growth Phenotypes: While a Salmonella ubiK mutant showed growth deficiency under aerobic conditions, particularly at higher temperatures, and was defective for proliferation in macrophages and mice infection models , the specific growth phenotypes of UbiB-deficient strains remain to be characterized.

• Oxygen Dependence: Interestingly, UbiK was found to be dispensable for UQ biosynthesis under anaerobic conditions, despite being expressed in the absence of oxygen . This suggests that the requirement for certain components of the ubiquinone biosynthesis pathway, potentially including UbiB, may differ depending on oxygen availability.

How does UbiB compare functionally to homologous proteins in other bacterial species?

UbiB belongs to a family of proteins involved in ubiquinone biosynthesis that is widely distributed across bacterial species. Comparative analysis reveals:

What genetic approaches are most effective for studying UbiB function in vivo?

Several genetic strategies have proven effective for investigating the function of proteins involved in ubiquinone biosynthesis, which can be applied to UbiB studies:

• Gene Deletion/Knockout Studies:

  • Construction of clean ubiB deletion mutants using homologous recombination or CRISPR-Cas9 systems

  • Complementation with plasmid-borne wild-type ubiB to confirm phenotypes are specifically due to UbiB absence

  • Analysis of growth characteristics under various conditions (aerobic vs. anaerobic, different carbon sources, temperature stress)

• Site-Directed Mutagenesis:

  • Target conserved residues for modification based on sequence alignments

  • Create point mutations in catalytic domains to determine essential residues

  • Analyze the effects of mutations on protein function and ubiquinone production

• Fluorescent Protein Tagging:

  • Create C-terminal or N-terminal GFP fusions for localization studies

  • Ensure tags do not interfere with protein function through complementation assays

  • Use techniques similar to those employed for studying XopJ localization in X. campestris, which involved GFP fusions and confocal laser scanning microscopy

• Protein-Protein Interaction Analysis:

  • Implement bacterial two-hybrid systems with both T18 and T25 fusions to account for directional preferences in interactions

  • Use pull-down assays with MBP-tagged UbiB to identify interaction partners

  • Apply crosslinking approaches to capture transient interactions

What analytical methods should be used to quantify ubiquinone and its intermediates when studying UbiB function?

To effectively analyze ubiquinone and its precursors in UbiB functional studies, researchers should employ these analytical techniques:

• High-Performance Liquid Chromatography (HPLC) with Electrochemical Detection (ECD):

  • Has been successfully used to quantify UQ₈ levels in bacterial samples

  • Provides sensitive detection of ubiquinone and related quinones

  • Can be optimized to detect specific wavelengths (UQ₈ at specific wavelength, DMK₈ and MK₈ at 247 nm)

• HPLC coupled with UV-Visible Detection:

  • Useful for detecting ubiquinone intermediates that absorb at specific wavelengths

  • Capable of detecting accumulated intermediates like octaprenylphenol at 275 nm

• Liquid Chromatography-Mass Spectrometry (LC-MS):

  • Provides both quantification and structural identification of intermediates

  • Enables detection of novel or unexpected metabolites

  • Allows for isotope labeling experiments to track metabolic flux

• Sample Preparation Protocol:

  • Extract bacterial cultures in late exponential phase

  • Use lipid extraction methods with appropriate organic solvents (e.g., hexane/petroleum ether)

  • Include internal standards for accurate quantification

  • Concentrate samples under nitrogen to prevent oxidation

What are the most effective approaches for studying UbiB in the context of bacterial pathogenesis?

To investigate UbiB's potential role in pathogenesis, researchers should consider these approaches:

• Infection Models:

  • Develop plant infection assays using ubiB mutant strains of X. campestris

  • Compare disease progression and bacterial proliferation between wild-type and mutant strains

  • Use microscopy to track bacterial colonization patterns

• Cellular Assays:

  • Assess bacterial survival in plant cells or tissues

  • Evaluate the ability of ubiB mutants to trigger defense responses

  • Study whether UbiB affects type III secretion system functionality, given the importance of this system in X. campestris pathogenicity

• Metabolic Analysis During Infection:

  • Monitor ubiquinone levels during different stages of infection

  • Assess whether host conditions affect ubiquinone biosynthesis

  • Determine if environmental stresses encountered during infection alter UbiB expression or function

• Comparative Studies with Related Proteins:

  • Based on findings that a Salmonella ubiK mutant was defective for proliferation in macrophages and mice infection , examine whether UbiB deficiency produces similar effects

  • Investigate potential connections between ubiquinone biosynthesis and virulence factor expression

What are common issues encountered when working with recombinant UbiB and how can they be addressed?

Researchers working with recombinant UbiB may encounter several challenges:

• Protein Solubility Issues:

  • Problem: UbiB may form inclusion bodies when overexpressed

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration, or use solubility-enhancing fusion tags (MBP, SUMO)

  • Alternatively, consider refolding protocols if inclusion bodies form

• Protein Stability Concerns:

  • Problem: UbiB may degrade during purification or storage

  • Solution: Include protease inhibitors, optimize buffer conditions (pH, salt concentration), and store in 50% glycerol at -20°C or -80°C as indicated in product information

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Activity Loss During Purification:

  • Problem: UbiB may lose activity during purification steps

  • Solution: Minimize purification steps, maintain reducing conditions if cysteines are present, and assess functionality after each purification step

• Interaction Detection Challenges:

  • Problem: Some protein-protein interactions may be missed due to directional preferences

  • Solution: Test interactions with tag fusions at both N-terminus and C-terminus, as demonstrated by the directional preference observed in UbiB-UbiK interactions

How should researchers interpret conflicting data regarding UbiB function?

When faced with contradictory results regarding UbiB function, researchers should apply these analytical strategies:

• Experimental Design Analysis:

  • Evaluate differences in experimental conditions (bacterial strains, growth conditions, oxygen availability)

  • Consider the effects of different protein tags or fusion constructs

  • Assess whether complementation controls were appropriately implemented

• Contextual Factors:

  • Examine oxygen-dependent effects, as some Ubi proteins function differently under aerobic versus anaerobic conditions

  • Consider strain-specific differences, as UbiB function may vary between different bacterial species or even pathovars

  • Evaluate potential compensatory mechanisms that might mask phenotypes

• Integration of Multiple Approaches:

  • Compare in vitro biochemical data with in vivo phenotypic analyses

  • Corroborate protein-protein interaction results using multiple methodologies

  • Validate genetic findings with direct protein function measurements

• Pathway Context:

  • Consider UbiB's role within the larger ubiquinone biosynthesis pathway

  • Assess effects on upstream and downstream metabolites

  • Examine potential redundancy with other proteins in the pathway

What are the most promising research avenues for elucidating UbiB's precise biochemical function?

Several research directions hold particular promise for uncovering UbiB's specific role:

• Structural Biology Approaches:

  • Determine UbiB's three-dimensional structure through X-ray crystallography or cryo-EM

  • Identify potential active sites or substrate binding pockets

  • Perform structure-guided mutagenesis to test functional hypotheses

• Biochemical Activity Assays:

  • Develop in vitro assays to test specific enzymatic activities

  • Explore potential kinase, ATPase, or oxidoreductase functions

  • Identify potential substrates through metabolite profiling of mutants

• Systems Biology Approaches:

  • Apply transcriptomics and proteomics to understand the broader effects of UbiB deficiency

  • Use metabolic flux analysis to track changes in ubiquinone biosynthesis pathway dynamics

  • Implement network analysis to position UbiB within the broader cellular metabolism

• Protein Complex Analysis:

  • Investigate whether UbiB forms part of a larger complex similar to the UbiK-UbiJ complex

  • Apply blue native PAGE, size exclusion chromatography, and cross-linking mass spectrometry to characterize potential multiprotein assemblies

  • Determine stoichiometry and stability of protein complexes involving UbiB

How might UbiB be exploited as a potential antibiotic target?

The essential role of ubiquinone in bacterial respiration makes UbiB a potential antibiotic target worthy of exploration:

• Target Validation Approaches:

  • Confirm essentiality of UbiB under various growth conditions

  • Determine whether chemical inhibition of UbiB produces bacteriostatic or bactericidal effects

  • Assess species specificity to identify differences between bacterial and host pathways

• Inhibitor Discovery Strategies:

  • Develop high-throughput screening assays for UbiB activity

  • Implement structure-based virtual screening once protein structure is available

  • Design peptidomimetics that disrupt essential protein-protein interactions

• Therapeutic Potential Assessment:

  • Evaluate inhibitor efficacy in cellular and animal infection models

  • Assess potential for resistance development

  • Determine spectrum of activity across different bacterial pathogens

• Combination Therapy Approaches:

  • Explore synergistic effects with existing antibiotics

  • Target multiple steps in the ubiquinone biosynthesis pathway simultaneously

  • Investigate potential to enhance host immune clearance through metabolic weakening