Recombinant Erwinia tasmaniensis Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Erwinia tasmaniensis and what is its taxonomic classification?

Erwinia tasmaniensis is a non-pathogenic bacterium belonging to the genus Erwinia within the family Enterobacteriaceae. The complete taxonomic classification is as follows:

Erwinia tasmaniensis strain Et1/99 is the type strain, isolated from apple flowers in Tasmania, Australia . The organism represents an epiphytic plant bacterium that is phylogenetically related to pathogenic species such as E. amylovora and E. pyrifoliae, which cause fire blight and Asian pear shoot blight, respectively . Unlike its pathogenic relatives, E. tasmaniensis is considered non-pathogenic to plants and may even compete with pathogenic bacteria during initial colonization, potentially serving as a biological control agent against fire blight .

What is the genomic structure of Erwinia tasmaniensis?

Erwinia tasmaniensis Et1/99 has a fully sequenced genome consisting of a 3.9 Mb circular chromosome and five plasmids . The genome sequencing was completed by the Max Planck Institute for Molecular Genetics and the National Center for Biotechnology Information in 2007-2008 . The complete genome has been assigned multiple accession numbers (NC_010693, NC_010694, NC_010695, NC_010696, NC_010697, NC_010699), reflecting its chromosome and plasmid components .

A distinguishing genomic feature of E. tasmaniensis Et1/99 is the complete absence of the sorbitol operon, which may contribute to its inability to invade fire blight host plants, unlike E. amylovora which relies on sorbitol utilization for virulence . The genome also contains secretion systems including the hypersensitive response type III pathway commonly found in many plant pathogens, though differences in virulence-related factors differentiate it from pathogenic Erwinia species .

What is the function of ubiquinone biosynthesis protein UbiB?

The UbiB protein plays a critical role in ubiquinone (coenzyme Q) biosynthesis. Ubiquinone is an essential component of the electron transport chain in bacterial respiration. The probable ubiquinone biosynthesis protein UbiB (ubiB) from Erwinia tasmaniensis specifically participates in the early steps of ubiquinone synthesis.

Functionally, UbiB is believed to act as a kinase-like protein that facilitates hydroxylation reactions in the ubiquinone biosynthetic pathway. The protein contains characteristic domains including a kinase-like region and is involved in the conversion of early ubiquinone precursors. Research on homologous UbiB proteins in other bacterial species suggests it plays a crucial role in aerobic respiration and oxidative stress responses, making it essential for bacterial adaptation to oxygen-rich environments.

How can I optimize expression and purification of recombinant UbiB protein?

Optimizing expression and purification of recombinant UbiB requires a systematic approach. Design of Experiments (DoE) methodology is particularly valuable for this purpose, as it allows for the simultaneous evaluation of multiple parameters while minimizing experimental runs.

When optimizing UbiB expression and purification, consider the following approach:

• Expression system selection: E. coli BL21(DE3) is commonly used for recombinant protein expression, but alternative systems may be more suitable depending on your specific research needs.

• DoE implementation: Identify key factors affecting expression and purification. For UbiB protein, critical factors typically include:

  • Induction temperature (typically 16-37°C)

  • Induction time (2-24 hours)

  • Inducer concentration (e.g., IPTG at 0.1-1.0 mM)

  • Media composition (LB, TB, or defined media)

• Experimental design: A Definitive Screening Design (DSD) with center points is recommended to efficiently identify significant factors with minimal experimental runs . This approach allows evaluation of main effects and two-way interactions while requiring fewer resources than a full factorial design.

• Purification optimization: Similar DoE approaches can be applied to purification steps, evaluating factors such as:

  • Buffer composition and pH

  • Imidazole concentration (for His-tagged proteins)

  • Flow rate and binding time

  • Elution parameters

JMP software can be used to create appropriate experimental designs and analyze results, as demonstrated in protein purification studies where such approaches have improved yields significantly .

What analytical methods are appropriate for assessing UbiB protein quality and activity?

Multiple analytical methods should be employed to comprehensively assess UbiB protein quality and activity:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (expected >90% purity)

  • Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC)

  • Western blotting with anti-UbiB or anti-tag antibodies

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Thermal shift assays to assess protein stability

  • Dynamic light scattering (DLS) to assess aggregation state

• Functional activity:

  • ATPase activity assays (UbiB exhibits kinase-like activity)

  • Coupling with downstream ubiquinone biosynthesis enzymes

  • Complementation assays in UbiB-deficient bacterial strains

• Mass spectrometry approaches:

  • Intact mass analysis to confirm molecular weight

  • Peptide mass fingerprinting to verify sequence coverage

  • Hydrogen-deuterium exchange to probe structural dynamics

For activity assays specifically, develop a standardized protocol monitoring ATP hydrolysis using either colorimetric methods (e.g., malachite green) or coupled enzyme assays. Establish positive and negative controls, and validate results against established parameters from homologous proteins in related species.

How can I perform reliable structure-function studies of UbiB protein?

To perform reliable structure-function studies of UbiB protein, implement the following comprehensive approach:

• Computational structure prediction:

  • Use AlphaFold2 or RoseTTAFold to generate theoretical structural models

  • Perform comparative modeling based on homologous proteins

  • Validate predicted structures using ProCheck and VERIFY3D

• Site-directed mutagenesis:

  • Target conserved residues in the predicted ATP-binding domain

  • Modify putative catalytic residues identified through alignment

  • Create a systematic alanine-scanning library focusing on the following regions:

    • GQMMSTR motif (positions 63-69)

    • VWIKFG sequence (positions 57-62)

    • C-terminal membrane-association domain

• Functional assays for mutants:

  • Measure kinetic parameters (Km, Vmax) for each mutant

  • Assess thermal stability changes using differential scanning fluorimetry

  • Determine binding affinities to substrates and cofactors

• Structural validation:

  • X-ray crystallography (challenging but gold standard)

  • Cryo-electron microscopy for larger complexes

  • Small-angle X-ray scattering (SAXS) for solution structure

• In vivo functional complementation:

  • Express wild-type and mutant UbiB in UbiB-deficient strains

  • Measure growth rates under aerobic conditions

  • Quantify ubiquinone production using LC-MS/MS

Develop a standardized workflow that allows for direct comparison of structural changes with functional outcomes to establish clear structure-function relationships.

How does UbiB from E. tasmaniensis compare to homologs in other bacterial species?

UbiB from Erwinia tasmaniensis shares significant sequence identity with homologs in other bacterial species, but contains distinct features that may influence its function and activity. Comparative analysis reveals:

Despite these differences, all UbiB homologs share conserved domains required for kinase-like activity and ubiquinone biosynthesis. E. tasmaniensis UbiB contains the characteristic nucleotide-binding domain and alpha-helical clusters typical of this protein family.

Functionally, E. tasmaniensis UbiB appears most similar to homologs from other plant-associated Erwinia species, consistent with their close phylogenetic relationship . The protein functions within a similar metabolic context across species, though expression levels and regulation may differ based on ecological niches.

When designing experiments targeting UbiB, these interspecies differences should be considered, particularly when extrapolating findings from model organisms to E. tasmaniensis.

What is the role of UbiB in bacterial metabolism and stress responses?

UbiB plays a multifaceted role in bacterial metabolism and stress response pathways:

• Primary metabolic functions:

  • Essential for aerobic respiration through ubiquinone biosynthesis

  • Influences energy production efficiency through electron transport chain optimization

  • Participates in regulatory networks controlling central carbon metabolism

• Oxidative stress response:

  • Ubiquinone acts as a lipid-soluble antioxidant in bacterial membranes

  • UbiB expression typically increases under oxidative stress conditions

  • Mutants lacking functional UbiB show increased sensitivity to oxidizing agents

• Environmental adaptation:

  • In plant-associated bacteria like E. tasmaniensis, UbiB activity may correlate with colonization success

  • Expression patterns shift during transition from epiphytic to endophytic growth

  • May influence competitive fitness in natural habitats

• Potential role in plant interactions:

  • Non-pathogenic E. tasmaniensis utilizes UbiB-dependent metabolic pathways during plant colonization

  • Unlike pathogenic relatives, E. tasmaniensis lacks sorbitol utilization capabilities

  • UbiB-dependent metabolism may contribute to the non-pathogenic phenotype

Research approaches to study these roles should include comparative transcriptomics under different stress conditions, metabolomic profiling of ubiquinone intermediates, and in vivo studies using UbiB mutants in varying environmental conditions.

How can I use bioinformatics approaches to identify potential functional partners of UbiB?

Comprehensive bioinformatic analysis can reveal potential functional partners of UbiB through the following approaches:

• Co-expression network analysis:

  • Analyze RNA-seq data from E. tasmaniensis under various conditions

  • Identify genes with expression patterns correlated with ubiB

  • Construct weighted gene co-expression networks using WGCNA or similar tools

• Protein-protein interaction prediction:

  • Use STRING database to identify known and predicted interactions

  • Apply interolog mapping from well-studied species like E. coli

  • Implement machine learning approaches trained on validated bacterial protein-protein interactions

• Genomic context analysis:

  • Examine gene neighborhood conservation across Erwinia species

  • Identify conserved operonic structures containing ubiB

  • Apply phylogenetic profiling to find genes with similar evolutionary patterns

• Structural docking simulations:

  • Generate models of UbiB using AlphaFold2

  • Perform systematic docking with potential partners identified above

  • Validate high-confidence interactions with molecular dynamics simulations

• Functional enrichment analysis:

  • Categorize potential partners by Gene Ontology terms

  • Identify KEGG pathways enriched among predicted interactors

  • Construct functional interaction networks

The results should be organized into a prioritized list of candidate partners for experimental validation using techniques such as bacterial two-hybrid assays, co-immunoprecipitation, or proximity-dependent biotin labeling.

How can I address contradictions in experimental data related to UbiB studies?

When encountering contradictions in UbiB experimental data, implement a structured approach based on contradiction pattern analysis:

• Classify the contradiction pattern:

  • Determine the number of interdependent items (α)

  • Identify the number of contradictory dependencies (β)

  • Calculate the minimal number of required Boolean rules (θ)

• Common contradiction patterns in UbiB research:

  • Activity vs. expression level discrepancies (α=2, β=1, θ=1)

  • Structure-function relationship inconsistencies (α=3, β=2, θ=1)

  • Multi-parameter contradictions in optimization studies (α=4, β=6, θ=2)

• Resolution strategies:

  • For simple contradictions (α=2), direct experimental repetition with controlled variables

  • For complex patterns (α≥3), implement Boolean minimization techniques to identify critical variables

  • Design verification experiments specifically targeting the minimal set of Boolean rules

• Data quality assessment framework:

  • Implement systematic metadata documentation for all experiments

  • Standardize experimental protocols to minimize technical variations

  • Apply statistical methods appropriate for the contradiction pattern

An example of resolving contradictions in UbiB activity data:

Analysis using Boolean minimization reveals that the buffer type is the critical factor in this contradiction pattern rather than pH as initially suspected. This approach allows for efficient resolution of complex contradictions in UbiB experimental data .

What quality control measures should be implemented in UbiB research?

Comprehensive quality control for UbiB research should include:

• Protein-specific quality control:

  • Batch-to-batch consistency validation using standardized activity assays

  • Regular verification of protein stability during storage (-20°C or -80°C)

  • Monitoring of freeze-thaw cycles with activity retention requirements (>90%)

• Experimental design validation:

  • Implementation of positive and negative controls in all assays

  • Use of center points in DoE studies to detect non-linear effects

  • Regular calibration of equipment used for UbiB characterization

• Data integrity measures:

  • Establishment of standard operating procedures (SOPs) for all UbiB protocols

  • Implementation of electronic laboratory notebooks with version control

  • Development of specific validation criteria for each assay type

• Statistical quality control:

  • Power analysis before experimental design to ensure sufficient replication

  • Application of appropriate statistical tests based on data distribution

  • Implementation of outlier detection algorithms with clear justification for exclusions

• Reporting standards:

  • Complete documentation of protein production conditions

  • Detailed methodology including buffer compositions

  • Comprehensive reporting of all experimental parameters, including failures

A standardized UbiB quality control checklist should be developed and consistently applied across all experiments to ensure reproducibility and reliability of results.

How can I troubleshoot low expression or activity of recombinant UbiB?

When encountering low expression or activity of recombinant UbiB, implement this systematic troubleshooting approach:

• Expression troubleshooting:

  • Verify plasmid sequence integrity through complete sequencing

  • Optimize codon usage for the expression host

  • Test multiple promoter systems (T7, tac, araBAD)

  • Evaluate different fusion tags (His, GST, MBP) for improved solubility

  • Screen expression conditions using a DoE approach as outlined in section 2.1

• Purification troubleshooting:

  • Implement on-column refolding for inclusion body recovery

  • Add stabilizing agents to buffers (glycerol, specific ions, reducing agents)

  • Optimize imidazole concentration to minimize non-specific binding

  • Test multiple chromatography techniques for improved selectivity

• Activity troubleshooting:

  • Verify cofactor requirements (ATP, Mg²⁺, Mn²⁺)

  • Assess buffer composition effects on activity

  • Evaluate the impact of reducing agents on enzyme activity

  • Test substrate quality and purity

• Analysis-based approach:

  • Implement thermal shift assays to identify stabilizing conditions

  • Use size exclusion chromatography to assess oligomeric state

  • Apply mass spectrometry to verify post-translational modifications

  • Conduct limited proteolysis to identify structural domains

For particularly challenging cases, consider using cell-free expression systems or alternative hosts like Bacillus or Pichia pastoris.

What are the potential applications of E. tasmaniensis UbiB in biocontrol strategies?

E. tasmaniensis has emerged as a potential biocontrol agent against phytopathogenic bacteria, particularly fire blight-causing E. amylovora . UbiB's role in this application builds on several key aspects:

• Competitive colonization mechanisms:

  • UbiB supports metabolic functions that allow E. tasmaniensis to establish in plant environments

  • The protein contributes to respiratory efficiency during epiphytic colonization

  • UbiB-dependent metabolism may provide competitive advantages in resource-limited niches

• Stress resistance in field applications:

  • UbiB-mediated ubiquinone biosynthesis enhances oxidative stress resistance

  • This improved stress tolerance increases biocontrol agent persistence

  • Engineering approaches targeting UbiB pathways could enhance performance

• Interaction with plant defense responses:

  • Unlike pathogenic relatives, E. tasmaniensis triggers minimal plant defense responses

  • UbiB's role in cell metabolism may contribute to this non-pathogenic interaction

  • Comparative studies with pathogenic Erwinia species can reveal key differences

• Genetic stability considerations:

  • UbiB gene conservation across E. tasmaniensis strains suggests functional importance

  • Long-term stability of biocontrol traits depends on metabolic gene maintenance

  • Monitoring UbiB sequence in field applications could serve as stability indicator

Research approaches should include field trials comparing wild-type and UbiB-modified strains, transcriptomic analysis of UbiB expression during plant colonization, and competitive assays against pathogenic Erwinia species under varying environmental conditions.

How can structural biology approaches advance our understanding of UbiB function?

Advanced structural biology approaches can significantly enhance our understanding of UbiB function through:

• High-resolution structure determination:

  • X-ray crystallography of purified UbiB with and without substrates/cofactors

  • Cryo-electron microscopy for larger UbiB-containing complexes

  • NMR spectroscopy for dynamic regions and ligand interactions

  • Integrative structural biology combining multiple experimental approaches

• Structural dynamics investigations:

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Single-molecule FRET to observe real-time dynamics

  • Molecular dynamics simulations to predict conformational ensembles

  • Time-resolved structural methods to capture catalytic intermediates

• Structure-guided functional studies:

  • Identification of catalytic residues and binding pockets

  • Design of specific inhibitors based on structural features

  • Engineering of UbiB variants with altered specificity or activity

  • Correlation of structural features with evolutionary conservation

• Complex formation studies:

  • Structural characterization of UbiB interactions with other ubiquinone biosynthesis enzymes

  • Mapping of membrane interaction domains

  • Identification of protein-protein interaction interfaces

  • Validation of predicted complexes using cross-linking mass spectrometry

These approaches should be integrated with biochemical and genetic studies to develop a comprehensive model of UbiB function within the ubiquinone biosynthetic pathway in E. tasmaniensis.