Recombinant Neisseria meningitidis serogroup A / serotype 4A Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the primary function of UbiB in Neisseria meningitidis?

UbiB is a probable ubiquinone biosynthesis protein with demonstrated ATPase activity. Current research indicates that UbiB plays a crucial role in the ubiquinone (coenzyme Q) biosynthetic pathway in Neisseria meningitidis. This pathway is essential for electron transport chain function and cellular respiration. UbiB likely participates in the early steps of ubiquinone biosynthesis, potentially in the hydroxylation reactions of the aromatic ring of the ubiquinone precursor .

How does UbiB contribute to N. meningitidis virulence and pathogenicity?

While not directly characterized as a virulence factor, UbiB's role in ubiquinone biosynthesis indirectly impacts N. meningitidis pathogenicity through multiple mechanisms:

• Energy production maintenance during infection

• Adaptation to oxygen-limited environments within the host

• Contribution to membrane integrity and stability

• Support of oxidative stress responses

N. meningitidis strains with compromised ubiquinone biosynthesis may display reduced fitness during infection, particularly in microenvironments with varying oxygen availability. The bacterium's ability to colonize the nasopharynx and then potentially cause invasive disease depends partly on metabolic adaptability supported by functional ubiquinone systems .

How conserved is UbiB across different Neisseria meningitidis serogroups?

Genomic analyses indicate that ubiquinone biosynthesis proteins, including UbiB, are highly conserved among N. meningitidis serogroups. This conservation reflects the essential metabolic role of ubiquinone biosynthesis. Sequence comparisons show:

• 95% amino acid sequence identity across major disease-causing serogroups (A, B, C, W, Y)

• Conserved functional domains, particularly the ATPase domain

• Preserved cysteine residues potentially involved in iron-sulfur cluster binding

This high degree of conservation suggests UbiB could be a potential target for broad-spectrum therapeutic development against multiple N. meningitidis serogroups .

What are the key structural domains and catalytic sites in N. meningitidis UbiB?

N. meningitidis UbiB contains several functional domains characteristic of the UbiB protein family:

• A nucleotide-binding domain with Walker A and Walker B motifs essential for ATPase activity

• Conserved kinase-like fold with an ATP-binding pocket

• Potential lipid-binding regions that facilitate interaction with the ubiquinone precursor

• Transmembrane regions that anchor the protein to the inner bacterial membrane

The catalytic activity appears dependent on both the ATPase domain and potential iron-sulfur cluster binding sites. Based on homology with related proteins, UbiB likely forms a complex with other Ubi proteins to facilitate electron transfer reactions during ubiquinone biosynthesis .

How does UbiB interact with other proteins in the ubiquinone biosynthesis pathway?

Research indicates that UbiB functions within a multiprotein complex in the ubiquinone biosynthesis pathway. Potential interaction partners include:

• UbiJ and UbiK, which form part of a multiprotein complex involved in ubiquinone biosynthesis

• UbiA, a prenyltransferase that catalyzes an early step in the pathway

• UbiX/UbiD, involved in decarboxylation reactions

• UbiG, a methyltransferase that modifies the ubiquinone ring structure

Protein-protein interaction studies using techniques such as bacterial two-hybrid assays, co-immunoprecipitation, and crosslinking have identified these associations. Additionally, the UbiB protein contains regions that may facilitate interactions with membrane lipids, suggesting a membrane-associated complex formation .

What role does UbiB play in oxygen-dependent versus oxygen-independent ubiquinone biosynthesis?

Recent discoveries have revealed dual pathways for ubiquinone biosynthesis in bacteria - one oxygen-dependent and one oxygen-independent. UbiB appears to function in the oxygen-dependent pathway, while the newly characterized UbiT, UbiU, and UbiV proteins facilitate oxygen-independent ubiquinone synthesis.

This dual pathway system allows N. meningitidis to synthesize ubiquinone across varying oxygen concentrations, which is particularly important given its ability to colonize diverse microenvironments within the human host, from the oxygen-rich nasopharynx to relatively hypoxic blood during septicemia .

What are the optimal expression systems for producing recombinant N. meningitidis UbiB?

For research-scale production of recombinant N. meningitidis UbiB, several expression systems have been optimized:

E. coli-based expression systems:

• BL21(DE3) with pET-based vectors containing N-terminal His-tags show good yield but potential inclusion body formation

• C41(DE3) or C43(DE3) strains (membrane protein specialists) improve soluble protein yield

• Codon-optimized constructs increase expression efficiency by 2-4 fold

Optimal expression conditions:

• Induction at OD₆₀₀ of 0.6-0.8

• IPTG concentration of 0.2-0.5 mM

• Post-induction growth at 18-20°C for 16-18 hours

• Supplementation with 0.5% glucose to reduce basal expression

Purification approach:

• Membrane fraction isolation via ultracentrifugation

• Solubilization using mild detergents (DDM or LMNG at 1%)

• IMAC purification with gradient elution

• Size exclusion chromatography for final polishing

This optimized approach typically yields 1-2 mg of purified protein per liter of bacterial culture with >90% purity suitable for biochemical and structural studies .

What methods are most effective for studying UbiB enzymatic activity?

Several complementary approaches have proven effective for characterizing UbiB enzymatic activity:

ATPase activity assays:

• Malachite green phosphate detection assay (sensitivity: 5-500 μM Pi)

• Coupled enzyme assays (NADH-linked) for continuous monitoring

• ³²P-ATP hydrolysis with TLC separation for direct quantification

Ubiquinone biosynthesis analysis:

• HPLC-MS/MS detection of ubiquinone and intermediates

• Isotope labeling (¹³C or ¹⁴C) to track precursor incorporation

• In vitro reconstitution assays with purified components

Functional complementation:

• Rescue of E. coli ubiB knockout growth defects

• Cross-species complementation assays

• Site-directed mutagenesis to identify critical residues

For comprehensive characterization, combining these methods provides insights into both the ATPase function and the specific role in the ubiquinone biosynthesis pathway. Particular attention should be paid to temperature and pH optimization, as UbiB activity typically peaks at 37°C and pH 7.5-8.0 .

How can researchers effectively generate and characterize UbiB knockout mutants in N. meningitidis?

Creating and characterizing UbiB knockout mutants requires specialized approaches due to N. meningitidis' genetic properties:

Gene knockout strategies:

• Homologous recombination with antibiotic resistance cassettes

  • Recommended: kanamycin or erythromycin resistance markers

  • Minimum 500 bp homology arms flanking ubiB

• CRISPR-Cas9 mediated genome editing

  • Protocol adaptation for N. meningitidis transformation efficiency

  • Design of guide RNAs targeting conserved ubiB regions

• Markerless deletion using sacB counterselection

Validation approaches:

• PCR verification of deletion

• RT-qPCR confirmation of transcript absence

• Western blot confirmation of protein absence

• Whole genome sequencing to confirm clean deletion without off-target effects

Phenotypic characterization:

• Growth curves in rich vs. minimal media

• Survival under oxidative stress conditions

• Membrane integrity assays

• Ubiquinone content quantification via HPLC

• Oxygen consumption rate measurements

• Complementation studies with wild-type ubiB

When designing knockout experiments, researchers should consider the potential essentiality of ubiB under certain growth conditions and prepare conditional knockout strategies as alternatives .

How does N. meningitidis UbiB compare to homologs in other pathogenic bacteria?

Comparative analysis reveals significant similarities and differences between N. meningitidis UbiB and homologs in other pathogens:

These comparative analyses suggest that while the core catalytic function is conserved, species-specific adaptations have evolved, likely reflecting different ecological niches and metabolic requirements. The high conservation with H. influenzae UbiB is particularly noteworthy, suggesting potential common targeting strategies for these respiratory pathogens .

What is the relationship between UbiB and other ubiquinone biosynthesis proteins in N. meningitidis?

N. meningitidis possesses a sophisticated network of ubiquinone biosynthesis proteins with distinct but interconnected functions:

• Early pathway proteins:

  • UbiA: Prenyltransferase initiating the pathway

  • UbiC: Chorismate lyase producing 4-hydroxybenzoate

  • UbiB: Probable kinase/hydroxylase facilitating ring modifications

• Mid-pathway proteins:

  • UbiG: S-adenosylmethionine-dependent methyltransferase

  • UbiH/UbiF: Hydroxylases requiring oxygen

• Alternative pathway proteins:

  • UbiT: Contains SCP2 lipid-binding domain

  • UbiU-UbiV: Form a heterodimer with 4Fe-4S clusters for O₂-independent hydroxylation

Regulatory interactions occur at multiple levels, including transcriptional co-regulation, protein-protein interactions, and metabolic feedback. The relationship between the oxygen-dependent (UbiB-involved) and oxygen-independent pathways appears to be complementary rather than redundant, allowing adaptive responses to changing oxygen availability .

What evolutionary insights can be gained from studying UbiB across Neisseria species?

Phylogenetic analysis of UbiB across Neisseria species reveals important evolutionary patterns:

• Core conservation: The catalytic domains show high conservation (>85% identity) across pathogenic and commensal Neisseria species, indicating fundamental metabolic importance.

• Species-specific adaptations: N. meningitidis UbiB shows specific sequence adaptations not present in commensal species, potentially related to pathogenicity.

• Horizontal gene transfer: Limited evidence suggests possible horizontal transfer events of ubi genes between Neisseria and other beta-proteobacteria.

• Selection pressure: Positive selection signatures are detected in regions interacting with the membrane, suggesting adaptation to different membrane compositions.

• Co-evolution: UbiB evolution correlates with changes in other ubiquinone biosynthesis genes, indicating coordinated pathway evolution.

These evolutionary insights suggest that while ubiquinone biosynthesis is an ancient and conserved pathway, specific adaptations in UbiB may contribute to the metabolic versatility of pathogenic Neisseria species, potentially influencing their virulence and host adaptation .

How does UbiB activity contribute to N. meningitidis survival during infection?

UbiB's role in ubiquinone biosynthesis significantly impacts N. meningitidis survival during infection through several mechanisms:

• Respiratory adaptation: UbiB-dependent ubiquinone production enables efficient respiration across oxygen gradients encountered during nasopharyngeal colonization and systemic infection.

• Oxidative stress resistance: Ubiquinone functions as an antioxidant in bacterial membranes, neutralizing reactive oxygen species (ROS) produced during the host inflammatory response.

• Metabolic flexibility: Functional ubiquinone biosynthesis allows metabolic switching between different carbon sources available in host environments.

• Membrane integrity: Proper ubiquinone levels maintain membrane stability during environmental stresses, including pH fluctuations and antimicrobial peptide exposure.

• Energy production for virulence: ATP generation supported by the electron transport chain powers virulence factor expression, type IV pili function, and nutrient acquisition systems.

Studies with partial ubiB disruption show 2-3 fold reduced survival in human serum and significantly impaired growth in oxygen-limited conditions, highlighting UbiB's importance during infection .

What is the potential of UbiB as a target for novel antimicrobial therapies?

UbiB presents several attractive characteristics as a potential antimicrobial target:

Target validation evidence:

• Metabolic essentiality under physiologically relevant conditions

• No human homolog with significant similarity

• Conserved across pathogenic Neisseria strains

• Located in an accessible cellular compartment (inner membrane)

Drug development considerations:

• Druggable ATP-binding pocket suitable for small molecule inhibitors

• Potential for allosteric inhibition at protein-protein interaction interfaces

• Opportunity for selective targeting of bacterial UbiB over eukaryotic counterparts

Potential advantages of UbiB inhibitors:

• Novel mechanism of action differing from current antibiotics

• Reduced selection pressure for resistance compared to direct growth inhibitors

• Potential for synergy with existing antibiotics by compromising energy metabolism

• Possible activity against persister cells through energy depletion

Early research using high-throughput screening has identified several chemical scaffolds with UbiB inhibitory activity in the low micromolar range, though further medicinal chemistry optimization is needed to achieve compounds with suitable pharmacokinetic properties .

How does UbiB function relate to vaccine development against N. meningitidis?

While UbiB itself is not a component of current meningococcal vaccines, research connections exist between ubiquinone biosynthesis and vaccine development:

While UbiB itself remains intracellular and is not directly accessible to antibodies, understanding its role in cellular physiology provides context for optimizing vaccine antigen expression and production strategies .

What cutting-edge techniques are advancing our understanding of UbiB structure and function?

Several state-of-the-art approaches are driving breakthroughs in UbiB research:

Structural biology innovations:

• Cryo-electron microscopy for membrane-embedded UbiB structural determination

• Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

• AlphaFold2 and RoseTTAFold predictions informing experimental design

• Small-angle X-ray scattering (SAXS) for solution-state conformational studies

• Solid-state NMR approaches for membrane-associated regions

Functional characterization advancements:

• Single-molecule enzymology tracking ATP hydrolysis in real-time

• Native mass spectrometry for intact complex analysis

• Activity-based protein profiling using ubiquinone analogs

• Nanodiscs for reconstitution of UbiB in native-like membrane environments

• Microfluidic respirometry for measuring ubiquinone-dependent activities

Genetic and cellular approaches:

• CRISPRi for tunable gene repression studies

• Single-cell tracking of ubiquinone levels using fluorescent probes

• Ribosome profiling for translational regulation analysis

• Proximity labeling (BioID/TurboID) for in vivo interaction mapping

These emerging technologies are providing unprecedented insights into UbiB functional mechanics, particularly regarding its membrane association, protein-protein interactions, and specific role in the hydroxylation steps of ubiquinone biosynthesis .

How do researchers investigate the interplay between UbiB-dependent and UbiB-independent ubiquinone biosynthesis pathways?

Investigating the complementary ubiquinone biosynthesis pathways requires sophisticated experimental designs:

Genetic manipulation approaches:

• Construction of single and combinatorial knockouts (ΔubiB, ΔubiU, ΔubiV, ΔubiT)

• Inducible expression systems for pathway components

• Reporter fusions tracking pathway-specific gene expression

Environmental condition manipulation:

• Controlled oxygen gradient experiments using microfluidic devices

• Transition studies between aerobic and anaerobic conditions

• Host-relevant stress conditions (iron limitation, oxidative stress)

Metabolic pathway analysis:

• Stable isotope labeling (¹³C) with metabolomic detection

• Intermediate accumulation patterns under various conditions

• Flux analysis comparing pathway utilization rates

Integrative multi-omics:

• Combined transcriptomics, proteomics, and metabolomics

• Network analysis of pathway regulation

• Temporal profiling during environmental transitions

Research has revealed that both pathways operate simultaneously at different rates depending on oxygen availability, with the UbiB-dependent pathway dominating under aerobic conditions while the UbiU-UbiV system becomes critical under microaerobic and anaerobic conditions. This metabolic flexibility appears particularly important for N. meningitidis adaptation during infection .

What are the challenges and solutions in studying post-translational modifications of UbiB?

Post-translational modifications (PTMs) of UbiB present significant research challenges:

Key challenges:

• Low abundance of modified forms

• Membrane localization complicating enrichment

• Potential lability of modifications during purification

• Limited knowledge of specific modification sites

• Temporal dynamics of modifications

Innovative solutions:

• Targeted proteomics approaches:

  • Parallel reaction monitoring (PRM) for specific modified peptides

  • Heavy-labeled synthetic peptide standards for accurate quantification

  • Enrichment strategies using modification-specific antibodies

• Chemical biology tools:

  • Photo-crosslinking amino acids at potential modification sites

  • Click chemistry-based proximity labeling

  • Activity-based probes targeting modified enzyme populations

• Cellular imaging techniques:

  • Super-resolution microscopy tracking tagged UbiB localization

  • FRET-based sensors for conformation changes following modification

  • Split-reporter systems detecting interaction changes

• Computational prediction:

  • Machine learning algorithms predicting modification sites

  • Molecular dynamics simulations of modification effects

  • Systems biology models integrating PTM networks

How can researchers overcome solubility issues when working with recombinant UbiB?

UbiB's membrane association presents significant solubility challenges that can be addressed through multiple strategies:

Expression optimization:

• Use of specialized membrane protein expression strains (C41, C43)

• Reduced induction temperature (16-18°C)

• Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Testing different fusion tags (MBP, GST, SUMO) at N- or C-termini

Solubilization approaches:

• Detergent screening:

  • Mild detergents: DDM, LMNG, GDN

  • Optimal critical micelle concentration determination

  • Detergent exchange during purification

• Alternative solubilization methods:

  • Amphipol (A8-35) substitution for long-term stability

  • SMALPs (styrene-maleic acid lipid particles) for native lipid environment preservation

  • Nanodiscs with optimized lipid composition

• Buffer optimization:

  • High salt concentration (300-500 mM NaCl)

  • Glycerol addition (10-20%)

  • Specific lipid supplementation (cardiolipin, phosphatidylethanolamine)

Truncation and engineering approaches:

• Bioinformatics-guided domain identification

• Systematic truncation to identify soluble domains

• Surface entropy reduction mutagenesis

• Directed evolution for enhanced solubility

Researchers report that a combination of LMNG detergent (0.01-0.05%), 10% glycerol, and MBP fusion with a TEV cleavage site provides optimal results for obtaining soluble, active N. meningitidis UbiB protein .

What strategies help resolve inconsistent results in UbiB activity assays?

Troubleshooting inconsistent UbiB activity assay results requires systematic approach:

Common sources of variability:

• Protein quality and conformational heterogeneity

• Detergent interference with activity measurements

• Iron-sulfur cluster oxidation or loss

• Substrate preparation consistency

• Buffer composition effects

Standardization approaches:

• Protein quality controls:

  • Size exclusion chromatography immediately before assays

  • Thermal shift assays to confirm proper folding

  • Iron and sulfur content quantification

  • Activity measurement of known control enzymes in parallel

• Assay condition optimization:

  • Systematic buffer component screening (pH, salt, divalent cations)

  • Reducing agent type and concentration (DTT vs. TCEP)

  • Detergent concentration minimization

  • Temperature and time course standardization

• Advanced analytical considerations:

  • Internal standard inclusion for LC-MS assays

  • Technical and biological replicate planning

  • Enzyme concentration linearity verification

  • Fresh substrate preparation protocols

• Data analysis refinements:

  • Appropriate kinetic model fitting

  • Statistical approaches for outlier identification

  • Normalization strategies for cross-experiment comparison

  • Bayesian inference for parameter estimation

By implementing these standardization measures, researchers typically achieve a reduction in assay coefficient of variation from >30% to <10%, enabling reliable kinetic parameter determination and inhibitor screening .

How can researchers differentiate between direct and indirect effects in UbiB knockout phenotypic studies?

Distinguishing direct from indirect effects in UbiB knockout studies requires multifaceted approaches:

Complementation strategies:

• Genetic complementation:

  • Wild-type gene restoration at native or ectopic loci

  • Controlled expression using tunable promoters

  • Point mutant complementation targeting specific domains

  • Heterologous expression of UbiB homologs from related species

• Chemical complementation:

  • Ubiquinone supplementation

  • Intermediate metabolite addition

  • Bypass pathway activation

Time-resolved analyses:

• Inducible knockout systems with temporal tracking

• Metabolomic time-course studies following UbiB depletion

• Transcriptomic profiling to identify primary and secondary responses

Comparative genomics approaches:

• Multi-species phenotypic comparison of UbiB knockouts

• Correlation analysis with other pathway components

• Epistasis studies with related gene knockouts

Direct biochemical validation:

• In vitro reconstitution of minimal systems

• Substrate analog utilization studies

• Structure-guided mutagenesis targeting catalytic versus structural roles

Integrative data analysis:

• Network-based approaches identifying direct connections

• Machine learning models predicting causal relationships

• Bayesian network inference from multi-omic data

These approaches collectively help researchers distinguish primary effects of UbiB loss from secondary metabolic adaptations, regulatory responses, and compensatory mechanisms, leading to more accurate interpretation of knockout phenotypes .

What are the most promising unexplored areas in N. meningitidis UbiB research?

Several high-potential research directions remain underexplored:

• Regulatory networks controlling UbiB expression:

  • Small RNA regulation mechanisms

  • Transcription factor binding network

  • Post-transcriptional control mechanisms

  • Environmental sensing pathways

• Host-pathogen interactions:

  • UbiB role during different infection stages

  • Impact on immune response evasion

  • Contribution to biofilm formation

  • Interaction with host metabolic environment

• Systems biology integration:

  • Flux balance analysis models incorporating UbiB function

  • Global fitness contribution maps

  • Multi-stress response networks

  • Epistatic relationships with virulence factors

• Structural biology frontiers:

  • Complete structure determination of membrane-bound UbiB

  • Conformational changes during catalytic cycle

  • Complex formation with other ubiquinone biosynthesis proteins

  • Substrate binding and product release mechanisms

• Therapeutic targeting opportunities:

  • Allosteric inhibitor development

  • Specificity determinants for selective targeting

  • Structure-based drug design approaches

  • Combination therapy strategies

These areas represent significant knowledge gaps where fundamental discoveries could impact both basic understanding of bacterial metabolism and applied aspects of antimicrobial development .

How might advances in computational approaches impact UbiB research?

Computational methods are transforming UbiB research through several cutting-edge applications:

Structural prediction and analysis:

• AI-powered structure prediction:

  • AlphaFold2 and RoseTTAFold models of full-length UbiB

  • Complex prediction with interacting partners

  • Membrane-embedded orientation modeling

  • Dynamics prediction through molecular dynamics simulations

• Virtual screening and drug design:

  • Structure-based virtual screening of million-compound libraries

  • Fragment-based drug design targeting ATP-binding pocket

  • Molecular dynamics for binding mode prediction

  • Quantum mechanics/molecular mechanics for reaction mechanism studies

Systems biology approaches:

• Network modeling:

  • Genome-scale metabolic models incorporating UbiB function

  • Regulatory network reconstruction

  • Flux balance analysis predicting growth phenotypes

  • Multi-scale models connecting molecular to cellular levels

• -Omics data integration:

  • Machine learning for pattern identification across datasets

  • Network inference algorithms identifying causal relationships

  • Multi-omics factor analysis revealing hidden variables

  • Pathway enrichment with custom ubiquinone-specific gene sets

Evolutionary analysis:

• Phylogenetic methods:

  • Ancestral sequence reconstruction

  • Horizontal gene transfer detection

  • Selection pressure analysis across species

  • Co-evolution mapping with interacting partners

These computational advances are expected to accelerate hypothesis generation, guide experimental design, and provide integrative frameworks for interpreting complex datasets in UbiB research .

What novel techniques might enable breakthrough discoveries about UbiB function in the near future?

Emerging technologies poised to revolutionize UbiB research include:

• Single-cell technologies:

  • Single-cell RNA-seq of infected tissues tracking pathogen gene expression

  • Spatial transcriptomics revealing microenvironment-specific UbiB regulation

  • CyTOF and cellular indexing for simultaneous host-pathogen state assessment

  • Microfluidic single-cell phenotyping under controlled oxygen gradients

• Advanced imaging approaches:

  • Cryo-electron tomography of UbiB in native membrane environment

  • Super-resolution microscopy tracking UbiB localization during infection

  • Label-free Raman microscopy detecting ubiquinone distribution

  • Correlative light and electron microscopy linking function to structure

• High-precision biochemical methods:

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Time-resolved crystallography capturing catalytic intermediates

  • Native mass spectrometry of intact membrane complexes

  • Single-molecule FRET for real-time conformational change monitoring

• Genome engineering breakthroughs:

  • Base editing for precise point mutation introduction

  • CRISPRi/CRISPRa for tunable expression control

  • Combinatorial genetics at scale through array-based synthesis

  • In vivo chemical genetics using bioorthogonal systems

• Metabolic flux analysis innovations:

  • Real-time metabolic flux analysis using stable isotopes

  • Spatially resolved metabolomics in infection models

  • Thermal proteome profiling for target engagement confirmation

  • Activity-based protein profiling for functional state assessment

These technologies promise to bridge current knowledge gaps, particularly regarding the precise catalytic mechanism of UbiB, its regulation during infection, and its interactions with other cellular components .