Recombinant Enterobacter sp. Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the role of UbiB in ubiquinone biosynthesis in Enterobacter species?

UbiB is a probable ubiquinone biosynthesis protein in Enterobacter species that plays a critical role in the oxygen-dependent pathway of ubiquinone (UQ) synthesis. Based on studies in related Enterobacteriaceae such as E. coli, UbiB likely functions as a kinase-like protein involved in one of the hydroxylation steps of UQ biosynthesis. Recent research has established that ubiquinone biosynthesis in Enterobacteriaceae involves both oxygen-dependent and oxygen-independent pathways, with UbiB participating in the aerobic route while proteins like UbiU, UbiV, and UbiT function in anaerobic conditions . Methodologically, researchers investigating UbiB function should consider comparative genomics approaches with well-characterized homologs in E. coli and targeted gene deletion studies to assess phenotypic changes in ubiquinone production.

How does UbiB function differ in aerobic versus anaerobic conditions?

UbiB primarily functions in aerobic conditions as part of the oxygen-dependent ubiquinone biosynthesis pathway. Under anaerobic conditions, Enterobacter species, like other Enterobacteriaceae, appear to utilize an alternative pathway involving UbiU, UbiV, and UbiT proteins that can perform hydroxylation reactions through an oxygen-independent process . To study these functional differences, researchers should employ both aerobic and anaerobic cultivation methods followed by analysis of UQ content using HPLC or LC-MS. Additionally, transcriptional analysis of ubiB expression under varying oxygen conditions can provide insights into its regulation. The oxygen-sensing transcriptional regulator Fnr, which controls the expression of ubiTUV genes in E. coli, may similarly regulate ubiB expression in response to oxygen availability .

What experimental approaches are recommended for purifying recombinant UbiB protein?

For purification of recombinant UbiB from Enterobacter species, a methodical approach incorporating the following steps is recommended:

• Clone the ubiB gene into an expression vector with a suitable tag (His6 or GST) for affinity purification

• Transform the construct into an E. coli expression strain optimized for membrane proteins (e.g., C41(DE3) or C43(DE3))

• Induce protein expression at lower temperatures (16-20°C) to improve folding

• Extract using mild detergents (DDM or LMNG) to solubilize the membrane-associated UbiB

• Purify using a two-step approach combining affinity chromatography and size exclusion chromatography

When designing experiments, researchers should consider that UbiB is likely membrane-associated, which may present solubility challenges. Expression trials comparing different solubilization conditions and detergents are essential for optimizing yield and activity of the purified protein.

How can researchers design experiments to elucidate UbiB's structural characteristics and functional domains?

Designing experiments to characterize UbiB's structure and functional domains requires a multi-technique approach:

• Perform sequence alignment analysis with homologous proteins to identify conserved domains and predict functional regions

• Use site-directed mutagenesis to create variants targeting predicted ATP-binding sites and potential catalytic residues

• Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic regions and potential substrate binding sites

• Apply cryo-electron microscopy or X-ray crystallography for structural determination, with particular attention to membrane preparation protocols

• Conduct in vitro kinase activity assays using purified UbiB with proposed substrates and ATP

Researchers should note that the membrane association of UbiB makes structural studies particularly challenging. Fusion protein approaches (e.g., with MBP or SUMO) may improve solubility while maintaining function. Additionally, complementation studies in ubiB-deficient strains can validate the functionality of mutant constructs. Success in structural studies may require detergent screening and lipid nanodisc reconstitution to maintain the native conformation of the protein.

What is the relationship between UbiB function and bacterial virulence in Enterobacter species?

The relationship between UbiB function and virulence in Enterobacter species appears to involve several interconnected pathways. Ubiquinone plays a crucial role in bacterial respiration and energy metabolism, which indirectly impacts virulence. Studies of related Enterobacteriaceae suggest that:

• Ubiquinone is essential for optimal growth and adaptation to changing oxygen levels encountered during host infection

• UQ-dependent respiration contributes to bacterial survival under oxidative stress conditions imposed by host immune responses

• Respiratory flexibility provided by UQ may enhance colonization capabilities in different host niches

Methodologically, researchers can investigate this relationship through:

• Creation of ubiB deletion mutants and assessment of virulence in models such as Galleria mellonella larvae or mouse infection models

• Transcriptomic analysis comparing wild-type and ubiB mutants during growth in human serum, which has been shown to induce significant metabolic adaptations in Enterobacter species

• Evaluation of bacterial survival under oxidative stress and serum bactericidal activity, which are characteristic virulence traits for septicemic pathogens like Enterobacter bugandensis

Multiple Enterobacter species are associated with nosocomial infections and can cause life-threatening sepsis, particularly in neonates and immunocompromised patients . Understanding UbiB's contribution to this virulence could identify potential therapeutic targets.

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

UbiB likely participates in a complex network of protein-protein interactions within the ubiquinone biosynthesis pathway. Based on research in related bacteria, potential interacting partners may include:

• Other Ubi proteins involved in hydroxylation steps (UbiI, UbiH)

• Oxygen-independent pathway components (UbiU, UbiV)

• Respiratory chain components that utilize ubiquinone

To methodically investigate these interactions, researchers should consider:

• Bacterial two-hybrid or split-GFP assays to screen for potential interactions

• Co-immunoprecipitation followed by mass spectrometry to identify interacting partners

• Proximity labeling approaches such as BioID or APEX to map the UbiB interactome

• In vitro reconstitution of partial pathways using purified components

The regulation of these interactions may differ between aerobic and anaerobic conditions, as Enterobacteriaceae employ distinct ubiquinone biosynthesis pathways depending on oxygen availability . Comprehensive interaction mapping should therefore include experiments under both conditions to fully understand UbiB's functional context.

What experimental controls should be included when studying recombinant UbiB expression and function?

When designing experiments for recombinant UbiB expression and functional analysis, researchers should include the following controls:

Expression Controls:

• Empty vector control to distinguish background from specific expression

• Wild-type UbiB expression for comparison with mutant variants

• Well-characterized protein with similar properties (size, membrane association) as a technical control

• Western blot verification of expression with tag-specific and UbiB-specific antibodies

Functional Assays Controls:

• UbiB knockout strain complemented with wild-type ubiB (positive control)

• UbiB knockout strain with empty vector (negative control)

• Kinase-dead UbiB mutant (predicted catalytic residue mutations)

• ATP depletion condition to verify dependency on ATP

Table 1: Essential Controls for UbiB Functional Studies

These controls ensure that observed phenotypes are specifically attributed to UbiB function rather than experimental artifacts or secondary effects.

How can researchers interpret conflicting data regarding UbiB function in different Enterobacter species?

When encountering conflicting data about UbiB function across different Enterobacter species, researchers should implement a systematic approach to data reconciliation:

• Phylogenetic Analysis: Construct phylogenetic trees of UbiB sequences from different Enterobacter species to identify evolutionary relationships and potential functional divergence.

• Comparative Genomics: Examine the genomic context of ubiB in different species, noting variations in gene neighborhoods that might suggest functional adaptations.

• Cross-Species Complementation: Test whether UbiB from one species can complement a ubiB deletion in another species, which can reveal functional conservation or specialization.

• Growth Condition Standardization: Ensure experimental conditions are standardized across studies, as differences in media composition, oxygen levels, or growth phase can significantly affect ubiquinone metabolism.

• Multi-omics Integration: Combine transcriptomic, proteomic, and metabolomic data to build a comprehensive picture of UbiB's role in different species.

Conflicting data may reflect genuine biological differences rather than experimental errors. Enterobacter species inhabit diverse environments and may have evolved species-specific adaptations in ubiquinone biosynthesis pathways. The oxygen-independent pathway involving UbiU, UbiV, and UbiT discovered in E. coli illustrates how closely related bacteria can develop alternative metabolic strategies for the same biosynthetic goal .

What are the methodological considerations for studying UbiB in the context of antibiotic resistance?

Studying UbiB in relation to antibiotic resistance requires careful methodological consideration due to the complex interplay between respiratory metabolism and resistance mechanisms. Enterobacter species are increasingly associated with multiple-drug resistance, particularly to cephalosporins . Researchers should consider:

• Strain Selection: Use clinically relevant, well-characterized Enterobacter strains with defined antibiotic resistance profiles. Include both sensitive and resistant isolates for comparison.

• Resistance Mechanism Characterization: Determine whether resistance is plasmid-mediated or chromosomally encoded. In Enterobacter bugandensis, for example, antibiotic resistance genes were found exclusively on a 299 kb IncHI plasmid, while virulence properties were chromosomally encoded .

• UbiB Manipulation Approaches:

  • Generate ubiB deletion mutants in resistant strains to assess impact on resistance

  • Create overexpression constructs to evaluate dosage effects

  • Introduce site-directed mutations in functional domains

• Phenotypic Assessments:

  • Determine minimum inhibitory concentrations (MICs) for relevant antibiotics

  • Measure growth curves under antibiotic stress conditions

  • Assess biofilm formation capacity, which can contribute to antibiotic tolerance

• Mechanistic Investigations:

  • Evaluate membrane potential and permeability, which ubiquinone may influence

  • Measure reactive oxygen species (ROS) production, as altered respiratory function can affect antibiotic-induced oxidative stress

  • Analyze expression of efflux pumps, which may be energetically linked to respiratory function

The emergence of multiple-drug resistance in Enterobacter has substantially limited therapeutic options, with carbapenems or fluoroquinolones remaining the most predictively active options, although resistance to both classes has been observed . Understanding how UbiB and ubiquinone metabolism intersect with resistance mechanisms could potentially identify novel therapeutic strategies.

What are the major challenges in expressing and purifying functionally active recombinant UbiB?

Researchers face several significant challenges when expressing and purifying functionally active recombinant UbiB:

• Membrane Association: UbiB is likely membrane-associated, making solubilization and purification technically challenging.
  Solution: Screen multiple detergents (DDM, LMNG, digitonin) at various concentrations to identify optimal solubilization conditions. Consider lipid nanodisc reconstitution for maintaining native conformation.

• Protein Instability: Kinase-like proteins can be inherently unstable when removed from their native environment.
  Solution: Include stabilizing agents (glycerol, specific lipids) in all buffers and consider purification at lower temperatures (4°C). Use thermal shift assays to identify stabilizing buffer conditions.

• Low Expression Levels: Membrane proteins often express poorly in heterologous systems.
  Solution: Test different expression systems (E. coli, yeast), promoters, and fusion tags (MBP, SUMO) to improve yield. Consider specialized E. coli strains designed for membrane protein expression.

• Proper Folding: Ensuring the recombinant protein adopts its native conformation.
  Solution: Express at lower temperatures (16-20°C) and lower inducer concentrations. Consider co-expression with chaperones (GroEL/ES, DnaK/J) to aid folding.

• Functional Verification: Confirming that purified UbiB retains its native activity.
  Solution: Develop robust activity assays based on predicted function (kinase activity, ATP hydrolysis). Include appropriate positive controls and substrate candidates.

Table 2: Optimization Strategies for Recombinant UbiB Expression

By systematically addressing these challenges, researchers can improve the likelihood of obtaining functionally active UbiB for structural and biochemical studies.

How can researchers effectively study UbiB in both aerobic and anaerobic conditions?

Studying UbiB under both aerobic and anaerobic conditions requires specialized approaches to capture its differential function across varying oxygen levels:

• Experimental Setup:

  • Use modular anaerobic chambers or bioreactors with precise oxygen control

  • Employ oxygen sensors to continuously monitor conditions

  • Prepare all media and reagents with appropriate degassing for anaerobic work

  • Consider microfluidic devices for real-time imaging of bacterial responses to oxygen shifts

• Transcriptional Analysis:

  • Monitor ubiB expression using qRT-PCR or RNA-Seq across oxygen gradients

  • Investigate the role of oxygen-sensing regulators (e.g., Fnr) in controlling ubiB expression

  • Compare with expression patterns of the oxygen-independent pathway genes (ubiT, ubiU, ubiV)

• Protein Function Assessment:

  • Develop in vitro activity assays compatible with anaerobic conditions

  • Use oxygen-scavenging enzyme systems to maintain anaerobiosis during biochemical assays

  • Consider radioisotope labeling techniques that can function in both conditions

• Metabolic Analysis:

  • Quantify ubiquinone and its precursors using LC-MS under both conditions

  • Employ 18O2 labeling to track oxygen incorporation in aerobic conditions

  • Use metabolic flux analysis to determine pathway utilization

Research in E. coli has demonstrated that bacteria can switch between oxygen-dependent and oxygen-independent ubiquinone biosynthesis pathways depending on environmental conditions, with the latter involving UbiU, UbiV, and UbiT proteins . Similar pathway switching likely occurs in Enterobacter species, making it essential to compare UbiB function across oxygen conditions to fully understand its metabolic context.

What analytical methods provide the most reliable quantification of ubiquinone production in UbiB studies?

For reliable quantification of ubiquinone production in UbiB studies, researchers should consider these analytical approaches, ranked by reliability and information content:

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

  • Most reliable for absolute quantification of ubiquinone and intermediates

  • Provides structural information to identify modified or unusual forms

  • Can detect and quantify biosynthetic intermediates that accumulate in mutants

  • Enables isotope labeling studies (e.g., with 18O2) to trace oxygen incorporation

  Method considerations: Use internal standards (ideally isotopically labeled ubiquinone), optimize extraction with appropriate solvents (typically hexane/ethanol or methanol/petroleum ether mixtures), and employ reverse-phase HPLC with electrospray ionization mass spectrometry.

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

  • Good for relative quantification and comparing strains

  • More accessible than LC-MS for many laboratories

  • Can be conducted with standard HPLC equipment

  Method considerations: UV detection at 275 nm for ubiquinone, coupled with diode array detection to capture full absorption spectrum for verification.

• Electrochemical Detection Methods:

  • Highly sensitive for ubiquinone's redox-active properties

  • Can distinguish between oxidized and reduced forms

  • Useful for studying ubiquinone's functional state in membranes

  Method considerations: Cyclic voltammetry or chronoamperometry with appropriate reference electrodes.

• Spectrophotometric Assays:

  • Simpler but less specific than chromatographic methods

  • Useful for high-throughput screening of mutants

  • Limited ability to distinguish ubiquinone from other quinones

  Method considerations: Difference spectroscopy between oxidized and reduced samples.

Table 3: Comparison of Analytical Methods for Ubiquinone Quantification

To ensure reliable results, extraction efficiency should be validated using recovery controls, and method precision should be established through repeated analyses of the same samples. Cross-validation using multiple analytical techniques is recommended for critical experiments.

What are the promising approaches for elucidating the catalytic mechanism of UbiB?

Elucidating the catalytic mechanism of UbiB requires a multifaceted approach combining structural biology, biochemistry, and genetic techniques:

• Structural Studies:

  • Cryo-electron microscopy to determine the three-dimensional structure, particularly focusing on potential substrate binding sites and catalytic regions

  • X-ray crystallography of UbiB in complex with substrates or substrate analogs

  • NMR studies of specific domains to understand dynamic aspects of catalysis

  • Computational modeling and molecular dynamics simulations to predict catalytic mechanisms

• Biochemical Approaches:

  • Enzyme kinetics with purified UbiB using potential substrates

  • Identification of reaction intermediates using rapid quench-flow techniques

  • Site-directed mutagenesis of predicted catalytic residues followed by activity assays

  • Chemical crosslinking studies to trap enzyme-substrate complexes

• Mechanistic Investigations:

  • ATP binding and hydrolysis assays to confirm kinase-like activity

  • Phosphate transfer studies using γ-32P-ATP to identify phosphorylated intermediates

  • Isotope labeling experiments to track atom transfer during catalysis

  • Investigation of potential redox function in addition to kinase activity

• Novel Technologies:

  • Single-molecule studies to observe conformational changes during catalysis

  • Time-resolved spectroscopy to capture transient catalytic states

  • Application of AlphaFold or similar AI-based structural prediction tools to generate working models

The mechanistic studies should build upon discoveries made with related proteins in the ubiquinone biosynthesis pathway. For example, research on UbiU and UbiV has demonstrated their involvement in oxygen-independent hydroxylation reactions in E. coli . UbiB may interact with these systems or employ parallel mechanisms in its function.

How might UbiB function be exploited for developing novel antimicrobial strategies?

UbiB's critical role in ubiquinone biosynthesis presents several potential avenues for antimicrobial development:

• Direct Inhibition Strategy:

  • Design competitive inhibitors targeting the ATP binding site of UbiB

  • Develop allosteric inhibitors that disrupt conformational changes necessary for catalysis

  • Create covalent inhibitors that irreversibly modify catalytic residues

  • Screen natural product libraries for compounds that specifically inhibit UbiB

• Pathway Disruption Approach:

  • Target the interaction between UbiB and other ubiquinone biosynthesis proteins

  • Develop compounds that prevent UbiB membrane association

  • Design inhibitors that disrupt both aerobic and anaerobic ubiquinone biosynthesis pathways to prevent metabolic adaptation

• Conditional Targeting:

  • Create oxygen-dependent prodrugs that specifically target Enterobacter in microaerobic infection sites

  • Develop inhibitors that selectively work under physiological conditions found during infection

  • Design compounds that synergize with the host immune response

• Combination Therapy Potential:

  • Identify synergistic interactions between UbiB inhibitors and existing antibiotics

  • Target both UbiB and components of the oxygen-independent pathway (UbiU, UbiV, UbiT) to prevent pathway switching

  • Combine UbiB inhibition with drugs that increase oxidative stress

Enterobacter species are increasingly associated with multiple-drug resistance, particularly to cephalosporins . As therapeutic options for multiply resistant strains become severely limited, with carbapenems or fluoroquinolones remaining the most predictively active options , novel targets like UbiB could provide alternative treatment strategies. The high virulence of certain Enterobacter species like E. bugandensis in infection models underscores the need for new antimicrobial approaches.

What is the current understanding of UbiB evolution and conservation across bacterial species?

The evolutionary history and conservation patterns of UbiB provide insights into its fundamental importance in bacterial metabolism:

• Phylogenetic Distribution:
  UbiB is widely distributed among proteobacteria but shows interesting patterns of conservation and variation. It is highly conserved in α-, β-, and γ-proteobacteria, including Enterobacteriaceae like Escherichia and Enterobacter. This conservation suggests essential functionality that has been maintained through evolutionary history. Comparative genomic analyses reveal that UbiB belongs to a superfamily of kinase-like proteins with members involved in various biosynthetic pathways.

• Domain Architecture:
  UbiB typically contains an N-terminal domain with kinase-like features and potential ATP-binding motifs. The C-terminal region often includes membrane-association elements that may be involved in substrate recognition or protein-protein interactions. This domain architecture is generally conserved across species, though specific sequence variations may reflect adaptations to different metabolic requirements or environmental niches.

• Co-evolution with Respiratory Systems:
  UbiB evolution appears linked to the development of respiratory flexibility in bacteria. Species like E. coli and Enterobacter that can grow under both aerobic and anaerobic conditions maintain both oxygen-dependent (UbiB-associated) and oxygen-independent (UbiUVT-associated) pathways for ubiquinone biosynthesis . This dual pathway system likely evolved to allow these bacteria to thrive in fluctuating oxygen environments, including those encountered during host infection.

• Functional Divergence:
  While UbiB's core function in ubiquinone biosynthesis is conserved, there is evidence for functional specialization across bacterial lineages. Some species show duplications of ubiB-like genes, suggesting sub-functionalization or neofunctionalization. Certain bacteria have evolved specialized versions of UbiB that may participate in modified pathways or interact with different sets of partner proteins.

Research on the oxygen-independent ubiquinone biosynthesis pathway involving UbiU, UbiV, and UbiT in E. coli has revealed how bacteria can develop alternative strategies for essential biosynthetic processes . Similar evolutionary innovations may have occurred with UbiB across different bacterial lineages, potentially explaining some of the functional variations observed between species.

How does UbiB function impact bacterial adaptation to different environments?

UbiB's role in ubiquinone biosynthesis significantly influences bacterial adaptation to diverse environmental conditions through several mechanisms:

This multifaceted role of UbiB in bacterial adaptation has significant implications for understanding the ecology and pathogenesis of Enterobacter species, which have been described as "pathogens poised to flourish" due to their adaptability .

What are the implications of UbiB research for understanding bacterial pathogenesis?

Research on UbiB provides several important insights into bacterial pathogenesis:

• Virulence Factor Expression:
  Efficient energy metabolism supported by UbiB-dependent ubiquinone production may be necessary for optimal expression of virulence factors. Enterobacter species, particularly E. bugandensis, have been shown to possess high virulence in both Galleria mellonella and mouse models of infection . The energy demands of virulence factor production may link UbiB function to pathogenesis through metabolic regulation.

• Serum Survival:
  Growth in human serum represents a critical virulence trait for septicemic pathogens. Enterobacter bugandensis has demonstrated the ability to grow in high concentrations (up to 90%) of human serum, with whole-genome transcriptome analysis revealing that approximately 7% of the genome is mobilized for serum growth . UbiB may contribute to this adaptation by supporting the metabolic changes required for serum survival.

• Host-Pathogen Interface:
  Ubiquinone biosynthesis influences bacterial responses at the host-pathogen interface in several ways:

  • Supporting energy generation for invasive processes

  • Contributing to defense against host-derived reactive oxygen species

  • Enabling adaptation to the shifting nutrient and oxygen landscapes encountered during infection

• Persistent Infection:
  The metabolic flexibility provided by UbiB-dependent and UbiB-independent ubiquinone biosynthesis pathways may facilitate persistent infection by allowing bacteria to adapt to changing conditions within the host. This adaptation capacity could contribute to the increasing incidence of Enterobacter infections in hospitals and communities .

• Antibiotic Tolerance:
  Metabolic state influences bacterial susceptibility to antibiotics, with respiratory activity affecting the efficacy of many antimicrobials. UbiB's role in energy metabolism may therefore influence antibiotic tolerance, potentially contributing to the concerning trend of multiple-drug resistance in Enterobacter species .

The emergence of highly virulent species like Enterobacter bugandensis, which has been associated with neonatal sepsis, underscores the importance of understanding metabolic factors that contribute to pathogenesis . UbiB research provides a window into how basic metabolic processes intersect with virulence in these opportunistic pathogens.

How can findings from UbiB studies be integrated into broader microbial physiology research?

Findings from UbiB studies can be meaningfully integrated into broader microbial physiology research in several ways:

• Systems Biology Frameworks:

  • Incorporate UbiB function into genome-scale metabolic models of Enterobacter species

  • Connect ubiquinone biosynthesis pathways to global regulatory networks

  • Develop predictive models of bacterial adaptation under varying environmental conditions

  • Use constraint-based modeling to predict the systemic effects of UbiB perturbation

• Comparative Physiology:

  • Contrast UbiB function across different bacterial species to identify common principles and unique adaptations

  • Examine how UbiB-dependent processes vary between pathogenic and non-pathogenic bacteria

  • Study how ubiquinone metabolism interfaces with other respiratory quinones like demethylmenaquinone (DMK)

  • Investigate evolutionary patterns in respiratory flexibility across bacterial lineages

• Environmental Microbiology:

  • Explore how UbiB function affects bacterial fitness in natural environments

  • Investigate the role of UbiB in microbial community interactions

  • Study how ubiquinone metabolism influences bacterial responses to environmental stressors

  • Examine UbiB expression patterns in environmental isolates under different conditions

• Translational Applications:

  • Apply knowledge of UbiB function to develop biosensors for environmental monitoring

  • Explore biotechnological applications of recombinant UbiB for specialty chemical production

  • Develop strategies to manipulate bacterial metabolism for bioremediation purposes

  • Investigate UbiB as a potential target for microbiome engineering

• Multi-omics Integration:

  • Combine transcriptomic, proteomic, and metabolomic approaches to build comprehensive models of UbiB function

  • Develop temporal multi-omics profiles to understand dynamic responses involving UbiB

  • Use multi-omics data to identify previously unknown connections between ubiquinone metabolism and other cellular processes

Table 4: Integration of UbiB Research with Broader Microbial Physiology Fields

Research on UbiU, UbiV, and UbiT in E. coli has revealed a novel anaerobic hydroxylation mechanism that contributes to bacterial adaptation to changing oxygen levels . Similar investigations of UbiB could uncover new principles of bacterial physiology with broad implications for understanding microbial life.