Recombinant Escherichia coli O9:H4 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is UbiB and what role does it play in ubiquinone biosynthesis?

UbiB is one of at least eleven proteins involved in ubiquinone (coenzyme Q) biosynthesis in Escherichia coli. It functions as a hydroxylase that introduces a hydroxyl group at specific positions of the benzene nucleus during ubiquinone synthesis . The UbiB protein is classified as a "probable ubiquinone biosynthesis protein," indicating its essential role in the electron transport chain when oxygen or nitrate serves as the electron acceptor .

Structurally, E. coli UbiB is a 546-amino acid protein that belongs to a family of proteins involved in energy metabolism . The ubiquinone pathway in E. coli involves multiple sequential reactions, and UbiB specifically participates in one of the three hydroxylation steps required for the modification of the aromatic ring structure of ubiquinone precursors .

Where is the ubiB gene located in the E. coli genome?

The ubiB gene is located at the 87-minute position on the Escherichia coli linkage map . This chromosomal location is significant for understanding the genetic organization of ubiquinone biosynthesis genes in E. coli. Interestingly, genetic mapping studies have shown that another ubiquinone biosynthesis gene, ubiD, is closely linked to ubiB . This genetic proximity suggests potential co-regulation or functional relationships between these genes in the ubiquinone biosynthetic pathway.

How does UbiB interact with other Ubi proteins in the biosynthetic pathway?

UbiB functions within a complex network of proteins involved in ubiquinone biosynthesis. Research using bacterial two-hybrid systems has demonstrated that UbiB can interact with UbiK . Specifically, UbiK was found to interact with UbiB when UbiK was fused to the T18 moiety in bacterial two-hybrid reporter systems .

This interaction appears to be part of a larger interaction network, as UbiK has been shown to interact with multiple other Ubi proteins including UbiE, UbiF, UbiG, UbiH, UbiI, UbiJ, and UbiX . These interactions suggest that ubiquinone biosynthesis likely involves multiprotein complexes rather than isolated enzymatic reactions, which may be important for channeling intermediates through the pathway efficiently.

What phenotypes are associated with ubiB mutations in E. coli?

Mutations in the ubiB gene lead to deficiencies in ubiquinone production in E. coli. These mutants exhibit impaired growth on substrates that require a functional electron transport chain, such as succinate . Since ubiquinone plays an essential role in aerobic and nitrate respiration, ubiB mutants show growth defects under conditions where these respiratory pathways are required.

Complementation studies have demonstrated that introducing a functional ubiB gene into mutant strains restores their ability to synthesize ubiquinone and grow on respiratory substrates . This confirms the direct involvement of UbiB in the ubiquinone biosynthetic pathway and its importance for respiratory metabolism in E. coli.

What is the mechanism of UbiB's hydroxylase activity in ubiquinone biosynthesis?

The exact molecular mechanism of UbiB's hydroxylase activity remains incompletely characterized. UbiB is involved in introducing a hydroxyl group to the benzene nucleus of ubiquinone precursors, but the specific biochemical details of this reaction have not been fully elucidated . Unlike typical monooxygenases that require molecular oxygen, there is evidence suggesting that UbiB may participate in an oxygen-independent hydroxylation process under certain conditions .

Recent research on UbiUV-dependent ubiquinone synthesis has shown that some hydroxylation steps in the ubiquinone pathway can occur through unique O₂-independent processes . Genetic studies and ¹⁸O₂ labeling experiments have revealed that proteins like UbiUV contribute to the hydroxylation of ubiquinone precursors in the absence of oxygen . This raises intriguing questions about whether UbiB might also have dual functionality under different oxygen conditions, potentially utilizing alternative electron acceptors or hydroxyl group sources when oxygen is limited.

How does UbiB function differ under aerobic versus anaerobic conditions?

This anaerobic pathway represents a novel mechanism for ubiquinone synthesis in the absence of oxygen. Research has shown that UbiUV-dependent ubiquinone synthesis is essential for nitrate respiration and uracil biosynthesis under anaerobiosis, and it contributes to bacterial multiplication in the mouse gut . The relationship between the traditional UbiB-dependent aerobic pathway and this alternative anaerobic pathway remains an active area of research. Understanding how these pathways interact and are regulated under different oxygen conditions is crucial for comprehending E. coli's metabolic flexibility.

What is the relationship between UbiB and bacterial pathogenesis in facultative anaerobes?

The connection between ubiquinone biosynthesis and bacterial pathogenesis is an emerging area of research. Studies have shown that in Salmonella enterica, another factor involved in ubiquinone biosynthesis (UbiK) is required for proliferation in macrophages and virulence in mice . Given UbiB's critical role in ubiquinone production and its interaction with UbiK, it likely also contributes to pathogenesis.

Ubiquinone biosynthesis appears to be a major driver in the capacity of E. coli to multiply in gut microbiota and of facultative anaerobic pathogens to multiply in their hosts . The ability to synthesize ubiquinone under both aerobic and anaerobic conditions may provide metabolic flexibility that contributes to bacterial survival and virulence in host environments. This link between respiratory mechanisms and phenotypic adaptation has implications for understanding how pathogenic bacteria adapt to changing oxygen levels during infection.

How do post-translational modifications affect UbiB function?

While the specific post-translational modifications of UbiB have not been extensively characterized in the available search results, understanding such modifications is critical for fully elucidating UbiB's function. Research on other Ubi proteins suggests that protein modifications and protein-protein interactions play important roles in regulating ubiquinone biosynthesis.

For example, studies have shown that UbiK forms a complex with UbiJ, and this interaction involves the C-terminal part of UbiJ . Similar interactions or modifications might regulate UbiB activity. Investigating potential phosphorylation, acetylation, or other modifications of UbiB under different growth conditions would provide insights into how the ubiquinone biosynthetic pathway is regulated at the post-translational level.

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

For expressing and purifying recombinant UbiB protein, researchers should consider the following methodological approach:

• Expression system selection: E. coli BL21(DE3) or similar strains are commonly used for expressing bacterial proteins. For UbiB, which is a membrane-associated protein, specialized expression strains designed for membrane proteins may yield better results.

• Vector design: Based on available information about UbiB, using vectors with inducible promoters (like T7) and appropriate fusion tags (such as His₆, MBP, or GST) can facilitate purification . The full-length UbiB protein (amino acids 1-546) should be expressed .

• Culture conditions: Initial expression tests should compare different induction temperatures (16°C, 25°C, 30°C), IPTG concentrations (0.1-1.0 mM), and induction times (4-24 hours) to optimize protein yield and solubility.

• Purification strategy: For His-tagged UbiB, nickel-nitrilotriacetic acid resin purification in buffer containing detergents (such as DDM or LDAO) is recommended to maintain protein solubility . Size-exclusion chromatography can be used as a polishing step.

• Storage conditions: Purified UbiB should be stored in Tris-based buffer with 50% glycerol at -20°C for short-term storage or -80°C for extended storage, avoiding repeated freeze-thaw cycles .

What methods are most effective for assessing UbiB enzymatic activity?

Assessing UbiB enzymatic activity requires specialized approaches due to its involvement in hydroxylation reactions within the ubiquinone biosynthetic pathway:

• Substrate preparation: Synthetic or isolated ubiquinone precursors such as 2-octaprenylphenol or related intermediates should be prepared as substrates .

• Activity assays: Hydroxylase activity can be measured using:

  • HPLC or LC-MS analysis to detect conversion of substrate to hydroxylated products

  • Oxygen consumption measurements for aerobic conditions

  • ¹⁸O₂ labeling experiments to track oxygen incorporation into products

• Detection methods: UV-visible spectrophotometry at specific wavelengths characteristic of ubiquinone intermediates, or mass spectrometry to identify specific hydroxylated products.

• Controls: Include positive controls (known hydroxylases) and negative controls (heat-inactivated enzyme) to validate assay performance.

• Cofactor requirements: Assess dependence on potential cofactors such as NAD(P)H, FAD, iron, or other metal ions that might be required for hydroxylase activity.

How can researchers generate and characterize ubiB mutants?

To generate and characterize ubiB mutants, researchers can employ the following methodological approaches:

• Mutagenesis strategies:

  • CRISPR-Cas9 genome editing for precise modifications

  • Lambda Red recombineering for gene deletion or replacement

  • Site-directed mutagenesis for specific amino acid changes

  • Random mutagenesis using error-prone PCR followed by selection

• Screening methods:

  • Growth on minimal media with succinate as the sole carbon source (ubiB mutants will show impaired growth)

  • HPLC analysis of cellular quinone content

  • Mass spectrometry to identify accumulated intermediates

• Complementation assays:

  • Transform mutants with plasmids carrying wild-type ubiB

  • Assess restoration of growth on respiratory substrates

  • Measure ubiquinone levels after complementation

• Phenotypic characterization:

  • Measure growth rates under various conditions (aerobic, anaerobic, different carbon sources)

  • Assess resistance to oxidative stress

  • Evaluate electron transport chain activity using respiration measurements

  • Determine nitrate reductase activity for assessing anaerobic respiration

• Biochemical analysis:

  • Identify accumulated ubiquinone precursors using techniques like nuclear magnetic resonance and mass spectrometry

  • Compare metabolic profiles between wild-type and mutant strains

What techniques can be used to study UbiB protein-protein interactions?

Several techniques can be employed to study UbiB protein-protein interactions in the ubiquinone biosynthetic pathway:

• Bacterial two-hybrid (BACTH) system:

  • This method has successfully identified interactions between UbiK and UbiB, as well as other Ubi proteins

  • It allows detection of both direct and indirect interactions through functional complementation of adenylate cyclase fragments

• Co-immunoprecipitation (Co-IP):

  • Express epitope-tagged UbiB (e.g., His₆-tag, FLAG-tag) in E. coli

  • Isolate protein complexes using tag-specific antibodies

  • Identify interacting partners by mass spectrometry or western blotting

• Pull-down assays:

  • Fuse UbiB to MBP, GST, or His₆ tags for affinity purification

  • Incubate with cell lysates or purified potential partners

  • Elute and analyze bound proteins

• Yeast two-hybrid system:

  • Can help identify direct interactions and pinpoint interaction domains

  • Has been successful in characterizing other Ubi protein interactions, such as UbiK-UbiJ

• Cross-linking coupled with mass spectrometry:

  • Treat cells or purified proteins with cross-linking agents

  • Digest and analyze by mass spectrometry to identify interaction sites

  • Provides spatial information about protein proximities

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

Differentiating between direct and indirect effects of ubiB mutations requires systematic analytical approaches:

• Complementation analysis:

  • Transform ubiB mutants with plasmids expressing wild-type UbiB

  • Quantify the extent of phenotype rescue

  • Use domain-specific or site-directed mutants to map functional regions

• Metabolomic profiling:

  • Compare metabolite profiles of wild-type, ubiB mutant, and complemented strains

  • Identify specific accumulation of ubiquinone precursors

  • Look for changes in related metabolic pathways

• Global gene expression analysis:

  • Conduct RNA-seq to identify transcriptional changes in ubiB mutants

  • Look for compensatory responses or stress signatures

  • Compare with expression profiles of other ubi gene mutants

• Suppressor mutation analysis:

  • Isolate suppressor mutations that restore growth in ubiB mutants

  • Identify genes that can bypass UbiB function

  • Map genetic interactions within the ubiquinone pathway

• Time-course experiments:

  • Monitor phenotypic and metabolic changes at different time points after shifting to restrictive conditions

  • Distinguish primary (rapid) from secondary (delayed) effects of ubiB inactivation

What bioinformatic tools are most useful for analyzing UbiB structure and function?

Several bioinformatic tools and approaches are valuable for analyzing UbiB structure and function:

• Sequence analysis tools:

  • Multiple sequence alignment (MUSCLE, Clustal Omega) to identify conserved residues across species

  • Phylogenetic analysis to trace evolutionary relationships among UbiB homologs

  • Domain prediction (PFAM, InterPro) to identify functional domains

• Structure prediction:

  • AlphaFold2 or RoseTTAFold for generating protein structure models

  • Molecular dynamics simulations to study protein flexibility and substrate interactions

  • Docking simulations to predict protein-substrate or protein-protein interactions

• Functional prediction:

  • Gene ontology (GO) enrichment analysis

  • Protein-protein interaction network analysis using STRING or similar databases

  • Co-expression analysis to identify functionally related genes

• Comparative genomics:

  • Analysis of gene neighborhoods across bacterial species

  • Identification of conserved operons or regulons

  • Correlation of UbiB presence with ubiquinone production across bacterial taxa

• Motif analysis:

  • Identification of conserved sequence motifs related to enzymatic activity

  • Prediction of catalytic sites and residues

  • Analysis of potential post-translational modification sites

How can researchers distinguish between aerobic and anaerobic ubiquinone biosynthesis pathways?

Distinguishing between aerobic and anaerobic ubiquinone biosynthesis pathways requires specialized experimental approaches:

• Isotope labeling experiments:

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

  • Compare isotope incorporation patterns between aerobic and anaerobic growth

  • Identify the source of oxygen atoms in hydroxylated intermediates

• Genetic approaches:

  • Generate mutants in aerobic pathway genes (ubiB, ubiH) and anaerobic pathway genes (ubiT, ubiU, ubiV)

  • Assess growth and ubiquinone production under both aerobic and anaerobic conditions

  • Perform epistasis analysis using double mutants

• Analytical methods:

  • Use HPLC, LC-MS, or GC-MS to separate and identify pathway-specific intermediates

  • Develop targeted assays for specific pathway markers

  • Compare metabolite profiles between conditions using metabolomics

• Time-course analysis:

  • Monitor changes in gene expression and metabolite levels during transition from anaerobic to aerobic conditions

  • Focus on the role of regulatory elements like Fnr in pathway switching

• Functional analysis:

  • Assess the contribution of each pathway to specific cellular functions (e.g., nitrate respiration, pyrimidine synthesis)

  • Measure fitness effects in different environments, including animal models

What statistical approaches are appropriate for analyzing UbiB expression data across different conditions?

When analyzing UbiB expression data across different experimental conditions, researchers should consider these statistical approaches:

• Differential expression analysis:

  • For RNA-seq data: DESeq2, edgeR, or limma-voom

  • For protein expression: limma for proteomics data

  • Appropriate normalization methods based on data distribution

• Multiple testing correction:

  • Benjamini-Hochberg procedure for controlling false discovery rate

  • Bonferroni correction for stringent control of family-wise error rate

  • q-value estimation for large-scale analyses

• Correlation analysis:

  • Pearson correlation for parametric relationships

  • Spearman or Kendall correlation for non-parametric relationships

  • Correlation networks to identify co-expression patterns with other Ubi proteins

• Time-series analysis:

  • Smoothing techniques for temporal expression data

  • Autoregressive integrated moving average (ARIMA) models

  • Principal component analysis for identifying major sources of variation

• Multivariate analysis:

  • Principal component analysis (PCA) or t-SNE for dimension reduction

  • Hierarchical clustering to identify condition-specific expression patterns

  • Partial least squares discriminant analysis (PLS-DA) for separating experimental groups

What are the current gaps in our understanding of UbiB function?

Despite significant progress in understanding ubiquinone biosynthesis, several critical gaps remain in our knowledge of UbiB function:

• The precise biochemical mechanism of UbiB-mediated hydroxylation remains incompletely characterized. While UbiB is known to be involved in hydroxylation reactions during ubiquinone synthesis, the exact catalytic mechanism, including cofactor requirements and reaction intermediates, requires further investigation .

• The three-dimensional structure of UbiB has not been determined experimentally, limiting our understanding of its catalytic site and substrate interactions. Structural studies would provide valuable insights into how UbiB recognizes and processes its substrates.

• The regulatory mechanisms controlling UbiB expression and activity under different environmental conditions remain poorly understood. While some ubiquinone biosynthesis genes are known to be regulated by oxygen-sensing factors like Fnr , the specific regulation of UbiB requires further clarification.

• The relationship between UbiB and the anaerobic ubiquinone biosynthesis pathway mediated by UbiUVT needs further exploration. Understanding how these pathways interact or compensate for each other would provide a more complete picture of ubiquinone biosynthesis regulation .

• The potential role of UbiB in bacterial pathogenesis and host-microbe interactions remains an understudied area that warrants further investigation, particularly given the importance of ubiquinone in bacterial adaptation to diverse environments .

What emerging technologies might advance research on UbiB and ubiquinone biosynthesis?

Several emerging technologies hold promise for advancing our understanding of UbiB and ubiquinone biosynthesis:

• Cryo-electron microscopy could enable determination of the three-dimensional structure of UbiB and its complexes with other Ubi proteins, providing insights into the molecular organization of the ubiquinone biosynthetic machinery.

• Single-cell metabolomics techniques would allow researchers to investigate cell-to-cell variability in ubiquinone biosynthesis and its regulation under different environmental conditions.

• Genome-wide CRISPR screens could identify previously unknown genes affecting ubiquinone biosynthesis and reveal synthetic interactions with ubiB and other ubi genes.

• Advanced protein labeling and imaging techniques, such as proximity labeling combined with mass spectrometry, would provide detailed maps of the protein interaction networks involving UbiB in living cells.

• Systems biology approaches integrating multi-omics data (transcriptomics, proteomics, metabolomics) could reveal how UbiB function is coordinated with broader cellular processes and how ubiquinone biosynthesis responds to environmental changes.