Recombinant Salmonella enteritidis PT4 Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the structural composition and function of ubiquinone biosynthesis protein UbiB in Salmonella enteritidis PT4?

UbiB is a 546-amino acid protein (Uniprot: B5QW75) involved in the biosynthesis pathway of ubiquinone (coenzyme Q), an essential electron carrier in bacterial respiratory chains. In Salmonella enteritidis PT4 (strain P125109), UbiB is classified as a "probable ubiquinone biosynthesis protein," with the gene designated as SEN3767 . The full protein sequence reveals a complex structure with multiple hydrophobic regions that likely facilitate membrane association, consistent with its role in ubiquinone metabolism.

The functional significance of UbiB lies in its participation in the electron transport chain, where ubiquinone serves as an electron shuttle. Based on comparative analysis with E. coli, UbiB likely plays a crucial role in bacterial adaptation to different oxygen environments, as ubiquinone (UQ) is primarily utilized under aerobic conditions, while demethylmenaquinones (DMK) function predominantly in anaerobic environments .

The protein contains several key structural elements:

• Multiple hydrophobic regions suggesting membrane association

• Potential kinase-like domains involved in phosphorylation reactions

• Conserved motifs across related bacterial species

• Probable binding sites for pathway intermediates

Research approaches to further characterize UbiB's structure-function relationship should include crystallography, site-directed mutagenesis, and functional complementation studies.

How does Salmonella enteritidis PT4 UbiB differ from homologous proteins in other bacterial species?

The ubiquinone biosynthesis pathway in closely related E. coli involves both oxygen-dependent and oxygen-independent mechanisms, with the latter controlled by ubiT, ubiU, and ubiV genes . This suggests Salmonella may possess similar pathway flexibility, with UbiB potentially serving different functions depending on environmental oxygen availability.

Methodologically, researchers investigating interspecies differences should:

• Perform comprehensive sequence alignments between Salmonella UbiB and homologs

• Conduct heterologous complementation experiments testing if UbiB from other species can restore function in Salmonella ubiB mutants

• Compare enzyme kinetics and substrate specificity using purified recombinant proteins

• Analyze expression patterns under identical growth conditions

Understanding these differences has implications for evolutionary studies and potentially for developing species-specific antimicrobial targets.

What methods can be used to efficiently express and purify Salmonella enteritidis PT4 UbiB protein for research applications?

Efficient expression and purification of recombinant Salmonella enteritidis PT4 UbiB requires careful consideration of its biochemical properties and potential membrane association. The following methodological approach is recommended:

Expression system optimization:

• Vector selection: pET-based vectors with tightly regulated T7 promoters are appropriate for potentially toxic metabolic proteins

• Host strain: BL21(DE3) or derivatives with additional features like pLysS for tighter expression control

• Expression conditions: Lower temperatures (16-20°C) often improve solubility of membrane-associated proteins

• Induction parameters: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8 for 4-16 hours

Purification strategy:

• Cell lysis: Gentle methods preserving protein structure (enzymatic lysis with lysozyme followed by mild sonication)

• Initial capture: Affinity chromatography using engineered tags (His6, GST)

• Intermediate purification: Ion exchange chromatography

• Polishing: Size exclusion chromatography

Buffer optimization:
Based on information from commercially available recombinant UbiB, the protein is stable in Tris-based buffer with 50% glycerol . A recommended buffer composition would be:

• 50 mM Tris-HCl, pH 7.5-8.0

• 150-300 mM NaCl

• 5-10% glycerol during purification steps

• 50% glycerol for long-term storage

• 1-5 mM reducing agent (DTT or β-mercaptoethanol)

Storage considerations:
Store the purified protein at -20°C for extended periods, with -80°C recommended for very long-term storage. Avoid repeated freeze-thaw cycles by preparing single-use aliquots .

How can site-directed mutagenesis be employed to identify critical functional domains in Salmonella enteritidis UbiB?

Site-directed mutagenesis represents a powerful approach to dissect the functional architecture of UbiB. Based on the available amino acid sequence , researchers should implement the following comprehensive mutagenesis strategy:

Target selection rationale:

• Conserved residues identified through multiple sequence alignments of UbiB across bacterial species

• Predicted active sites based on structural models or homology with characterized proteins

• Putative membrane-interacting regions (hydrophobic patches)

• Potential substrate-binding pockets

• Regions implicated in protein-protein interactions with other ubiquinone biosynthesis enzymes

Systematic mutation approach:

Functional validation methods:

• Enzymatic activity assays measuring ubiquinone production

• Complementation of UbiB-deficient strains

• Protein-protein interaction analysis with other pathway components

• Structural integrity assessment via circular dichroism or thermal shift assays

This approach systematically maps structure-function relationships while avoiding potential artifacts from more dramatic genetic manipulations.

What experimental designs are most effective for investigating UbiB's role in Salmonella pathogenesis and virulence?

Investigating UbiB's role in Salmonella pathogenesis requires a multi-faceted experimental approach combining genetic, biochemical, and infection models. The following research design provides comprehensive insights while controlling for experimental variables:

Genetic manipulation strategies:

• Generate precise ubiB deletion mutants using λ-Red recombination

• Create conditional expression strains using tetracycline-regulatable promoters

• Develop complemented strains expressing wild-type or mutant UbiB variants

• Engineer reporter fusions (luciferase/GFP) to monitor ubiB expression during infection

In vitro infection models:

• Epithelial cell invasion assays (measuring adherence, invasion efficiency)

• Macrophage survival assays (quantifying intracellular replication)

• Neutrophil killing resistance tests

• Biofilm formation capacity assessment

In vivo infection models:

• Mouse typhoid model (systemic infection)

• Streptomycin-pretreated mouse colitis model (gastrointestinal infection)

• Competitive index assays co-infecting with wild-type and ubiB mutants

• Organ bacterial burden quantification (liver, spleen, mesenteric lymph nodes)

Mechanistic investigations:

• Ubiquinone quantification during different infection stages

• Transcriptional profiling of wild-type vs. ubiB mutants during infection

• Metabolomic analysis of central carbon metabolism

• Assessment of resistance to host-derived antimicrobial factors

This design is particularly relevant considering that Salmonella enteritidis is implicated in 60% of salmonellosis cases in Europe and is globally recognized as a leading cause of this disease . Understanding metabolic adaptations during infection could reveal new intervention strategies.

How does the ubiquinone biosynthesis pathway in Salmonella adapt to changing oxygen conditions, and what role does UbiB play in this adaptation?

Salmonella's ability to transition between oxygen-rich and oxygen-limited environments is critical for its success as a pathogen. The ubiquinone biosynthesis pathway, including UbiB, plays a central role in this adaptation. Based on research in related bacteria, we can outline the following methodological approach to investigate this process:

Experimental design for studying oxygen-dependent adaptation:

• Growth condition matrix:

  • Strictly aerobic (vigorous shaking, high oxygen transfer)

  • Microaerobic (limited oxygen transfer)

  • Anaerobic (oxygen-free atmosphere)

  • Oxygen shift experiments (transition between conditions)

• Analytical methods:

  • Quantitative analysis of ubiquinone and menaquinone content by HPLC or LC-MS

  • Transcriptional profiling of ubiB and related genes using RT-qPCR

  • Protein expression analysis via Western blotting

  • Bacterial growth kinetics under each condition

• Genetic approach:

  • Compare wild-type, ΔubiB, and complemented strains

  • Create reporter constructs to monitor pathway regulation

From E. coli studies, we know that ubiquinone (UQ) is predominantly used under aerobic conditions while demethylmenaquinones (DMK) function under anaerobic conditions . Notably, an anaerobic O2-independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes exists in E. coli , suggesting similar mechanisms may operate in Salmonella.

The research should focus on:

• Determining if UbiB is differentially expressed under varying oxygen tensions

• Quantifying the metabolic impact of UbiB deficiency under each condition

• Identifying if UbiB interacts with the anaerobic UQ biosynthesis machinery

• Measuring the kinetics of adaptation during environmental transitions

These studies will provide insights into how Salmonella modulates its respiratory chain during infection as it encounters diverse oxygen environments within the host.

What are the most effective experimental controls when working with recombinant Salmonella enteritidis UbiB protein?

Rigorous experimental controls are essential when working with recombinant Salmonella enteritidis UbiB to ensure data validity and reproducibility. The following comprehensive control framework should be implemented:

For protein expression and purification:

For functional studies:

• Enzymatic activity controls:

  • Substrate-free reactions to establish baseline

  • Heat-inactivated enzyme controls

  • Reactions with known inhibitors

  • Concentration gradients to establish linearity

• Binding assay controls:

  • Non-specific binding surfaces

  • Competitive binding with unlabeled ligands

  • Tests with known interaction partners and non-interactors

• Structural analysis controls:

  • Reference proteins with known properties

  • Multiple buffer conditions

  • Technical replicates across different protein preparations

• In vivo complementation controls:

  • Empty vector complementation

  • Complementation with unrelated proteins

  • Partial complementation with truncated variants

The storage buffer for recombinant UbiB should contain 50% glycerol in a Tris-based buffer as indicated by commercial preparations , which serves as a valuable reference point for maintaining protein stability during experimental manipulations.

What analytical techniques provide the most informative data on UbiB's role in ubiquinone biosynthesis in Salmonella?

A comprehensive analytical approach combining multiple techniques yields the most informative data on UbiB's role in ubiquinone biosynthesis. These methodologies should be implemented sequentially to build a complete functional profile:

1. Biochemical activity characterization:

• Enzyme kinetics: Measure reaction rates under varying substrate concentrations to determine Km, Vmax, and catalytic efficiency

• Substrate specificity: Test activity with various pathway intermediates to identify precise reaction step

• Cofactor requirements: Systematically test effects of potential cofactors (ATP, metals, etc.)

• pH and temperature optima: Establish conditions for maximum activity

2. Metabolite analysis:

• HPLC quantification: Measure ubiquinone content in wild-type vs. ΔubiB strains

• LC-MS/MS identification: Detect and quantify pathway intermediates that accumulate in mutants

• Isotope labeling: Track incorporation of labeled precursors to determine flux through the pathway

• Metabolic flux analysis: Quantify changes in central carbon metabolism feeding into ubiquinone synthesis

3. Structural biology approaches:

4. Interaction studies:

• Affinity purification coupled with mass spectrometry: Identify interaction partners

• Bacterial two-hybrid screening: Map protein-protein interactions within the pathway

• Surface plasmon resonance: Determine binding kinetics with substrates or other proteins

5. In vivo approaches:

• Reporter gene fusions: Monitor expression under different conditions

• Complementation analysis: Test ability of mutant variants to restore function

• Phenotypic microarrays: Assess growth across hundreds of conditions

This multilayered analytical framework provides complementary data points that, when integrated, offer a comprehensive understanding of UbiB's precise biochemical function and regulatory context.

How can researchers effectively investigate the relationship between UbiB function and antibiotic resistance in Salmonella enteritidis?

The potential relationship between UbiB function and antibiotic resistance represents an important research direction, particularly given that "Multidrug-resistant (MDR) due to Salmonella is known as a major public health problem around the world" . A systematic research approach should include:

1. Susceptibility profiling:

2. Mechanistic investigations:

• Measure membrane potential in wild-type vs. ΔubiB strains using fluorescent probes

• Quantify intracellular antibiotic accumulation using radiolabeled or fluorescent antibiotics

• Assess expression of efflux pump components with and without functional UbiB

• Monitor ATP levels to correlate energy production with resistance mechanisms

• Measure oxidative stress responses following antibiotic exposure

3. Genetic approaches:

• Create libraries of UbiB point mutants with varying levels of function

• Measure mutation frequency to antibiotic resistance in UbiB-deficient backgrounds

• Perform transcriptome analysis comparing wild-type and ΔubiB responses to antibiotics

• Screen for suppressors that restore antibiotic sensitivity in UbiB mutants

4. Clinical relevance assessment:

• Compare UbiB sequence variations in antibiotic-sensitive vs. resistant clinical isolates

• Test clinical isolates for correlations between ubiquinone production and resistance profiles

• Evaluate synergy between metabolic inhibitors and conventional antibiotics

5. Pharmacological intervention:

• Develop UbiB inhibitors and test as antibiotic adjuvants

• Test existing metabolic inhibitors for ability to potentiate antibiotic activity

This research framework systematically explores how energy metabolism through the ubiquinone pathway may contribute to antibiotic resistance mechanisms, potentially revealing new therapeutic strategies.

How has the UbiB protein evolved across different Salmonella serovars, and what does this reveal about its functional importance?

Evolutionary analysis of UbiB across Salmonella serovars provides crucial insights into its functional constraints and adaptive potential. A comprehensive research approach should incorporate:

1. Phylogenetic analysis methods:

• Sequence acquisition from multiple Salmonella serovars, including Enteritidis and Typhimurium (highlighted in search result )

• Multiple sequence alignment using MUSCLE or MAFFT algorithms

• Phylogenetic tree construction using maximum likelihood methods

• Calculation of sequence conservation metrics (percent identity, similarity)

• Analysis of selection pressure using dN/dS ratios to identify regions under purifying or positive selection

2. Structural conservation analysis:

• Homology modeling of UbiB from different serovars

• Structural superimposition to identify conserved three-dimensional elements

• Mapping of conserved residues onto structural models

• Prediction of functionally important regions based on conservation patterns

3. Functional domain comparison:

• Identification of conserved catalytic residues across serovars

• Analysis of potential substrate binding sites

• Comparison of predicted membrane-interacting regions

• Evaluation of protein-protein interaction interfaces

4. Experimental validation approaches:

• Cross-complementation experiments testing if UbiB from different serovars can functionally substitute

• Biochemical characterization of UbiB variants from diverse serovars

• Creation of chimeric proteins to map functionally important domains

• Correlation of sequence variations with phenotypic differences

This evolutionary perspective is particularly relevant given that Salmonella enterica serovar Enteritidis and Typhimurium are the most important strains affecting humans , with Enteritidis implicated in 60% of European salmonellosis cases and being the world's leading cause of this disease .

The high degree of expected conservation in UbiB across serovars would reflect its essential role in energy metabolism, while variations might indicate adaptations to different host environments or ecological niches.

What is the relationship between the UbiB protein and the anaerobic ubiquinone biosynthesis pathway identified in other bacteria?

The relationship between UbiB and the anaerobic ubiquinone biosynthesis pathway represents an intriguing research question with implications for understanding Salmonella's metabolic versatility. Based on information from search result , we can outline a research approach to investigate this connection:

1. Comparative genomics approach:

• Identify homologs of the E. coli ubiT, ubiU, and ubiV genes (which control anaerobic UQ biosynthesis) in Salmonella enteritidis

• Analyze gene neighborhoods and potential operon structures

• Compare regulatory elements controlling expression of these genes

• Conduct phylogenetic analysis to determine evolutionary relationships

2. Expression analysis methods:

• Perform RT-qPCR to quantify expression of ubiB versus anaerobic pathway genes under varying oxygen conditions

• Use reporter gene fusions to visualize expression patterns in single cells

• Conduct Western blotting to measure protein levels

• Implement ChIP-seq to identify transcription factors controlling these pathways

3. Functional interaction studies:

• Create single and combination gene knockouts (ΔubiB, ΔubiTUV, double mutants)

• Measure ubiquinone production under aerobic and anaerobic conditions

• Perform bacterial two-hybrid or co-immunoprecipitation experiments to detect protein-protein interactions

• Conduct metabolomic profiling to identify pathway intermediates

4. Phenotypic characterization:

• Compare growth kinetics of mutants under varying oxygen concentrations

• Assess respiratory capacity using oxygen consumption measurements

• Determine electron transport chain composition and activity

• Evaluate adaptation to oxygen fluctuations through transition experiments

This investigation would clarify whether UbiB functions exclusively in the traditional aerobic pathway or also contributes to the oxygen-independent mechanism, providing insights into how Salmonella adapts its energy metabolism during host colonization where it encounters varying oxygen tensions.

What are the most promising approaches for developing targeted inhibitors of UbiB as potential antimicrobial agents?

The development of UbiB inhibitors as antimicrobial agents represents a promising research direction, particularly given the rising concern of multidrug-resistant Salmonella . A comprehensive drug discovery pipeline would include:

1. Target validation strategies:

• Confirm essentiality of UbiB in Salmonella growth and virulence through conditional knockouts

• Assess phenotypic consequences of UbiB inhibition in relevant infection models

• Evaluate potential for resistance development through directed evolution experiments

• Determine if human homologs exist that might cause off-target effects

2. High-throughput screening approaches:

• Develop biochemical assays suitable for compound library screening

• Implement whole-cell screening using reporter strains

• Design counterscreens to eliminate non-specific inhibitors

• Establish clear selection criteria for hit compounds

3. Structure-based drug design methodology:

• Generate high-quality structural data through X-ray crystallography or cryo-EM

• Identify druggable pockets through computational analysis

• Conduct virtual screening of compound libraries

• Implement fragment-based approaches to identify chemical scaffolds

4. Medicinal chemistry optimization:

• Establish structure-activity relationships through systematic modifications

• Optimize for potency, selectivity, and physicochemical properties

• Implement iterative design-synthesis-testing cycles

• Address potential pharmacokinetic limitations

5. Compound characterization and validation:

• Determine mechanism of action through biochemical and genetic approaches

• Assess spectrum of activity against various Salmonella strains

• Evaluate activity against other bacterial pathogens

• Measure in vitro toxicity against mammalian cells

6. Preclinical evaluation:

• Test efficacy in animal models of Salmonella infection

• Determine pharmacokinetic and pharmacodynamic parameters

• Assess potential for resistance development

• Evaluate safety and toxicology profiles

This systematic approach leverages UbiB's essential role in energy metabolism to develop novel antimicrobials that could help address the significant public health burden of Salmonella infections, including the 43% of poultry isolates identified as S. Enteritidis in the referenced study .

How might advanced techniques like CRISPR-Cas9 genome editing and cryo-electron microscopy advance our understanding of UbiB function in Salmonella?

Emerging technologies offer unprecedented opportunities to deepen our understanding of UbiB function. A forward-looking research program would leverage these advanced techniques:

1. CRISPR-Cas9 genome editing applications:

• Precise genomic modifications:

  • Generate scarless mutations in ubiB with single nucleotide resolution

  • Create libraries of UbiB variants with systematic mutations across the protein

  • Introduce tagged versions of UbiB at the native locus

  • Implement CRISPRi for tunable gene expression control

• High-throughput functional genomics:

  • Conduct genome-wide screens to identify genetic interactions with ubiB

  • Perform tiling mutagenesis across the ubiB locus to map functional elements

  • Create double-mutant libraries to identify synthetic lethal interactions

  • Implement base editing for precise amino acid substitutions

2. Cryo-electron microscopy approaches:

• Structural determination:

  • Achieve high-resolution structures of UbiB in different functional states

  • Visualize UbiB in complex with interaction partners

  • Capture conformational changes during catalytic cycles

  • Examine UbiB integration into membrane complexes

• In situ structural biology:

  • Visualize UbiB localization within bacterial cells

  • Study native membrane associations through cellular tomography

  • Observe changes in distribution under different growth conditions

  • Examine reorganization during stress responses

3. Integrative multi-omics strategies:

• Combine transcriptomics, proteomics, and metabolomics data

• Implement systems biology modeling to predict UbiB function in metabolic networks

• Correlate structural insights with functional genomics data

• Develop predictive models of ubiquinone biosynthesis regulation

4. Single-cell technologies:

• Monitor UbiB expression heterogeneity in bacterial populations

• Track real-time changes in ubiquinone production at the single-cell level

• Correlate metabolic state with antibiotic susceptibility

• Observe adaptation to environmental changes in real-time

These advanced techniques will transform our understanding of how Salmonella modulates ubiquinone biosynthesis during infection processes, potentially revealing new intervention strategies against this important pathogen that causes significant disease burden worldwide .