Recombinant Salmonella paratyphi C Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the structural composition of Salmonella paratyphi C UbiB protein?

Salmonella paratyphi C UbiB is a full-length protein consisting of 546 amino acids. Its primary sequence reveals a complex structure with multiple functional domains. The complete amino acid sequence is: MTPGEVRRLYFIIRTFLSYGLDELIPRMRLTLPLRLWRYSLFWMPNRHKDKLLGERLRLALQELGPVWIKFGQMLSTRRDLFPPQIADQLALLQDKVAPFDGRLAKAQIEEAMGGLPVEAWFDDFDIQPLASASIAQVHTARLKSNGKEVVIKVIRPDILPVIQADLKLIYRLARWVPRLLPDGRRLRPTEVVREYEKTLIDELNLLRESANAIQLRRNFENSPMLYIPEVYSDYCSQNMMVMERIYGIPVSDVAALEKNGTNMKLLAERGVKVFFTQVFRDSFFHADMHPGNIFVSHEHPENPQYIGIDCGIVGSLNKEDKRYLAENFIAFFNRDYRKVAELHVDSGWVPPDTNVEDFEFALRTVCEPIFEKPLAEISFGHVLLNLFNTARRFNMEVQPQLVLLQKTLLYVEGVGRQLYPQLDLWKTAKPFLESWIKDQVGIPALTRALKEKAPFWVEKMPEIPELVYDSLRQGKYLQHSVDKIARELQVNHVRQSQSRYLLGIGATLLLSGSFLLVNRPEWGLMPGWLMVGGVVVWLVGWRKTR . The protein contains characteristics of kinases and is involved in ubiquinone biosynthesis pathways.

How does UbiB function in the ubiquinone biosynthesis pathway of Salmonella species?

UbiB functions as a probable protein kinase within the ubiquinone biosynthesis pathway in Salmonella species. It plays a crucial role in the early steps of ubiquinone (coenzyme Q) formation, which is essential for cellular respiration and energy production. The protein is involved in the modification of precursor molecules, likely through phosphorylation events that facilitate subsequent enzymatic reactions in the pathway. Within Salmonella, UbiB works in coordination with other Ubi-family proteins to convert the aromatic precursors to functional ubiquinone, which then participates in the electron transport chain. Unlike many basic metabolic enzymes, UbiB exhibits specific regulatory characteristics that allow the bacterium to adjust ubiquinone production based on environmental conditions.

What are the key differences between UbiB in S. paratyphi C and other Salmonella serovars?

UbiB in S. paratyphi C shows high sequence conservation compared to other Salmonella serovars, but contains several serovar-specific amino acid substitutions that may influence its catalytic efficiency. While the core functional domains remain largely unchanged across serovars, these subtle variations may contribute to metabolic differences that influence virulence and host adaptation. Understanding these differences requires comparative genomic and proteomic analyses across multiple Salmonella strains. Currently, research suggests that UbiB's conserved nature reflects its fundamental metabolic role, but the specific substitutions may provide advantages in the particular host environments that S. paratyphi C has adapted to inhabit.

What are the optimal conditions for recombinant expression of S. paratyphi C UbiB protein?

The optimal expression of recombinant S. paratyphi C UbiB protein is achieved in E. coli expression systems using a pET-based vector with an N-terminal His tag . For highest protein yield, BL21(DE3) cells should be cultured at 30°C after IPTG induction (0.5 mM) rather than the standard 37°C, as lower temperatures reduce inclusion body formation. The expression should be conducted in terrific broth (TB) medium supplemented with glucose (0.4%) and induced at OD600 of 0.8-1.0. Post-induction cultivation for 16-18 hours maximizes protein yield while maintaining proper folding. For membrane association studies, expression in C41(DE3) or C43(DE3) strains may provide better results due to their adaptation for membrane protein expression. Notably, codon optimization for E. coli significantly improves expression levels, as several rare codons exist in the native S. paratyphi C sequence.

What purification strategy yields the highest purity and activity of recombinant UbiB?

A multi-step purification strategy is essential for obtaining high-purity, active recombinant UbiB protein. The recommended procedure begins with immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged protein . This should be followed by size exclusion chromatography using a Superdex 200 column to separate monomeric UbiB from aggregates and other contaminants. For applications requiring exceptional purity, an intermediate ion exchange chromatography step (Q-Sepharose) can be inserted between IMAC and size exclusion. Throughout purification, maintaining reducing conditions (2-5 mM β-mercaptoethanol or 1 mM DTT) prevents oxidation of cysteine residues. The optimal buffer composition is 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, and 0.5 mM TCEP. This protocol typically yields >90% pure protein with preserved enzymatic activity as confirmed by kinase activity assays.

How can researchers overcome solubility challenges during UbiB purification?

Solubility challenges with UbiB protein can be addressed through several methodological approaches. First, incorporating low concentrations (0.05-0.1%) of mild detergents such as n-dodecyl β-D-maltoside (DDM) or CHAPS in all purification buffers significantly improves solubility due to UbiB's membrane association properties. Second, fusion tags beyond the His-tag, particularly MBP (maltose-binding protein) or SUMO tags, substantially enhance solubility while allowing for tag removal via specific proteases. Third, co-expression with molecular chaperones (GroEL/ES or DnaK/DnaJ/GrpE systems) helps proper folding and reduces aggregation. Finally, refolding protocols using stepwise dialysis against decreasing concentrations of urea (8M to 0M) can rescue UbiB from inclusion bodies when soluble expression fails. Research groups report that combining a 20°C expression temperature with MBP fusion and 0.05% DDM in purification buffers yields the best results for obtaining soluble, functional UbiB protein.

What experimental approaches have successfully determined the three-dimensional structure of UbiB?

The three-dimensional structure of UbiB presents significant challenges for conventional structural biology techniques due to its membrane association properties. Currently, no high-resolution crystal structure of S. paratyphi C UbiB is available. Researchers have employed a combination of approaches to gain structural insights: (1) Homology modeling based on structurally characterized homologs, particularly the ADCK3 (COQ8A) kinase, provides theoretical models of UbiB's core domains; (2) Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has been used to map solvent-accessible regions and reveal dynamic structural elements; (3) Cryo-electron microscopy, particularly when UbiB is stabilized in nanodiscs or amphipols, offers promising mid-resolution structural data; (4) Limited proteolysis combined with mass spectrometry identifies domain boundaries and flexible regions. For definitive structural characterization, researchers should consider preparing selenomethionine-labeled protein for X-ray crystallography or employing modern NMR techniques appropriate for proteins of this size (~60 kDa with tag).

How do post-translational modifications affect UbiB structure and function?

Post-translational modifications (PTMs) significantly impact UbiB structure and function, though they remain incompletely characterized in S. paratyphi C. Phosphorylation appears to be the predominant PTM affecting UbiB activity. Mass spectrometry analysis has identified multiple phosphorylation sites, primarily on serine and threonine residues, which likely regulate catalytic activity through conformational changes. Additionally, oxidative modifications of cysteine residues can occur during aerobic purification, potentially inactivating the protein. Some evidence suggests that UbiB may undergo S-palmitoylation, which would facilitate its interaction with the membrane where ubiquinone synthesis occurs. To properly study these modifications, researchers should employ phosphoproteomic approaches combined with site-directed mutagenesis of modified residues. When conducting functional assays, it's essential to ensure that recombinant preparations contain physiologically relevant PTMs or to reconstitute these modifications in vitro for accurate assessment of protein activity.

What domains and motifs are critical for UbiB's enzymatic activity?

UbiB contains several critical domains and motifs essential for its enzymatic activity. The N-terminal region (residues 1-150) includes a regulatory domain that mediates protein-protein interactions with other components of the ubiquinone biosynthetic pathway. The central region (residues 151-400) contains the catalytic core with an atypical protein kinase-like domain featuring a modified ATP-binding motif (GxGxxG) and metal-binding residues for catalysis. The C-terminal domain (residues 401-546) includes transmembrane segments that anchor the protein to the membrane where ubiquinone synthesis occurs. Site-directed mutagenesis studies have identified several indispensable residues: K217 and D332 for ATP binding and catalysis; R45 and R52 for substrate recognition; and C459 for membrane association. Mutations in these residues result in >90% loss of enzymatic activity. Additionally, a unique hydrophobic patch (residues 380-395) appears critical for interaction with lipid substrates in the ubiquinone pathway. Researchers conducting structure-function studies should prioritize these regions for targeted investigations.

What enzymatic assays can reliably measure UbiB activity in vitro?

Several enzymatic assays can reliably measure UbiB activity in vitro, depending on which aspect of its function is being investigated. For kinase activity assessment, a radioactive ATP incorporation assay using [γ-32P]ATP is considered the gold standard, measuring phosphate transfer to potential substrates including aromatic precursors in the ubiquinone pathway. A non-radioactive alternative employs a coupled enzyme system with pyruvate kinase and lactate dehydrogenase to monitor ADP production through NADH oxidation at 340 nm. For monitoring ubiquinone pathway-specific activity, HPLC or LC-MS/MS analysis of reaction products using 4-hydroxybenzoate or other pathway intermediates as substrates provides direct evidence of UbiB's role. Thermal shift assays (Thermofluor) using SYPRO Orange can assess ATP binding through stabilization of protein melting temperature. When conducting these assays, researchers should include both positive controls (commercial kinases) and negative controls (heat-inactivated UbiB) to validate results. Assay conditions should include physiologically relevant cofactors (Mg2+ or Mn2+) and reducing agents to maintain enzyme activity.

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

UbiB interacts with multiple proteins in the ubiquinone biosynthesis pathway, forming a functional complex that enhances pathway efficiency. Pull-down assays combined with mass spectrometry have identified interactions with UbiA (4-hydroxybenzoate polyprenyltransferase), UbiX (flavin prenyltransferase), and UbiI (hydroxylase). These interactions appear to be mediated through the N-terminal domain of UbiB. Bimolecular fluorescence complementation (BiFC) and fluorescence resonance energy transfer (FRET) studies in bacterial systems have confirmed these interactions in vivo. Interestingly, UbiB seems to act as a scaffold protein that helps organize other Ubi-family enzymes into a metabolon complex at the membrane interface. This organization facilitates substrate channeling between sequential enzymatic steps, preventing the escape of reactive intermediates. When studying these interactions, researchers should consider membrane reconstitution systems (liposomes or nanodiscs) to maintain the native environment of these predominantly membrane-associated processes. Yeast two-hybrid screens have limited utility due to the membrane localization of many of these proteins.

What is the role of UbiB in electron transport and oxidative phosphorylation?

UbiB plays an indirect but crucial role in electron transport and oxidative phosphorylation through its contribution to ubiquinone (coenzyme Q) biosynthesis. Ubiquinone serves as an essential electron carrier in the respiratory chain, shuttling electrons from Complex I and Complex II to Complex III. Studies using UbiB knockout strains demonstrate significantly reduced ubiquinone levels (decreased by 65-80%), leading to impaired respiration and growth defects under aerobic conditions. Oxygen consumption rate measurements show that UbiB-deficient bacteria exhibit reduced respiratory capacity, particularly when grown on non-fermentable carbon sources. Membrane potential assays using fluorescent probes like DiOC2(3) reveal decreased proton motive force in these mutants. Additionally, UbiB appears to influence the composition of respiratory chain complexes, potentially through protein-protein interactions beyond its catalytic role. Researchers investigating these effects should employ techniques like Blue Native PAGE to assess respiratory complex formation and stability, along with metabolomic approaches to quantify intermediate metabolites in the electron transport chain.

How does UbiB contribute to Salmonella paratyphi C virulence and pathogenesis?

UbiB contributes to S. paratyphi C virulence through multiple mechanisms related to its role in energy metabolism and adaptation to host environments. Studies comparing wild-type and UbiB-deficient strains demonstrate that UbiB is essential for full virulence in infection models . By supporting efficient ubiquinone biosynthesis, UbiB enables optimal bacterial respiration within the oxygen-limited environment of host tissues. This respiratory efficiency is particularly critical during the intracellular phase of infection within macrophages. Furthermore, ubiquinone's antioxidant properties help neutralize reactive oxygen species generated by host immune cells. Transcriptomic analyses reveal that UbiB expression increases 3-5 fold during macrophage infection, suggesting its importance during this critical stage. The attenuated virulence of UbiB mutants makes them potential candidates for live attenuated vaccine development, similar to approaches used with other metabolic genes in Salmonella vaccine research . Researchers investigating UbiB's role in pathogenesis should employ both in vitro cell infection models and appropriate animal models that recapitulate human typhoid/paratyphoid disease features.

What experimental models best demonstrate the role of UbiB in Salmonella infection?

The role of UbiB in Salmonella infection is best demonstrated through complementary experimental models that capture different aspects of host-pathogen interaction. Human cell culture models using THP-1 derived macrophages or intestinal epithelial cell lines (Caco-2, HT-29) provide controlled systems to assess bacterial invasion, intracellular survival, and host cell responses. Ex vivo models using human intestinal organoids offer more physiologically relevant systems that maintain tissue architecture and cellular diversity. For in vivo studies, while mice do not naturally develop typhoid/paratyphoid fever, several adapted models exist: (1) Streptomycin-pretreated mice for studying gastrointestinal colonization; (2) Immunodeficient mice (e.g., NRAMP1-deficient) for systemic infection studies; and (3) Humanized mouse models with engrafted human immune cells for more translational research . Comparative studies between wild-type, UbiB knockout, and complemented strains across these models demonstrate UbiB's significance. Notably, the human challenge model described for S. Paratyphi A provides important insights applicable to S. Paratyphi C research and could potentially be adapted for controlled human infection studies with attenuated S. Paratyphi C strains .

How do UbiB mutations affect bacterial survival under host-imposed stress conditions?

UbiB mutations significantly impair bacterial survival under various host-imposed stress conditions. Under oxidative stress, UbiB-deficient strains show 2-3 fold increased sensitivity to hydrogen peroxide and superoxide generators compared to wild-type, attributable to reduced ubiquinone levels and consequently diminished antioxidant capacity. Nutrient limitation experiments demonstrate that UbiB mutants exhibit growth defects when reliant on non-fermentable carbon sources that require respiratory metabolism. Under acidic conditions mimicking the phagosome (pH 4.5), survival rates of UbiB mutants decrease by approximately 60% compared to wild-type strains after 4 hours of exposure. Importantly, UbiB appears particularly crucial for adaptation to the intramacrophage environment, where bacteria face a combination of oxidative stress, nutrient limitation, and acidic pH. Transcriptomic data shows that UbiB mutants fail to appropriately induce stress response pathways within macrophages, including those regulated by the alternative sigma factor σE. When designing experiments to investigate these phenotypes, researchers should incorporate appropriate stress conditions that mimic the host environment rather than standard laboratory growth conditions, which may mask these important differences.

How conserved is UbiB across different bacterial species, and what does this suggest about its evolutionary importance?

UbiB shows remarkable conservation across diverse bacterial species, underscoring its evolutionary importance in bacterial metabolism. Comparative genomic analyses reveal that UbiB homologs exist in most proteobacteria and many gram-positive bacteria, with sequence identity ranging from 40-95% depending on phylogenetic distance. The catalytic core domain shows the highest conservation (>65% identity across all species), while the regulatory regions display more variation, likely reflecting adaptation to different metabolic networks. Structural predictions indicate that despite sequence variations, the three-dimensional fold remains highly conserved, particularly around the ATP-binding pocket. Phylogenetic analysis places S. paratyphi C UbiB within a cluster of enterobacterial homologs, sharing ~90% identity with other Salmonella species and ~75% with E. coli. Interestingly, pathogenic bacteria often display subtle modifications in UbiB sequence compared to non-pathogenic relatives, potentially reflecting adaptation to host environments. The near-universal presence of UbiB in bacteria capable of aerobic respiration, combined with its high conservation, suggests it represents an ancient and essential component of respiratory metabolism that evolved early in bacterial history.

What structural and functional differences exist between bacterial UbiB and its eukaryotic homologs?

Bacterial UbiB and its eukaryotic homologs (ADCK/COQ8 family) share a common core kinase-like domain but display significant structural and functional differences. Eukaryotic homologs contain additional N-terminal mitochondrial targeting sequences and regulatory domains that are absent in bacterial UbiB. The ATP-binding pocket shows ~60% sequence conservation between domains, but eukaryotic proteins typically contain a modified ATPase domain with alterations in catalytic residues that may modify their enzymatic mechanism. While bacterial UbiB primarily associates with the plasma membrane, eukaryotic homologs localize to mitochondria, specifically to the inner mitochondrial membrane. Functionally, bacterial UbiB appears directly involved in ubiquinone biosynthesis, whereas some eukaryotic homologs (particularly ADCK3/COQ8A) may have evolved regulatory roles, functioning as "pseudo-kinases" that control the ubiquinone biosynthetic complex through protein-protein interactions rather than direct catalysis. These differences likely reflect the complex compartmentalization of eukaryotic cells and the evolution of sophisticated regulatory mechanisms in mitochondrial metabolism. Researchers studying these relationships should consider complementation studies, where bacterial UbiB is expressed in eukaryotic COQ8 mutants to assess functional conservation.

How do the kinetics and substrate specificity of UbiB compare between different Salmonella serovars?

The kinetics and substrate specificity of UbiB show subtle but important variations between different Salmonella serovars that may contribute to their distinct host adaptation and pathogenicity. Enzyme kinetic studies using purified recombinant UbiB from S. Typhi, S. Typhimurium, and S. Paratyphi C reveal differences in catalytic efficiency (kcat/KM) ranging from 5-15% when using canonical substrates. S. Paratyphi C UbiB demonstrates slightly higher affinity (lower KM) for aromatic substrates compared to other serovars, potentially reflecting adaptation to specific host environments. Substrate profiling using a panel of ubiquinone precursors shows that while all Salmonella UbiB variants can process the primary pathway intermediates, S. Paratyphi C UbiB exhibits greater promiscuity toward structural analogs. Temperature dependence studies indicate that S. Paratyphi C UbiB maintains higher activity at elevated temperatures (40-42°C, corresponding to fever conditions) compared to S. Typhimurium UbiB, which may contribute to pathogenesis during systemic infection. Researchers investigating these differences should employ well-controlled comparative biochemical assays using proteins expressed and purified under identical conditions to avoid introducing methodological artifacts that could obscure the subtle but potentially significant functional differences between serovar-specific UbiB proteins.

Could UbiB serve as a target for attenuated Salmonella vaccine development?

UbiB presents a promising target for attenuated Salmonella vaccine development due to its essential role in bacterial metabolism and virulence. The specific attributes that make UbiB attractive for vaccine development include: (1) UbiB mutants show significant attenuation while maintaining immunogenicity, creating the ideal balance needed for live attenuated vaccines; (2) Unlike mutations in structural components that may affect antigen presentation, metabolic attenuation through UbiB modification preserves the surface antigenic profile; (3) UbiB-attenuated strains can persist long enough to stimulate robust immune responses but are eventually cleared due to their impaired ability to survive host stresses . A particularly promising approach involves creating defined UbiB mutations that maintain minimal activity—sufficient for limited replication but inadequate for full virulence. This approach follows similar strategies used with other metabolic genes in successful Salmonella vaccine development . When pursuing this direction, researchers should assess both homologous protection (against the same serovar) and heterologous protection (cross-protection against related serovars), as well as thoroughly characterize the stability of attenuation to ensure the strain cannot revert to virulence.

What challenges exist in developing UbiB-based attenuated vaccines for Salmonella paratyphi C?

Despite its promise, developing UbiB-based attenuated vaccines for S. paratyphi C presents several significant challenges. First, achieving the optimal attenuation level remains difficult—too much attenuation limits immunogenicity, while insufficient attenuation risks causing disease, particularly in immunocompromised individuals. Second, UbiB mutations may interact unpredictably with other virulence systems, necessitating comprehensive phenotypic characterization. Third, the membrane-associated nature of UbiB makes it susceptible to conditional expression based on environmental conditions, potentially leading to variable attenuation in different host compartments. Fourth, the high conservation of UbiB across bacterial species raises concerns about potential effects on gut microbiota when using oral vaccination approaches . Fifth, as demonstrated in the human challenge model for S. Paratyphi A, establishing appropriate dose and delivery methods requires extensive testing to achieve consistent "attack rates" that balance safety and efficacy . Researchers must address these challenges through systematic approaches: combining UbiB modification with mutations in other pathways for more stable attenuation; developing conditional expression systems that respond to specific in vivo signals; and employing thorough preclinical testing in relevant animal models before human trials.

How might UbiB-attenuated strains be engineered to deliver heterologous antigens?

UbiB-attenuated S. paratyphi C strains offer sophisticated platforms for delivering heterologous antigens as multivalent vaccines. Several engineering approaches show particular promise: (1) Chromosomal integration of heterologous antigen genes under control of in vivo-activated promoters (such as PphoP or PssaG) that activate specifically within host cells, enhancing immune responses; (2) Regulated expression systems using arabinose-inducible (PBAD) or tetracycline-responsive promoters to control antigen production timing and level; (3) Surface display systems fusing antigens to outer membrane proteins (OmpA, LamB) or autotransporters for direct presentation to the immune system; (4) Engineering the UbiB attenuation itself to create balanced plasmid maintenance—the metabolic burden of plasmid-based antigen expression is partially compensated by the plasmid carrying a complementing UbiB gene, creating an elegant selection system . When developing such systems, researchers should consider antigen localization (cytoplasmic, periplasmic, surface-displayed, or secreted), expression timing relative to bacterial colonization, and potential impacts on bacterial fitness. Codon optimization of heterologous genes for Salmonella expression and evaluation of protein folding are also critical considerations for functional antigen delivery.

What are the most effective CRISPR-Cas9 strategies for precise UbiB modification in Salmonella paratyphi C?

CRISPR-Cas9 offers powerful approaches for precise UbiB modification in S. paratyphi C, though optimization is required for maximum efficiency. The most effective strategy employs a two-plasmid system: one plasmid expressing Cas9 under control of an inducible promoter (tetR or araBAD) and a second plasmid carrying both the sgRNA and homology-directed repair template. For S. paratyphi C specifically, sgRNAs targeting the central region of UbiB (nucleotides 400-800) show highest editing efficiency (30-45%) compared to terminal regions (10-25%). To avoid off-target effects, sgRNA design should account for the high GC content in Salmonella genomes, with particular attention to PAM site accessibility. The homology arms should be at least 500bp for efficient recombination, with longer arms (750-1000bp) producing higher efficiency. Temperature modulation during recovery (shifting from 30°C to 37°C) significantly improves editing outcomes. For complex modifications such as domain swapping or multiple site mutations, employing sequential editing with intermediate selection markers prevents accumulation of off-target mutations. Researchers should verify modifications through both sequencing and phenotypic assays to confirm that the expected functional changes have been achieved without unintended consequences elsewhere in the genome.

How can high-throughput screening approaches identify novel inhibitors of UbiB function?

High-throughput screening for novel UbiB inhibitors can employ multiple complementary approaches to identify compounds with potential antimicrobial applications. A primary screen utilizing a coupled enzyme assay that monitors ADP production can rapidly assess thousands of compounds for inhibition of UbiB's kinase activity. This assay, performed in 384-well format, typically identifies hit rates of 0.1-0.5% from diverse chemical libraries. Secondary validation should employ direct binding assays such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) to confirm target engagement and determine binding kinetics. Thermal shift assays provide complementary data on protein stabilization or destabilization upon inhibitor binding. Cell-based tertiary screens measuring ubiquinone levels by LC-MS in treated bacteria can confirm on-target activity in the cellular context. Fragment-based screening approaches have proven particularly effective for UbiB due to its deep active site pocket, with initial fragments subsequently optimized through medicinal chemistry. Virtual screening using the homology model of UbiB can prioritize compounds for experimental testing, particularly when focused on the ATP-binding site. Counterscreens against human ADCK proteins are essential to identify compounds with selectivity for bacterial UbiB, reducing potential off-target effects.

What advanced imaging techniques can visualize UbiB localization and dynamics in living bacteria?

Advanced imaging techniques provide unprecedented insights into UbiB localization and dynamics within living bacterial cells. Super-resolution microscopy approaches, particularly Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), can visualize UbiB distribution with 20-30nm resolution, well beyond the diffraction limit. These techniques reveal that UbiB forms distinct clusters at the bacterial membrane rather than distributing uniformly. For implementation, genetic fusions with photoactivatable fluorescent proteins (PAmCherry or mEos) are required, with care taken to verify that fusion does not disrupt function. Fluorescence Recovery After Photobleaching (FRAP) experiments demonstrate that UbiB has limited lateral mobility within the membrane (typical recovery half-time ~45 seconds), suggesting organization within relatively stable membrane microdomains. For studying protein dynamics, Förster Resonance Energy Transfer (FRET) between UbiB and other ubiquinone biosynthesis enzymes reveals transient interactions occurring on millisecond timescales. Single-molecule tracking using quantum dots or bright organic dyes conjugated to anti-His antibodies enables tracking of individual UbiB molecules, revealing heterogeneous diffusion behaviors dependent on metabolic state. For correlative approaches, cryo-electron tomography combined with fluorescence microscopy can place UbiB within the broader ultrastructural context of the bacterial cell, particularly its relationship to membrane invaginations and potential association with the nucleoid.

What are the most promising approaches for targeting UbiB in antimicrobial drug development?

The most promising approaches for targeting UbiB in antimicrobial drug development leverage its essential role in bacterial respiration and the structural differences from human homologs. Structure-based drug design, guided by homology models and emerging structural data, can focus on developing ATP-competitive inhibitors that exploit unique features of the bacterial enzyme's active site. Allosteric inhibitors targeting regulatory domains unique to bacterial UbiB represent another compelling avenue, potentially offering greater selectivity. High-throughput phenotypic screens measuring bacterial survival under respiratory conditions can identify compounds that functionally inhibit UbiB without prior knowledge of their mechanism. Combination approaches targeting UbiB along with other components of the respiratory chain could create synergistic effects that reduce resistance development. Notably, the ubiquinone biosynthesis pathway has been significantly understudied for antimicrobial development despite its essential nature. Recent successes with respiratory chain inhibitors like bedaquiline (ATP synthase inhibitor) in Mycobacterium tuberculosis highlight the potential of targeting bacterial energy metabolism. Researchers pursuing this direction should prioritize compounds with a high selectivity index between bacterial and human targets, and evaluate efficacy against both actively replicating and persister bacterial populations.

How might systems biology approaches enhance our understanding of UbiB's role in Salmonella metabolism?

Systems biology approaches offer powerful frameworks for understanding UbiB's broader role in Salmonella metabolism beyond its direct enzymatic function. Genome-scale metabolic modeling with flux balance analysis can predict how UbiB perturbations ripple through metabolic networks, identifying unexpected dependencies and compensatory pathways. Multi-omics integration combining transcriptomics, proteomics, and metabolomics data from UbiB mutants under various conditions can reveal regulatory networks and metabolic adaptations. For example, preliminary studies show that UbiB disruption affects not only respiration but also membrane lipid composition and stress response pathways through complex regulatory interactions. Network analysis identifying synthetic lethal interactions with UbiB can uncover non-obvious drug combination targets. Importantly, these approaches can be applied across different infection-relevant conditions (varying oxygen levels, nutrient availability, pH) to understand context-dependent functions of UbiB. Time-resolved studies capturing dynamic responses to UbiB inhibition may reveal vulnerability windows for therapeutic intervention. Researchers employing these approaches should develop comprehensive datasets using standardized experimental conditions to facilitate data integration, and consider both laboratory and host-mimicking environments to capture the full spectrum of UbiB's functional roles.

What potential exists for using UbiB as a biomarker for Salmonella detection in clinical and environmental samples?

UbiB holds significant potential as a biomarker for Salmonella detection in clinical and environmental samples due to several advantageous characteristics. The sequence conservation within Salmonella serovars coupled with distinct differences from other bacterial genera enables development of highly specific detection methods. PCR-based approaches targeting unique regions of the UbiB gene show sensitivity down to 10-100 CFU/mL in complex matrices like food homogenates or stool samples. Aptamer-based biosensors recognizing the UbiB protein demonstrate rapid detection capabilities (15-30 minutes) with minimal sample processing. Mass spectrometry approaches targeting UbiB-specific peptides after tryptic digestion can identify Salmonella in polymicrobial samples without cultivation. For environmental monitoring, quantitative PCR targeting UbiB correlates well with traditional culture methods while providing results in hours rather than days. Notably, UbiB's stable expression across various growth conditions makes it a more reliable target than environmentally regulated virulence factors. For implementation in resource-limited settings, isothermal amplification methods (LAMP) targeting UbiB, coupled with colorimetric detection, offer equipment-free approaches with sensitivity comparable to conventional PCR. Researchers developing such detection methods should validate performance across diverse Salmonella serovars and in realistic sample matrices to ensure robust performance in field applications.

What methods best assess the impact of point mutations on UbiB function?

Assessment of point mutations on UbiB function requires a multi-level experimental approach that combines in vitro biochemical characterization with cellular and in vivo phenotyping. At the biochemical level, thermal stability assays comparing wild-type and mutant proteins provide initial insights into structural perturbations, while enzyme kinetics measurements (kcat, KM) quantify specific catalytic effects. Binding studies using isothermal titration calorimetry or microscale thermophoresis can assess how mutations affect interactions with substrates, ATP, and potential protein partners. At the cellular level, complementation studies where mutant UbiB variants are expressed in UbiB-knockout strains allow assessment of functional rescue through growth curves, respiratory capacity measurements, and ubiquinone quantification. Membrane association assays using fractionation and Western blotting determine if mutations affect subcellular localization. For more detailed analysis, hydrogen-deuterium exchange mass spectrometry comparing wild-type and mutant proteins can map conformational changes beyond the immediate mutation site. When designing mutation studies, researchers should prioritize residues in key functional regions: the ATP-binding pocket, predicted catalytic sites, potential regulatory domains, and membrane-interaction surfaces. A comprehensive mutational analysis should include conservative and non-conservative substitutions at each position of interest to establish structure-function relationships with high resolution.

How can protein-protein interactions of UbiB be comprehensively mapped in the bacterial cell?

Comprehensively mapping UbiB's protein-protein interactions in bacterial cells requires integrated approaches that capture both stable and transient interactions in their native context. In vivo crosslinking with formaldehyde or photo-reactive amino acid analogs incorporated into UbiB can freeze interactions for subsequent analysis. Proximity-dependent labeling methods, particularly APEX2 or TurboID fused to UbiB, enable biotinylation of proximal proteins that can be identified by mass spectrometry, providing a dynamic interactome map. For targeted validation, bacterial two-hybrid systems adapted for membrane proteins (BACTH system using T18/T25 fragments) can confirm specific interactions. Co-immunoprecipitation using epitope-tagged UbiB under gentle solubilization conditions preserves physiologically relevant complexes. Label-free quantitative proteomics comparing wild-type and UbiB knockout strains can identify proteins whose stability depends on UbiB interaction. For spatial organization of these interactions, fluorescence lifetime imaging microscopy (FLIM) combined with FRET can map interaction networks within living cells with nanometer resolution. When analyzing the resulting interactome data, researchers should employ computational approaches to distinguish true interactors from contaminants, using appropriate statistical methods and controls. Integration with functional genomics data can place these interactions in broader biological context, potentially revealing unexpected roles for UbiB beyond ubiquinone biosynthesis.

What new approaches could improve the stability and yield of recombinant UbiB for structural studies?

Improving stability and yield of recombinant UbiB for structural studies requires innovative approaches addressing its challenging properties as a membrane-associated protein. Fusion with thermostable proteins (T4 lysozyme or rubredoxin) inserted into flexible loops can rigidify the structure and provide crystal contact points without disrupting the core fold. Systematic screening of expression constructs with varying N- and C-terminal boundaries can identify minimal functional domains with improved expression characteristics. For stability enhancement, directed evolution approaches using random mutagenesis followed by screening for thermostable variants have yielded UbiB proteins with 7-10°C higher melting temperatures while maintaining native activity. Codon harmonization (rather than simple codon optimization) adjusting the codon usage to match the natural translational rhythm of the source organism improves folding efficiency during heterologous expression. For membrane association challenges, amphipathic polymers like styrene maleic acid (SMA) can extract UbiB with its native lipid environment intact, potentially preserving structural features lost during detergent solubilization. Novel purification approaches using engineered binding proteins (nanobodies or affimers) targeting conformational epitopes can selectively capture properly folded protein. For crystallography attempts, in situ proteolysis during crystallization trials can remove flexible regions that hinder crystal packing. Researchers pursuing structural studies should consider parallel approaches including X-ray crystallography, cryo-EM, and NMR, as each method has complementary strengths for addressing different aspects of UbiB structure.