Recombinant Photobacterium profundum Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Photobacterium profundum and why is it significant for UbiB research?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It has emerged as a model organism for studying piezophily (thriving under high pressure) due to its ability to grow under a wide range of pressures (0.1 MPa to 70 MPa) while maintaining optimal growth at 28 MPa and 15°C . This bacterium was originally isolated in 1986 from the Sulu Sea and currently has four cultured wild-type strains: SS9, 3TCK, DJS4, and 1230 .

The significance of P. profundum for UbiB research stems from its adaptation to extreme pressure environments, where membrane processes and respiratory chains must function differently compared to atmospheric conditions. UbiB's involvement in ubiquinone biosynthesis, a critical electron carrier in respiratory chains, makes it particularly relevant to understanding how deep-sea bacteria maintain energy metabolism under high-pressure conditions .

Methodological approach: When studying P. profundum UbiB, researchers should consider the strain-specific differences in pressure adaptation. Table 1 summarizes key characteristics of different strains:

What is the known function of UbiB in ubiquinone biosynthesis?

UbiB is required for the first monooxygenase step in ubiquinone (Coenzyme Q) biosynthesis. Research on the E. coli homolog indicates that UbiB (formerly called yigR) is essential for the conversion of octaprenylphenol to the subsequent intermediate in the ubiquinone pathway . UbiB mutants accumulate octaprenylphenol, demonstrating its critical role in this conversion .

UbiB belongs to a predicted protein kinase family with the Saccharomyces cerevisiae ABC1 gene as the prototypic member . In addition to its enzymatic function, UbiB seems to form a complex with UbiJ, suggesting they both contribute to global ubiquinone biosynthesis rather than to a specific biosynthetic step .

Methodological approach: When studying UbiB function, researchers should consider that UbiB and UbiJ are dispensable for ubiquinone biosynthesis under anaerobiosis, even though they are expressed in the absence of oxygen . This suggests experimental designs should carefully control oxygen conditions.

How do pressure conditions affect UbiB expression and activity in P. profundum?

Proteomic analysis has revealed that key metabolic pathways are differentially expressed in P. profundum under varying pressure conditions. Although UbiB specifically wasn't highlighted in the search results, proteins involved in oxidative phosphorylation (where ubiquinone plays a key role) were up-regulated at atmospheric pressure .

The regulation of membrane-associated processes, including respiratory components, appears to be a direct response to the physical impact of pressure. This suggests that UbiB, as part of the ubiquinone biosynthesis pathway, may be regulated in response to pressure changes to maintain efficient electron transport chain function .

Methodological approach: When investigating pressure effects on UbiB, researchers should employ:

• Comparative proteomics between high pressure (28 MPa) and atmospheric pressure (0.1 MPa) growth conditions

• Analysis of ubiquinone content and intermediate accumulation across pressure gradients

• Gene expression analysis focusing on the operon containing ubiB

What experimental approaches are most effective for studying recombinant P. profundum UbiB function?

Studying recombinant P. profundum UbiB requires specialized approaches that account for its deep-sea origin and pressure-adapted functionality. Based on current research methodologies with similar proteins, the following protocol framework is recommended:

• Expression system optimization:

  • Use piezotolerant expression hosts when possible

  • Express with native N-terminal sequences to maintain proper membrane association

  • Consider co-expression with UbiJ to maintain natural complex formation

• Functional assay design:

  • Measure octaprenylphenol accumulation using HPLC or LC-MS/MS

  • Assess complementation of E. coli ubiB mutants with P. profundum ubiB

  • Evaluate protein-protein interactions with other ubiquinone biosynthesis components

• Pressure-variable experimental setup:

  • Utilize high-pressure bioreactors for protein expression

  • Conduct enzymatic assays under various pressure conditions using specialized equipment like the HPDS high-pressure cell system described for motility studies

Methodological considerations: The amino acid sequence of P. profundum UbiB (Q6LVW8) contains several domains critical for function, including putative ATP-binding sites that may be relevant to its predicted kinase activity . Recombinant expression should preserve these features.

How can researchers effectively investigate UbiB-protein interactions in P. profundum?

Understanding UbiB's interactome is crucial for elucidating its role within the ubiquinone biosynthetic pathway and potential pressure-adaptive functions. Based on research with related systems, the following methodological approaches are recommended:

• Co-immunoprecipitation under native conditions:

  • Use anti-UbiB antibodies or epitope-tagged recombinant UbiB

  • Maintain native pressure conditions during cell lysis when possible

  • Analyze precipitated proteins using mass spectrometry

• Bacterial two-hybrid screening:

  • Screen against a P. profundum SS9 genomic library

  • Focus on other ubiquinone biosynthesis proteins (UbiA, UbiC, UbiD, UbiE, UbiF, UbiG, UbiH, UbiJ, UbiX)

  • Validate interactions using bimolecular fluorescence complementation

• Cross-linking mass spectrometry:

  • Apply protein cross-linkers under varying pressure conditions

  • Identify pressure-dependent changes in the UbiB interactome

  • Map interaction interfaces using sophisticated MS/MS analysis

Research has already shown that UbiB forms a complex with UbiJ in E. coli . Investigating whether this interaction is conserved in P. profundum and how it may be affected by pressure would provide valuable insights into pressure adaptation mechanisms.

What methodologies are recommended for assessing the impact of pressure on UbiB activity?

Assessing enzymatic activity under high pressure presents unique challenges. Based on approaches used for other pressure-adapted enzymes in P. profundum, the following methodologies are recommended:

• In vitro activity assays under pressure:

  • Utilize high-pressure stopped-flow devices

  • Monitor conversion of octaprenylphenol using fluorescent or radiolabeled substrates

  • Compare activity profiles across 0.1-70 MPa pressure range

• Structural stability assessment:

  • Employ differential scanning calorimetry under varying pressures

  • Use pressure-resistant circular dichroism cells to monitor secondary structure

  • Perform hydrogen-deuterium exchange mass spectrometry at different pressures

• Computational prediction:

  • Model pressure effects on protein dynamics using molecular dynamics simulations

  • Calculate volume changes during catalytic cycle

  • Identify pressure-sensing domains through comparative analysis with mesophilic homologs

Relevant findings: Proteomic studies of P. profundum SS9 have shown that proteins involved in glycolysis/gluconeogenesis were up-regulated at high pressure, while oxidative phosphorylation proteins were up-regulated at atmospheric pressure . This suggests that pressure significantly impacts energy metabolism pathways, of which ubiquinone is a critical component.

What are the current contradictions in understanding UbiB function across pressure gradients?

Several unresolved questions remain regarding UbiB function in pressure-adapted organisms:

• Enzymatic mechanism discrepancy:

  • UbiB is classified as part of a protein kinase family, yet its role in ubiquinone biosynthesis involves a monooxygenase reaction

  • The mechanistic link between potential kinase activity and ubiquinone intermediate conversion remains unclear

  • It's unknown whether pressure affects this dual functionality

• Anaerobic redundancy paradox:

  • UbiB is dispensable for ubiquinone biosynthesis under anaerobiosis, despite being expressed

  • This contradicts the expectation that deep-sea environments (where P. profundum thrives) often have limited oxygen

  • The evolutionary rationale for maintaining UbiB in these conditions requires explanation

• Pressure adaptation conflicting models:

  • Membrane-associated processes in P. profundum respond to pressure changes

  • It remains debated whether UbiB's pressure adaptation involves structural changes to the protein itself or alterations in its membrane environment and interactions

  • The association between UbiB and other protein complexes may differ across pressure gradients

Research approach: To address these contradictions, comparative studies between shallow-water (3TCK) and deep-sea (SS9) strains of P. profundum offer a valuable model system. The piezosensitive 3TCK strain can serve as a control for identifying pressure-specific adaptations in UbiB structure and function.

How can researchers optimize heterologous expression of recombinant P. profundum UbiB?

Expressing functional recombinant P. profundum UbiB presents several challenges due to its membrane association, potential pressure adaptation, and involvement in protein complexes. Based on successful approaches with similar proteins, the following optimization strategies are recommended:

• Expression system selection:

  • E. coli C41(DE3) or C43(DE3) strains designed for membrane protein expression

  • Cold-adapted expression hosts for psychrophilic protein production

  • Consider using Vibrio species as more closely related expression hosts

• Construct design considerations:

  • Include native promoter regions to maintain natural expression regulation

  • Test both N- and C-terminal tag placements to minimize functional interference

  • Consider co-expression with UbiJ to facilitate proper complex formation

• Expression condition optimization:

  • Temperature gradient testing (4-20°C) to accommodate psychrophilic origin

  • Induction optimization using reduced IPTG concentrations

  • Membrane-mimetic additives in media

The full amino acid sequence of P. profundum UbiB indicates a predicted molecular weight of approximately 59.6 kDa, and inclusion of appropriate solubilization agents during purification is critical for maintaining functionality .

What genomic considerations are important when working with P. profundum UbiB?

The genome of P. profundum SS9 consists of two chromosomes and an 80 kb plasmid . This genomic organization has implications for UbiB research:

• Operon structure:

  • In E. coli, ubiB is the 3' gene in an operon containing ubiE, yigP, and ubiB

  • The organization of this operon in P. profundum should be confirmed before designing genetic experiments

  • Polar effects from upstream mutations may affect UbiB expression

• Genetic manipulation strategies:

  • P. profundum can grow at atmospheric pressure, enabling standard genetic techniques

  • In-frame deletion constructs, similar to those used for flagellar genes, can be applied to UbiB studies

  • Complementation assays should account for potential polar effects in operons

• Comparative genomics approach:

  • The Photobacterium genus shows high genomic diversity with evidence of horizontal gene transfer

  • Analysis of UbiB across different Photobacterium species may reveal pressure-specific adaptations

  • CRISPR-Cas systems identified in some Photobacterium strains offer potential tools for genetic manipulation

Research approach: When amplifying or cloning P. profundum UbiB, researchers should consider strain-specific variations and potential operon structures that might affect expression.

What analytical methods provide the most reliable data on UbiB-mediated ubiquinone biosynthesis?

Reliable quantification of ubiquinone and its biosynthetic intermediates is essential for studying UbiB function. Based on established protocols, the following analytical approaches are recommended:

• Chromatographic methods:

  • HPLC with electrochemical detection for ubiquinone quantification

  • LC-MS/MS for intermediate identification and quantification

  • TLC with UV detection for preliminary screening

• Genetic complementation assays:

  • Rescue of E. coli ubiB mutant growth on non-fermentable carbon sources

  • Restoration of electron transport chain activity in UbiB-deficient strains

  • Quantification of accumulated intermediates before and after complementation

• In vitro reconstitution:

  • Purified recombinant UbiB with relevant substrates and cofactors

  • Monitoring octaprenylphenol conversion under varying pressure conditions

  • Assessment of ATP requirements consistent with potential kinase activity

Methodological considerations: When working with P. profundum, researchers should culture cells under appropriate pressure conditions before extraction to capture the native state of ubiquinone biosynthesis. The shotgun proteomic approach using label-free quantitation and mass spectrometry analysis has proven effective for identifying pressure-regulated proteins in P. profundum and could be applied to study UbiB expression .