Recombinant Hahella chejuensis Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Hahella chejuensis and why is its UbiB protein significant to researchers?

Hahella chejuensis is a Gram-negative, aerobic, rod-shaped and motile marine bacterium originally isolated from marine sediment collected from Marado, Cheju Island, Republic of Korea . This organism has attracted significant scientific attention due to two primary characteristics: its ability to produce abundant extracellular polysaccharides and a red pigment called prodigiosin that exhibits lytic activity against harmful algal bloom (HAB)-causing dinoflagellates, particularly Cochlodinium polykrikoides .

The UbiB protein in H. chejuensis is part of the ubiquinone biosynthesis pathway. Ubiquinone (UQ), also known as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes that primarily functions as an electron carrier in respiratory chains . The significance of studying H. chejuensis UbiB lies in understanding unique adaptations of ubiquinone biosynthesis in marine bacteria and potential applications in addressing harmful algal blooms through metabolic engineering approaches.

What is the genomic context of ubiB in Hahella chejuensis?

The ubiB gene in H. chejuensis is located within the 7.2-megabase genome as part of the ubiquinone biosynthesis gene cluster . Genomic analysis revealed that H. chejuensis is the first sequenced species in the Oceanospiralles clade, with sequence analysis showing its distant relationship to the Pseudomonas group . The genome contains approximately 69 genomic islands (GIs) constituting about 23.0% of the chromosome, with genes involved in various functions including biosynthesis of exopolysaccharides, toxins, polyketides, non-ribosomal peptides, and pigmentation .

The ubiB gene is designated as HCH_01080 in the ordered locus names system . This genomic context is important for understanding the regulation and evolutionary history of ubiquinone biosynthesis in this marine bacterium.

What are the optimal expression systems for producing recombinant H. chejuensis UbiB protein?

Based on research protocols, Escherichia coli expression systems have been successfully used to produce recombinant H. chejuensis proteins, including UbiB. The methods typically involve:

• Gene cloning: The ubiB gene (HCH_01080) is amplified from H. chejuensis genomic DNA using PCR with specifically designed primers that include appropriate restriction sites.

• Vector construction: The amplified gene is inserted into an expression vector containing a His-tag sequence to facilitate purification. Common vectors include pET series plasmids with T7 promoters.

• Host strain selection: E. coli strains such as BL21(DE3) or EPI300 have been successfully used for expression of H. chejuensis proteins . For UbiB specifically, E. coli expression has been demonstrated to yield functional protein .

• Expression conditions: Induction with IPTG at concentrations of 0.1-1.0 mM when cultures reach mid-log phase (OD600 = 0.6-0.8), followed by growth at lower temperatures (16-25°C) for several hours has proven effective for similar proteins.

• Purification: His-tagged proteins can be purified using nickel affinity chromatography with careful optimization of imidazole concentrations for washing and elution steps.

The expression of recombinant UbiB requires careful optimization as membrane-associated proteins can be challenging to express in soluble form. Addition of glycerol (typically 5-50%) to storage buffers helps maintain protein stability, with 50% being commonly used for long-term storage .

What analytical techniques are most effective for characterizing the function of recombinant UbiB protein?

Several complementary analytical approaches can be employed to characterize recombinant UbiB protein:

• Enzymatic activity assays: Since UbiB is involved in ubiquinone biosynthesis, enzymatic assays can measure ATP binding and kinase activity. These typically involve incubating purified UbiB with potential substrates and ATP, followed by detection of phosphorylated products using radioactive labeling (32P-ATP) or LC-MS/MS.

• Protein-protein interaction studies: Techniques such as pull-down assays, bacterial two-hybrid systems, and co-immunoprecipitation can identify interaction partners of UbiB within the ubiquinone biosynthesis pathway. This approach has been effectively used to identify protein complexes in similar systems, as demonstrated with UbiK-UbiJ complex in E. coli .

• Structural characterization: X-ray crystallography, cryo-EM, or NMR spectroscopy can provide insights into the three-dimensional structure of UbiB. This information is crucial for understanding substrate binding sites and catalytic mechanisms.

• Complementation assays: Functional characterization can be performed by testing whether H. chejuensis UbiB can complement ubiB-deficient E. coli mutants, restoring ubiquinone biosynthesis. Complementation can be assessed by measuring ubiquinone levels using HPLC or LC-MS/MS .

• Biophysical analysis: Thermal shift assays (TSA), circular dichroism (CD), and isothermal titration calorimetry (ITC) can provide information about protein stability, secondary structure, and binding affinities.

These techniques, when used in combination, provide comprehensive insights into UbiB function and its role in ubiquinone biosynthesis.

How does the mechanism of UbiB in H. chejuensis compare with UbiB in other bacterial species?

The UbiB protein in H. chejuensis shares functional similarities with UbiB homologs in other bacteria, particularly those in E. coli, but with some notable differences:

• Sequence conservation: The H. chejuensis UbiB (546 amino acids) contains conserved domains characteristic of the UbiB family, including regions associated with ATP binding. Alignment analysis shows moderate sequence identity with E. coli UbiB (approximately 40-50%), suggesting conservation of core functional domains while maintaining species-specific adaptations .

• Functional role: In E. coli, UbiB functions as an accessory factor necessary for ubiquinone biosynthesis rather than as a direct catalytic enzyme. Research indicates that UbiB likely acts as a protein kinase involved in regulating ubiquinone biosynthetic enzymes . Given the sequence similarities, H. chejuensis UbiB is predicted to perform analogous functions, though marine adaptations may influence its specific mechanism.

• Marine adaptations: H. chejuensis requires 2% NaCl for optimal growth, and its proteins, including UbiB, may have evolved unique structural features for function in high-salt environments. The bacterium's genome contains Na+/H+ antiporters and Na+-translocating respiratory NADH:ubiquinone oxidoreductase systems that generate sodium motive force for cellular processes in marine environments .

• Regulatory context: Unlike E. coli UbiB, the regulation of H. chejuensis UbiB may be influenced by the bacterium's unique two-component signal transduction systems. The genome contains a high number of two-component systems (47 sensors, 103 response regulators, and 23 sensor-response regulator hybrids), suggesting complex regulatory mechanisms that may affect ubiquinone biosynthesis .

These differences highlight the importance of studying UbiB across diverse bacterial species to understand how ubiquinone biosynthesis has evolved to function in different ecological niches.

What role might UbiB play in the production of secondary metabolites like prodigiosin in H. chejuensis?

The potential relationship between UbiB and secondary metabolite production, particularly prodigiosin, represents an intriguing research question:

• Metabolic interconnections: Ubiquinone biosynthesis, facilitated by UbiB, involves the electron transport chain and cellular respiration, which generate energy and redox cofactors. These processes may indirectly influence the biosynthesis of secondary metabolites like prodigiosin by affecting cellular energy status and redox balance.

• Regulatory overlap: Genome analysis of H. chejuensis has revealed that prodigiosin biosynthesis is regulated by a two-component signal transduction system involving hapXY genes . Given that ubiquinone biosynthesis may also be regulated by sensory systems responding to environmental cues, there could be regulatory overlap between these pathways.

• Experimental evidence: Studies on related organisms have shown connections between respiratory chain components and secondary metabolite production. For example, in Serratia marcescens, the loss of serine-type D-Ala-D-Ala carboxypeptidase DacA resulted in enhanced prodigiosin production . Similar connections might exist between UbiB function and prodigiosin synthesis in H. chejuensis.

• Respiratory adaptation: H. chejuensis grows optimally in 2% NaCl and has specific respiratory adaptations for marine environments, including Na+-translocating respiratory complexes . These adaptations may create unique metabolic conditions that influence both ubiquinone function and secondary metabolite production.

To conclusively establish connections between UbiB and prodigiosin biosynthesis, researchers would need to conduct gene knockout studies of ubiB in H. chejuensis and analyze the effects on prodigiosin production, similar to approaches used to study the hap cluster .

How can CRISPR-Cas9 genome editing be optimized for studying ubiB function in H. chejuensis?

Implementing CRISPR-Cas9 genome editing in H. chejuensis to study ubiB function requires careful optimization due to the unique characteristics of this marine bacterium:

• Design of delivery system:

  • Plasmid-based systems using broad-host-range vectors compatible with Gamma-Proteobacteria

  • Conjugation-based transfer from E. coli to H. chejuensis, similar to methods used for transposon mutagenesis

  • Alternative delivery methods such as electroporation with optimized buffers containing NaCl to maintain cell viability

• sgRNA design considerations:

  • Target sequences within the ubiB gene (HCH_01080) with minimal off-target effects

  • GC content optimization (H. chejuensis has a GC content of approximately 53.9%)

  • Use of codon-optimized Cas9 for expression in H. chejuensis

  • Design of appropriate homology-directed repair templates for gene replacement or modification

• Selection strategy:

  • Incorporate appropriate antibiotic resistance markers that function in H. chejuensis

  • Consider salt-tolerance when designing selection media (optimal growth at 2% NaCl)

  • Screen for phenotypes related to ubiquinone deficiency, such as altered growth rates or respiratory defects

• Validation protocols:

  • PCR verification of genomic modifications

  • Sequencing to confirm precise editing

  • Phenotypic assays including measurement of ubiquinone levels using HPLC or LC-MS/MS

  • Complementation studies with wild-type ubiB to confirm phenotype specificity

• Control experiments:

  • Creation of marker-only integration strains

  • Use of non-targeting sgRNAs as negative controls

  • Complementary approaches such as transposon mutagenesis to validate findings

This approach would build upon successful genetic manipulation methods previously used with H. chejuensis, such as those employed to study the prodigiosin biosynthetic pathway .

What experimental design would best elucidate the structure-function relationship of UbiB in H. chejuensis?

A comprehensive experimental design to investigate the structure-function relationship of UbiB should include:

• Structural analysis pipeline:

  • Expression and purification of full-length and truncated versions of UbiB with appropriate tags

  • Initial structural characterization using circular dichroism (CD) to assess secondary structure

  • X-ray crystallography or cryo-EM studies of purified protein

  • Molecular dynamics simulations to predict functional motions

  • Homology modeling using E. coli UbiB as a template if direct structural determination proves challenging

• Functional domain mapping:

  • Site-directed mutagenesis of conserved residues identified through sequence alignment with homologs

  • Creation of chimeric proteins combining domains from H. chejuensis and E. coli UbiB

  • Truncation analysis to identify minimal functional units

  • Assessment of ATP binding and potential kinase activity for each variant

• In vivo complementation assays:

  • Testing ability of wild-type and mutant H. chejuensis UbiB to complement E. coli ubiB mutants

  • Quantification of ubiquinone production in complemented strains using HPLC or LC-MS/MS

  • Growth rate analysis under various stress conditions that require functional ubiquinone

• Protein-protein interaction studies:

  • Pull-down assays to identify interaction partners

  • Bacterial two-hybrid or yeast two-hybrid screening

  • Cross-linking coupled with mass spectrometry to map interaction interfaces

  • Fluorescence resonance energy transfer (FRET) to assess dynamic interactions

• Enzymatic activity characterization:

  • Development of in vitro assays for potential kinase activity

  • Determination of substrate specificity

  • Kinetic analysis of wild-type and mutant proteins

  • Effects of marine-relevant conditions (salt concentration, pH) on activity

This multi-faceted approach would generate complementary data sets that together would elucidate how the structure of UbiB determines its function in ubiquinone biosynthesis in the marine environment.

Comparative Analysis Questions

Comparative analysis of ubiquinone biosynthesis across marine bacteria provides valuable insights into evolutionary adaptations and specialized mechanisms:

• Adaptation to marine environments:

  • Marine bacteria like H. chejuensis have adapted their metabolic pathways, including ubiquinone biosynthesis, to function optimally in high-salt environments

  • The requirement of 2% NaCl for optimal growth in H. chejuensis suggests ionic interactions may influence protein function and pathway regulation

  • Marine adaptations include Na+/H+ antiporters and Na+-translocating respiratory NADH:ubiquinone oxidoreductase, which may directly interact with the ubiquinone biosynthesis pathway

• Genomic context and regulation:

  • H. chejuensis contains approximately 69 genomic islands constituting about 23% of the chromosome, suggesting substantial horizontal gene transfer that may have influenced ubiquinone biosynthesis genes

  • The genome encodes numerous two-component regulatory systems (47 sensors, 103 response regulators, and 23 sensor-response regulator hybrids), potentially allowing for sophisticated environmental sensing and regulation of metabolic pathways including ubiquinone biosynthesis

  • Comparison with other marine bacteria could reveal conserved regulatory elements specific to marine adaptation

• Metabolic integration:

  • In H. chejuensis, the relationship between ubiquinone biosynthesis and secondary metabolite production (such as prodigiosin) may represent unique metabolic integration specific to this organism's ecological niche

  • Marine bacteria often have unique respiratory adaptations that may influence the function and regulation of ubiquinone in electron transport chains

  • Studies in related organisms have demonstrated connections between respiratory chain components and secondary metabolite production, suggesting similar links may exist in marine bacteria

• Evolutionary implications:

  • As the first sequenced species in the Oceanospiralles clade, H. chejuensis provides an important reference point for understanding the evolution of ubiquinone biosynthesis in diverse marine bacteria

  • Comparative genomic analysis has revealed that H. chejuensis is distantly related to the Pseudomonas group, suggesting potential divergence in metabolic pathways including ubiquinone biosynthesis

  • Phylogenetic analysis of ubiquinone biosynthesis genes across marine bacteria could reveal patterns of vertical inheritance versus horizontal gene transfer

These comparative insights provide a foundation for understanding how ubiquinone biosynthesis has adapted to diverse marine environments and ecological niches.

What are the major challenges in expressing and purifying functional recombinant H. chejuensis UbiB, and how can they be addressed?

Researchers face several technical challenges when working with recombinant H. chejuensis UbiB:

• Solubility issues:

  • Challenge: UbiB may form inclusion bodies or aggregate during expression due to improper folding

  • Solution: Optimize expression conditions by lowering induction temperature (16-20°C), reducing inducer concentration, or using specialized E. coli strains like Rosetta or Arctic Express

  • Alternative approach: Use solubility-enhancing fusion tags such as MBP, SUMO, or TrxA in addition to the His-tag

  • Validation: Compare activity of protein expressed under different conditions to confirm functionality

• Marine protein adaptation to expression host:

  • Challenge: H. chejuensis proteins are adapted to marine salt conditions that differ from standard E. coli cytoplasmic conditions

  • Solution: Supplement growth media with NaCl (approximately 2%) to mimic native conditions

  • Alternative approach: Use marine-derived expression hosts that naturally grow in high-salt environments

  • Validation: Compare protein stability and activity in buffers with varying salt concentrations

• Membrane association:

  • Challenge: UbiB may associate with membranes, complicating purification

  • Solution: Use gentle detergents (DDM, CHAPS, or digitonin) during cell lysis and purification

  • Alternative approach: Consider native membrane extraction methods followed by detergent solubilization

  • Validation: Perform subcellular fractionation to determine localization in expression system

• Protein stability during purification:

  • Challenge: UbiB may lose activity during purification due to removal from native environment

  • Solution: Include stabilizing agents such as glycerol (50% for long-term storage) , reducing agents, and appropriate cofactors

  • Alternative approach: Develop rapid purification protocols to minimize time between cell lysis and activity assays

  • Validation: Monitor activity at each purification step to identify points of activity loss

• Functional verification:

  • Challenge: Confirming that purified UbiB retains native functionality

  • Solution: Develop robust activity assays based on predicted kinase function

  • Alternative approach: Use complementation assays in ubiB-deficient E. coli strains

  • Validation: Compare activity of recombinant protein with activity in native H. chejuensis cell extracts

These strategies have been successfully applied to similar proteins and can be adapted specifically for H. chejuensis UbiB to overcome expression and purification challenges.

How can researchers effectively design control experiments to validate UbiB function in H. chejuensis?

Robust control experiments are essential for validating UbiB function and avoiding misinterpretation of results:

• Genetic manipulation controls:

  • Negative control: Generate a marker-only insertion strain that disrupts a non-coding region to control for effects of genetic manipulation

  • Positive control: Create a complemented strain where the disrupted ubiB gene is reintroduced on a plasmid or at a different genomic location

  • Specificity control: Disrupt other genes in the ubiquinone biosynthesis pathway to compare phenotypic effects

  • Implementation: Use established methods similar to those employed for prodigiosin biosynthesis gene analysis

• Biochemical assay controls:

  • Negative control: Use heat-inactivated UbiB protein in enzymatic assays

  • Substrate specificity control: Test activity with non-physiological substrates to confirm specificity

  • Known inhibitor control: If available, use known inhibitors of similar kinases to validate assay sensitivity

  • Implementation: Include controls in each experimental repetition to account for day-to-day variation

• Protein-protein interaction controls:

  • Non-specific binding control: Use unrelated proteins with similar properties (size, charge) in pull-down assays

  • Competition control: Perform competition assays with unlabeled protein to confirm specificity of interactions

  • Subcellular localization control: Confirm colocalization of interacting proteins in cellular compartments

  • Implementation: Use both positive and negative controls in each interaction experiment

• Physiological function controls:

  • Environmental control: Test phenotypes under various growth conditions (temperature, salt concentration, carbon source)

  • Chemical complementation: Attempt to rescue ubiquinone deficiency phenotypes by supplementing growth media with ubiquinone or precursors

  • Temporal control: Monitor gene expression and protein levels throughout growth phases

  • Implementation: Design factorial experiments testing multiple conditions simultaneously

• Data analysis controls:

  • Technical replicates: Perform multiple technical replicates to assess measurement variation

  • Biological replicates: Use independently derived mutant strains to confirm phenotypic consistency

  • Statistical analysis: Apply appropriate statistical tests with corrections for multiple comparisons

  • Implementation: Ensure sufficient replication to achieve statistical power for detecting relevant effect sizes

These control strategies would build on approaches successfully used to study other aspects of H. chejuensis biology, such as the screening for factors affecting prodigiosin biosynthesis and genomic analysis techniques .

What emerging technologies could advance our understanding of UbiB function in H. chejuensis?

Several cutting-edge technologies show promise for elucidating UbiB function in H. chejuensis:

• Cryo-electron microscopy (cryo-EM):

  • Application: Determine high-resolution structures of UbiB alone and in complex with interaction partners

  • Advantage: Requires less protein than X-ray crystallography and can capture dynamic conformations

  • Implementation strategy: Optimize sample preparation conditions for membrane-associated proteins using appropriate detergents or nanodiscs

  • Expected impact: Revealing atomic-level details of substrate binding sites and conformational changes

• Proximity-dependent labeling proteomics (BioID or APEX):

  • Application: Identify the complete interactome of UbiB in living H. chejuensis cells

  • Advantage: Captures transient and weak interactions in the native cellular environment

  • Implementation strategy: Generate fusion proteins with biotin ligase or peroxidase tags and optimize expression in H. chejuensis

  • Expected impact: Comprehensive mapping of the protein interaction network surrounding UbiB

• Single-molecule techniques:

  • Application: Observe real-time conformational changes and interactions of individual UbiB molecules

  • Advantage: Reveals heterogeneity and dynamic processes masked in bulk measurements

  • Implementation strategy: Develop fluorescently labeled UbiB constructs compatible with techniques like FRET or optical tweezers

  • Expected impact: Understanding the dynamic processes underlying UbiB function

• Metabolomics combined with stable isotope labeling:

  • Application: Track metabolic flux through ubiquinone biosynthesis pathway in wild-type and ubiB mutant strains

  • Advantage: Provides quantitative insights into pathway kinetics and bottlenecks

  • Implementation strategy: Culture H. chejuensis with isotope-labeled precursors and perform time-course sampling

  • Expected impact: Identifying metabolic consequences of UbiB dysfunction throughout the cellular metabolome

• AlphaFold and other AI-based structural prediction:

  • Application: Generate accurate structural models of UbiB and predict functional sites

  • Advantage: Rapidly provides structural insights that can guide experimental design

  • Implementation strategy: Utilize the latest versions of protein structure prediction algorithms with refinement based on experimental data

  • Expected impact: Accelerating structure-based functional studies and rational design of experiments

These technologies, when integrated with traditional biochemical and genetic approaches, have the potential to significantly advance our understanding of UbiB function in H. chejuensis and related organisms.

How might understanding UbiB function contribute to addressing harmful algal blooms through biotechnological approaches?

Understanding UbiB function could inform novel biotechnological strategies for addressing harmful algal blooms (HABs):

• Metabolic engineering of H. chejuensis:

  • Rationale: H. chejuensis produces prodigiosin, which shows algicidal activity against Cochlodinium polykrikoides at very low concentrations (1 ppb)

  • Connection to UbiB: If UbiB influences secondary metabolite production through effects on cellular energy metabolism, manipulating UbiB could potentially enhance prodigiosin production

  • Implementation approach: Use genome editing to optimize UbiB function or expression levels, potentially increasing algicidal compound yields

  • Potential impact: Development of enhanced H. chejuensis strains for biological control of HABs

• Synthetic biology applications:

  • Rationale: Understanding the relationship between primary metabolism (including ubiquinone biosynthesis) and secondary metabolism could enable design of synthetic pathways

  • Connection to UbiB: Knowledge of how UbiB regulates ubiquinone biosynthesis could inform design principles for metabolic channeling

  • Implementation approach: Create synthetic gene circuits that couple ubiquinone biosynthesis regulation to production of algicidal compounds

  • Potential impact: Development of engineered bacteria with improved specificity and efficacy against HAB-causing organisms

• Targeted enzyme inhibitors:

  • Rationale: If UbiB inhibition in harmful algae disrupts their growth or survival, it could represent a novel control strategy

  • Connection to UbiB: Structural and functional studies of UbiB could enable design of specific inhibitors

  • Implementation approach: Use structure-based drug design to develop compounds that target UbiB in harmful algae but not in beneficial organisms

  • Potential impact: Creation of environmentally friendly chemical controls for HABs

• Biosensors for early HAB detection:

  • Rationale: Early detection of HABs enables more effective intervention

  • Connection to UbiB: If UbiB or ubiquinone levels change in response to environmental conditions that predict HABs, this could serve as a biomarker

  • Implementation approach: Develop biosensors using UbiB-based recognition elements coupled to signal transduction systems

  • Potential impact: Improved monitoring systems for early warning of HAB development

• Ecological applications:

  • Rationale: Understanding the ecological role of H. chejuensis and its metabolites could inform ecosystem management approaches

  • Connection to UbiB: If UbiB function is linked to adaptation to specific marine environments, this could inform habitat restoration strategies

  • Implementation approach: Use knowledge of H. chejuensis physiology to design ecological interventions that promote natural control of HABs

  • Potential impact: Development of sustainable, ecosystem-based approaches to HAB management