Recombinant Citrobacter koseri Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is the role of UbiB in ubiquinone biosynthesis in Citrobacter koseri?

UbiB in C. koseri functions as an essential component in the oxygen-dependent pathway for ubiquinone (UQ) biosynthesis. The protein demonstrates ATPase activity and is specifically required for aerobic conditions of growth. When examining the metabolic pathway, UbiB participates in the hydroxylation reactions needed to modify the aromatic ring of 4-hydroxybenzoic acid (4-HB) during UQ synthesis .

Research methodologically demonstrates this role through knockout studies, where ΔubiB strains produce significantly reduced amounts of ubiquinone (UQ8) specifically under aerobic conditions . The experimental approach to confirm UbiB function typically involves:

• Generation of targeted gene deletions using λ-Red recombination

• Complementation studies with plasmid-expressed ubiB

• HPLC quantification of ubiquinone levels under varying oxygen conditions

• Assessment of growth rates in defined media with differential carbon sources

How is recombinant C. koseri UbiB typically expressed and purified for functional studies?

For optimal expression and purification of recombinant C. koseri UbiB, researchers should employ the following methodological approach:

• Cloning the ubiB gene with appropriate tags (typically N-terminal His6 or Strep-tag)

• Expression in E. coli BL21(DE3) or similar strain using an inducible system (IPTG-inducible T7 promoter)

• Growth at lower temperatures (16-20°C) after induction to enhance protein solubility

• Cell lysis under anaerobic conditions to maintain native protein structure

• Purification using affinity chromatography followed by size exclusion

Critical consideration must be given to buffer composition, as the ATPase activity of UbiB requires specific conditions:

• pH maintained at 7.5-8.0

• Inclusion of magnesium (1-5 mM MgCl₂)

• Addition of reducing agents (1-5 mM DTT or TCEP)

• Potential addition of ubiquinone precursors for stability

The purified protein can then be assessed for ATPase activity using malachite green phosphate detection assays or radioactive ATP hydrolysis assays.

What experimental systems are best suited for studying C. koseri UbiB function?

To effectively study C. koseri UbiB function, researchers should consider complementary experimental systems:

In vivo systems:

• C. koseri ubiB knockout strains complemented with wild-type or mutant ubiB variants

• E. coli ubiB mutants complemented with C. koseri ubiB for comparative studies

• Growth under varying oxygen concentrations (aerobic, microaerobic, anaerobic)

• Infection models in appropriate animals (neonatal rat and mouse models)

In vitro systems:

• Reconstituted ubiquinone biosynthesis assays with purified components

• ATPase activity measurements with varying substrates

• Protein-protein interaction studies with other ubiquinone biosynthesis components

• Membrane association assays to determine subcellular localization

Analytical approaches:

• LC-MS/MS for identification of reaction intermediates

• HPLC for quantification of ubiquinone and precursors

• RNA-seq for transcriptional analysis under varying conditions

• Protein co-immunoprecipitation to identify interaction partners

The combination of these systems provides comprehensive insights into UbiB function and regulation within the broader context of ubiquinone biosynthesis.

How does the function of UbiB differ between the aerobic and anaerobic ubiquinone biosynthesis pathways in C. koseri?

The function of UbiB shows striking pathway-specific activity in C. koseri ubiquinone biosynthesis. UbiB operates exclusively in the oxygen-dependent pathway, as demonstrated by the observation that ΔubiB strains produce limited ubiquinone only under aerobic conditions . In contrast, under anaerobic conditions, C. koseri employs an alternative O₂-independent pathway for ubiquinone biosynthesis that does not require UbiB.

Methodologically, this differential function can be investigated through:

• Metabolomic profiling of ubiquinone intermediates in wild-type vs. ΔubiB strains under varying O₂ conditions

• Isotope labeling studies using ¹³C-labeled precursors to track carbon flow through different pathways

• Comparative transcriptomics and proteomics of cells grown under aerobic vs. anaerobic conditions

• In vitro reconstitution of both pathways with purified components

Research findings indicate that while the O₂-dependent pathway utilizes UbiB for hydroxylation reactions, the O₂-independent pathway employs alternative hydroxylases (UbiU-UbiV) that utilize [4Fe-4S] clusters to catalyze similar reactions without molecular oxygen . This represents a remarkable example of metabolic plasticity that allows proteobacteria like C. koseri to synthesize ubiquinone across the entire range of environmental oxygen levels.

What structural and functional domains in C. koseri UbiB are critical for its role in ubiquinone biosynthesis?

C. koseri UbiB contains several crucial domains that define its function in ubiquinone biosynthesis:

• N-terminal nucleotide-binding domain: Contains the Walker A and Walker B motifs essential for ATP binding and hydrolysis

• C-terminal α-helical domain: Likely involved in protein-protein interactions with other ubiquinone biosynthesis enzymes

• Conserved LYK motif: Critical for catalytic activity, potentially involved in substrate recognition

• Membrane-association motifs: Hydrophobic patches that facilitate association with the inner membrane where ubiquinone synthesis occurs

Methodological approaches to investigate domain functionality include:

• Site-directed mutagenesis of key residues followed by complementation assays

• Truncation analysis to define minimal functional domains

• Domain-swapping experiments with homologous proteins from other species

• Hydrogen-deuterium exchange mass spectrometry to identify substrate-binding regions

• Crystallography or cryo-EM structural studies combined with molecular dynamics simulations

Functional analysis should employ activity assays measuring:

• ATP hydrolysis rates with purified protein

• Binding affinity for ubiquinone precursors

• Protein-protein interaction strength with other pathway components

• In vivo complementation efficiency of mutant variants

Research indicates that mutations in the Walker A motif (particularly the lysine residue) abolish ATPase activity and consequently ubiquinone biosynthesis, suggesting that ATP hydrolysis is coupled to the catalytic function of UbiB in the hydroxylation reactions of the ubiquinone pathway.

How does the expression of recombinant C. koseri UbiB impact bacterial virulence and pathogenicity?

The relationship between UbiB expression and C. koseri virulence presents an intriguing area of research. While not directly identified as a classical virulence factor, UbiB's role in ubiquinone biosynthesis indirectly affects bacterial pathogenicity through modulation of energy metabolism and stress responses.

Methodological approaches to investigate this relationship include:

• Infection models with UbiB variants:

  • Neonatal rat and mouse models of meningitis and brain abscess formation

  • Comparison of wild-type, ΔubiB, and complemented strains

  • Measurement of bacterial loads in blood and cerebrospinal fluid

  • Histopathological examination of infected tissues

• Virulence assays:

  • Survival within macrophages and brain microvascular endothelial cells

  • Biofilm formation capability

  • Resistance to oxidative and nitrosative stress

  • Growth in iron-limited conditions

• Transcriptional analysis:

  • RNA-seq comparing expression profiles of wild-type and ΔubiB strains during infection

  • ChIP-seq to identify regulators of ubiB expression under different conditions

  • qRT-PCR validation of key virulence genes

Research findings suggest that UbiB-dependent ubiquinone biosynthesis may be particularly important during certain phases of infection. Since C. koseri can cause meningitis and brain abscess in neonates and immunocompromised individuals , the ability to synthesize ubiquinone under varying oxygen tensions within host tissues is likely critical for sustained virulence. The brain environment, in particular, may present unique oxygen gradients that require metabolic flexibility.

The impact of UbiB on virulence should be considered alongside other identified C. koseri virulence factors, including those involved in flagellar apparatus biosynthesis (ompA, csg fimbriae, and the che operon) and iron uptake systems (chu, fep, and ent) .

What is the relationship between UbiB and the high-pathogenicity island (HPI) cluster in C. koseri?

While direct evidence for interaction between UbiB and the high-pathogenicity island (HPI) in C. koseri is limited, integrative analysis suggests potential functional relationships worthy of investigation.

The HPI cluster in C. koseri has been identified as a key virulence determinant, with deletion mutants showing significantly decreased virulence in animal models . This cluster is involved in iron acquisition, which is essential for bacterial pathogenesis. Ubiquinone biosynthesis and iron metabolism are interconnected through several mechanisms:

• Respiratory chain function: Ubiquinone-dependent respiration requires iron-containing proteins

• Oxidative stress resistance: Both systems contribute to managing oxidative stress

• Energy provision for iron transport: ATP generated via ubiquinone-dependent respiration powers iron acquisition systems

Methodological approaches to investigate this relationship include:

• Construction of double mutants (ΔubiB/ΔHPI) to assess synergistic effects

• Transcriptional analysis of iron uptake genes in ΔubiB strains

• Measurement of intracellular iron levels in various mutant backgrounds

• Assessment of ubiquinone levels in HPI mutants

Animal studies have demonstrated that HPI deletion severely attenuates C. koseri virulence in vivo, with mutants losing the ability to replicate in the brain . Similar studies with UbiB mutants could reveal whether ubiquinone biosynthesis defects result in comparable attenuation, potentially suggesting a functional link between these systems.

How do oxygen-sensing mechanisms regulate UbiB expression and function in C. koseri during environmental transitions?

The regulation of UbiB in response to environmental oxygen levels represents a sophisticated example of bacterial adaptation. Given that UbiB functions specifically in the O₂-dependent pathway for ubiquinone biosynthesis, its expression and activity must be precisely coordinated with oxygen availability.

Methodological approaches to investigate oxygen-dependent regulation include:

• Transcriptional analysis:

  • Promoter mapping using 5' RACE

  • Reporter fusion assays (lacZ, gfp) to monitor promoter activity

  • ChIP-seq to identify transcription factors binding the ubiB promoter

  • RNA-seq comparing expression under aerobic, microaerobic, and anaerobic conditions

• Protein-level regulation:

  • Western blotting to quantify UbiB levels under different oxygen tensions

  • Pulse-chase experiments to determine protein stability

  • Post-translational modification analysis using mass spectrometry

  • Protein-protein interaction screens to identify regulatory partners

• Functional assays:

  • Measurement of ATPase activity under different redox conditions

  • Assessment of membrane association as a function of oxygen availability

  • Determination of ubiquinone synthesis rates during oxygen transitions

Research findings suggest that bacteria equipped with both O₂-dependent and O₂-independent pathways, like C. koseri, have evolved sophisticated regulatory mechanisms to seamlessly transition between these pathways . This metabolic plasticity likely involves multiple layers of regulation, from transcriptional control to allosteric modulation of enzyme activity.

Potential oxygen-sensing mechanisms regulating UbiB may include:

• FNR-like transcription factors that respond directly to oxygen

• ArcAB two-component system sensing respiratory chain status

• SoxRS system responding to oxidative stress

• Iron-sulfur cluster-containing regulators sensitive to oxygen and iron availability