Recombinant Sodalis glossinidius Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Sodalis glossinidius and what is its ecological significance?

Sodalis glossinidius is a maternally transmitted secondary endosymbiont that establishes chronic, stable associations within tissues of tsetse flies (Glossina spp.) . This bacterium is of particular interest because it can reside both intracellularly and extracellularly throughout the fly . The ecological significance of S. glossinidius lies in its potential role in vector biology, particularly as it has been considered a platform for anti-Trypanosoma paratransgenesis strategies to reduce the transmission of African sleeping sickness .

Unlike primary obligate endosymbionts, S. glossinidius can be cultured in vitro on agar plates and in liquid cultures, making it an accessible model for studying host-symbiont interactions . Research has demonstrated that S. glossinidius has evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen, as evidenced by phylogenetic reconstructions that consistently place Sodalis in a clade with enteric pathogens Shigella and Salmonella .

What is the role of UbiB in ubiquinone biosynthesis?

UbiB is a critical protein required for ubiquinone (UQ, coenzyme Q) biosynthesis, specifically participating in the first monooxygenase step in this pathway . Ubiquinone is a lipophilic electron carrier molecule that plays an essential role in cellular bioenergetics, particularly in aerobic respiration as part of the electron transport chain.

Research has identified UbiB as a member of a predicted protein kinase family, of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member . The UbiB protein has been characterized as having ATPase activity and is one of eleven proteins known to participate in UQ biosynthesis in Escherichia coli .

When UbiB is disrupted or mutated, bacteria accumulate octaprenylphenol, an early intermediate in the UQ biosynthetic pathway, indicating that UbiB is involved in the conversion of this intermediate to the next compound in the pathway . This specific role in a hydroxylation reaction suggests UbiB may either directly catalyze this reaction or serve as an accessory factor for other biosynthetic enzymes.

How can researchers distinguish between UbiB and other Ubi proteins involved in ubiquinone biosynthesis?

Distinguishing between UbiB and other Ubi proteins requires a multi-faceted approach:

• Genetic analysis: Comparing phenotypes of various ubi gene mutants. UbiB mutants specifically accumulate octaprenylphenol, while mutations in other ubi genes result in the accumulation of different pathway intermediates .

• Protein domain analysis: UbiB contains domains characteristic of the predicted protein kinase family, which distinguishes it from other Ubi proteins with different functional domains .

• Complementation studies: Transforming mutant strains with plasmids containing different ubi genes can determine which gene rescues the wild-type phenotype .

• Functional analysis: Testing enzymatic activities in vitro using purified proteins can help determine the specific biochemical function of each Ubi protein .

• Expression pattern analysis: Examining gene expression under different environmental conditions (aerobic vs. anaerobic) can differentiate UbiB from other Ubi proteins that may be differentially regulated .

The discovery that the E. coli yigR gene (homolog of P. stuartii aarF) corresponds to ubiB, not the previously suspected fre gene, highlights the importance of proper identification and characterization of ubiquinone biosynthesis components .

How does ubiquinone biosynthesis differ between O₂-dependent and O₂-independent pathways?

Bacteria have developed two distinct pathways for ubiquinone biosynthesis to adapt to environments with different oxygen levels . These pathways differ in their enzymatic machinery and regulatory mechanisms:

O₂-dependent pathway:

• Requires molecular oxygen as a substrate for hydroxylation reactions

• Utilizes monooxygenase enzymes that incorporate oxygen atoms into the aromatic ring

• Key proteins include UbiI, UbiH, and UbiF, which are aerobic hydroxylases

• Function is limited to aerobic or microaerobic conditions

• Long established as the canonical pathway for UQ biosynthesis

O₂-independent pathway:

• Functions in the absence of molecular oxygen

• Relies on alternative hydroxylases that do not require O₂ as a substrate

• Involves UbiT, UbiU, and UbiV proteins

• UbiU and UbiV form a heterodimer, with each protein binding a 4Fe-4S cluster via conserved cysteines that are essential for activity

• Represents a novel class of O₂-independent hydroxylases

• Enables bacteria to synthesize ubiquinone across the entire O₂ range

Table 1: Comparison of key features of O₂-dependent and O₂-independent ubiquinone biosynthesis pathways

*Note: UbiK and UbiJ are dispensable for UQ biosynthesis under anaerobiosis, even though they are expressed in the absence of oxygen .

The dual pathway system allows bacteria like E. coli to synthesize ubiquinone across the entire oxygen range, thereby optimizing their metabolism for different environmental conditions .

What techniques are available for genetic manipulation of Sodalis glossinidius?

Several genetic manipulation techniques have been developed for S. glossinidius, addressing the technical challenges posed by its complex growth requirements and uncharacterized physiology :

• Transduction using bacteriophage P1:

  • P1 can infect, lysogenize, and promote transduction in Sodalis species

  • Enables generalized transduction of DNA fragments between cells

  • Can be used for the delivery of plasmids and suicide vectors

• Tn5 mutagenesis:

  • Creates random insertions in the bacterial genome

  • Has been used to identify genes involved in cell invasion, such as the type III secretion system gene invC

  • Combined with negative selection procedures to isolate specific mutants

• Conjugal DNA transfer:

  • Exploits natural mechanism of DNA transfer among bacteria

  • Can be used for both forward and reverse genetic experiments

  • Enables creation of targeted gene knockouts and insertions

  • The most efficient technique developed for genetic manipulation of S. glossinidius

• Lambda Red recombineering:

  • Although not directly mentioned for Sodalis in the search results, this technique has been used with closely related bacteria and could potentially be adapted

The development of these genetic tools opens possibilities for studying Sodalis-tsetse associations and evaluating S. glossinidius-based tsetse fly paratransgenesis strategies to combat the transmission of African trypanosomiasis .

How is the abundance of Sodalis influenced by insect social behavior?

Social interactions among insects can significantly influence the abundance and distribution of Sodalis symbionts, as demonstrated in a study of halictid bees . Key findings include:

• Transmission dynamics: Social interactions facilitate transmission of microbes between individuals, potentially reducing variation in gut communities within social groups .

• Differential abundance patterns: Sodalis dominates community differences between social forms (eusocial vs. solitary bees), with supervised learning classification identifying Sodalis OTUs as 9 of the 10 most important features distinguishing social behaviors .

• Frequency differences: In Lasioglossum albipes, a bee species with both solitary and eusocial forms, Sodalis was detected in significantly fewer eusocial samples (6 out of 75) compared to solitary samples (30 out of 75), with a χ² test yielding p = 1.1 × 10⁻⁵ .

• Evolutionary implications: The relationship between sociality and symbiont community composition suggests that the evolution of social behaviors and symbiont diversity may be tightly linked .

• Strain diversity: Multiple strains of Sodalis have independently colonized halictids at least three times, with these strains appearing to be mutually exclusive within individual bees, suggesting competition for hosts .

This research indicates that social behavior in insects can create specific ecological niches that influence the abundance and distribution of Sodalis symbionts, with potential implications for the evolution of both host and symbiont.

How can researchers design experiments to study UbiB function in Sodalis under different oxygen conditions?

Designing experiments to study UbiB function in Sodalis under varying oxygen conditions requires a comprehensive approach addressing multiple factors:

Experimental design framework:

• Strain construction and verification:

  • Generate ubiB knockout mutants using conjugation or P1 transduction methods

  • Create complementation strains expressing UbiB from plasmids

  • Verify mutations using PCR and sequencing

  • Confirm protein expression levels using Western blotting

• Oxygen gradient cultivation system:

  • Design a controlled gradient chamber to maintain precise O₂ levels

  • Use Definitive Screening Design (DSD) to optimize experimental parameters

  • Monitor oxygen levels continuously using Clark-type electrodes or optical sensors

  • Include anaerobic controls using chambers with oxygen scavengers

• Ubiquinone quantification:

  • Extract quinones using standardized lipid extraction protocols

  • Analyze using HPLC-MS to quantify ubiquinone and intermediates

  • Implement sensitive detection methods to identify even trace amounts of UQ

Table 2: Experimental factors for UbiB function analysis using Design of Experiments approach

• Metabolic profiling:

  • Measure electron transport chain activity using specific inhibitors

  • Assess growth rates under different oxygen conditions

  • Quantify ATP production to evaluate energetic consequences of UbiB function

  • Analyze metabolomic changes using LC-MS/MS

• Structure-function analysis:

  • Generate site-directed mutants targeting conserved residues

  • Express and purify recombinant UbiB protein for in vitro assays

  • Assess ATPase activity under varying oxygen conditions

  • Determine protein-protein interactions with other ubiquinone biosynthesis components

This experimental framework will enable researchers to characterize the specific role of UbiB in ubiquinone biosynthesis under different oxygen conditions and determine whether Sodalis utilizes both O₂-dependent and O₂-independent pathways for ubiquinone biosynthesis.

What are the requirements for expression and purification of recombinant UbiB from Sodalis glossinidius?

Expression and purification of recombinant UbiB from Sodalis glossinidius presents several challenges due to the protein's membrane association and potential cofactor requirements. The following methodological approach addresses these challenges:

Expression system selection:

• Host options:

  • E. coli expression systems (BL21(DE3), C41(DE3), C43(DE3))

  • Yeast systems (P. pastoris, S. cerevisiae)

  • Baculovirus-infected insect cells

  • Mammalian cell expression systems

• Vector design:

  • Include affinity tags (His6, GST, MBP) for purification

  • Consider fusion proteins to enhance solubility

  • Include TEV or PreScission protease sites for tag removal

  • Optimize codon usage for the chosen expression host

Expression optimization:

• Induction conditions:

  • Test various induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Optimize inducer concentration and induction timing

  • Consider auto-induction media for E. coli systems

• Solubility enhancement:

  • Include appropriate detergents for membrane protein solubilization

  • Test protein stabilizers (glycerol, reducing agents)

  • Consider co-expression with chaperones

Purification protocol:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Affinity chromatography based on fusion tags

  • Ensure buffers contain appropriate detergents

• Intermediate purification:

  • Ion exchange chromatography

  • Hydrophobic interaction chromatography

• Polishing step:

  • Size exclusion chromatography

  • Remove size exclusion step for large-scale production as it is not ideal for manufacturing scale

• Quality control:

  • SDS-PAGE analysis (>90% purity)

  • Mass spectrometry verification

  • Activity assays

Storage conditions:

• Store at -20°C or -80°C for long-term storage

• Working aliquots at 4°C for up to one week

• Include glycerol in storage buffer

• Avoid repeated freezing and thawing cycles

Implementing a Design of Experiments (DoE) approach can significantly improve optimization of the purification protocol, allowing for systematic evaluation of factors affecting protein yield and activity .

How can Sodalis glossinidius be used as a paratransgenesis platform to combat trypanosome transmission?

Sodalis glossinidius holds significant potential as a paratransgenesis platform to reduce Trypanosoma brucei transmission, which causes African sleeping sickness. The following methodological framework outlines the approach:

Fundamental principles:

• Biological basis:

  • S. glossinidius establishes chronic, stable associations in tsetse flies

  • The infection can be inherited through both vertical (maternal) and horizontal (paternal) transmission

  • S. glossinidius persists within tsetse fly populations, providing a self-sustaining delivery system

• Technical prerequisites:

  • Genetic manipulation capability through conjugation

  • Ability to express foreign proteins in Sodalis

  • Stable inheritance of genetic modifications

Methodological approach:

• Anti-trypanosomal effector selection:

  • Antimicrobial peptides targeting trypanosomes

  • Single-domain antibodies against trypanosome surface proteins

  • RNA interference constructs targeting essential trypanosome genes

  • CRISPR-Cas systems for targeted degradation of trypanosome DNA

• Construct design:

  • Promoter selection for tissue-specific expression

  • Codon optimization for efficient translation in Sodalis

  • Inclusion of secretion signals for effector export

  • Stability elements to ensure long-term expression

• Delivery system:

  • Engineered Sodalis strains can be introduced into tsetse flies through:
    a) Microinjection into adult flies
    b) Blood meal supplementation
    c) Larval exposure

  • Monitoring of bacterial colonization using fluorescent markers

• Efficacy assessment:

  • Measure trypanosome development in engineered flies

  • Determine transmission rates to mammals

  • Evaluate long-term stability of the engineered symbiont

  • Assess potential fitness effects on tsetse flies

• Field application considerations:

  • Population replacement models

  • Ecological impact assessment

  • Regulatory compliance strategy

  • Implementation and monitoring protocols

This approach leverages the natural biology of the Sodalis-tsetse symbiosis, particularly the recently developed tools for genetic manipulation of Sodalis, to create a sustainable solution for reducing trypanosome transmission and alleviating the burden of African sleeping sickness in affected regions .

What are the evolutionary implications of Sodalis' type III secretion system for symbiont-host interactions?

The presence of a functional type III secretion system (T3SS) in Sodalis glossinidius has significant evolutionary implications for symbiont-host interactions, revealing insights into the transition from pathogenesis to mutualism:

Evolutionary origins:

• Phylogenetic evidence:

  • Phylogenetic reconstructions based on inv/spa genes consistently place Sodalis in a well-supported clade with enteric pathogens like Shigella and Salmonella

  • This suggests that Sodalis evolved from an ancestor with a parasitic intracellular lifestyle

  • The T3SS of Sodalis shares high sequence identity with the T3SS encoded by Salmonella pathogenicity island 1

• Functional conservation:

  • Tn5 mutagenesis identified invC in Sodalis, which is essential for invasion of insect cells

  • The mutant strain with disrupted invC is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse flies

  • This demonstrates that the T3SS remains functional and necessary for host colonization

Implications for symbiont-host interactions:

• Mechanism of initial colonization:

  • The T3SS likely facilitates the initial entry of Sodalis into host cells

  • This suggests that the early stages of the symbiotic relationship may resemble pathogen invasion

• Tissue-specific localization:

  • Different components of the T3SS may have varying importance depending on the host cell type

  • Research has shown that ysaV and orgA, components of T3SS, are not required for Sodalis invasion of S2R+ cells, suggesting either different infection mechanisms for different cell types or that S2R+ cells readily endocytose Sodalis independent of its T3SS

• Evolution of mutualism:

  • The retention of a functional T3SS in a mutualistic symbiont supports the hypothesis of a parasitism-mutualism continuum

  • This suggests that vertically transmitted mutualistic endosymbionts may evolve from horizontally transmitted parasites

• Genomic consequences:

  • The comparison of T3SS genes between Sodalis and pathogens can reveal selection pressures acting on these genes during the transition to mutualism

  • Patterns of sequence conservation or divergence may indicate which aspects of the secretion system are essential for the symbiotic lifestyle

The study of Sodalis' T3SS provides a unique window into the evolutionary processes that shape the transition from pathogenesis to mutualism, offering insights into how bacterial symbionts evolve and adapt to their hosts over time.

What are the optimal conditions for culturing Sodalis glossinidius in laboratory settings?

Culturing Sodalis glossinidius presents unique challenges due to its status as a facultative endosymbiont with specific growth requirements. The following methodological guidelines address these challenges:

Growth media formulations:

• Liquid culture media:

  • BHI (Brain Heart Infusion) supplemented with:

    • 10% heat-inactivated fetal bovine serum

    • 0.1% yeast extract

    • Appropriate antibiotics for selection of transformed strains

  • Consider microaerophilic conditions (1-5% oxygen) for optimal growth

• Solid media:

  • BHI agar supplemented similarly to liquid media

  • Plates should be incubated at 25-27°C under microaerophilic conditions

  • Colony formation may take 3-7 days due to slow growth rate

Culture conditions optimization:

• Temperature:

  • Optimal growth occurs at 25-27°C

  • Higher temperatures (>30°C) may inhibit growth

  • Lower temperatures (<20°C) significantly slow growth rate

• Atmospheric conditions:

  • Growth can occur in both aerobic and anaerobic conditions

  • Microaerophilic conditions often yield better growth

  • Consider oxygen-controlled incubators for consistent results

• Growth monitoring:

  • Optical density (OD600) measurements may be less reliable due to clumping

  • Colony forming unit (CFU) determination on solid media provides more accurate quantification

  • Phase-contrast microscopy can help assess culture health

Troubleshooting common issues:

• Poor growth:

  • Check media composition and freshness

  • Verify incubation temperature and atmospheric conditions

  • Ensure inoculum is from a healthy, log-phase culture

• Contamination:

  • Implement strict aseptic technique

  • Include selective antibiotics if possible

  • Verify purity through microscopic examination and selective plating

• Loss of viability during storage:

  • Prepare glycerol stocks (25% final concentration) from log-phase cultures

  • Store at -80°C for long-term preservation

  • Minimize freeze-thaw cycles

These methods provide a foundation for successfully culturing Sodalis glossinidius in laboratory settings, enabling further research on this important endosymbiont.

How can researchers resolve contradictory data regarding UbiB function in different bacterial species?

Resolving contradictory data regarding UbiB function across different bacterial species requires a systematic approach to identify sources of variation and reconcile inconsistencies:

Sources of data contradiction:

• Genetic context variations:

  • Operon structure differences across species (e.g., the ubiE, yigP, and ubiB operon in E. coli)

  • Polar effects of mutations may cause misinterpretation of gene function

  • Presence of paralogs with redundant functions

• Methodological differences:

  • Variations in culture conditions affecting ubiquinone biosynthesis

  • Different analytical techniques for detecting ubiquinone and intermediates

  • Inconsistent mutant construction strategies

• Environmental factors:

  • Oxygen availability influencing pathway selection

  • Temperature effects on enzyme activity

  • Nutrient availability affecting metabolism

Systematic resolution approach:

• Standardized genetic analysis:

  • Create precise deletions avoiding polar effects on downstream genes

  • Confirm mutations by genome sequencing rather than just PCR verification

  • Perform complementation studies with well-characterized constructs

• Comparative genomic analysis:

  • Examine synteny of ubi genes across species

  • Identify conserved domains and motifs in UbiB homologs

  • Analyze evolutionary relationships using phylogenetic methods

• Unified experimental conditions:

  • Establish standardized growth conditions across species

  • Control oxygen levels precisely

  • Use consistent analytical methods for metabolite detection

• Cross-species functional testing:

  • Express UbiB from different species in a common host

  • Test functional complementation across species

  • Identify species-specific interacting partners

Table 3: Data resolution framework for UbiB function across bacterial species

• Statistical meta-analysis:

  • Combine data from multiple studies

  • Weight evidence based on methodological rigor

  • Identify patterns that explain apparent contradictions

This systematic approach can help reconcile contradictory data regarding UbiB function, leading to a more unified understanding of its role in ubiquinone biosynthesis across different bacterial species.

What are the promising new approaches for studying the functional interactions between UbiB and other proteins in the ubiquinone biosynthesis pathway?

Several innovative approaches hold promise for elucidating the functional interactions between UbiB and other proteins in the ubiquinone biosynthesis pathway:

• Cryo-electron microscopy of protein complexes:

  • Visualize the native architecture of UbiB-containing complexes

  • Determine the spatial arrangement of interacting proteins

  • Identify conformational changes upon substrate binding or protein-protein interactions

• Proximity-dependent biotin labeling:

  • Express UbiB fused to BioID or TurboID enzymes

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Map the protein interaction landscape during ubiquinone biosynthesis under various conditions

• Single-molecule techniques:

  • Apply fluorescence resonance energy transfer (FRET) to monitor protein interactions in real-time

  • Use optical tweezers to study the mechanical properties of protein complexes

  • Implement super-resolution microscopy to visualize nanoscale organization

• Integrative structural biology:

  • Combine X-ray crystallography, NMR, and molecular dynamics simulations

  • Create computational models of the entire ubiquinone biosynthesis complex

  • Predict functional interactions and test through targeted mutagenesis

• Synthetic biology approaches:

  • Engineer minimal ubiquinone biosynthesis systems with defined components

  • Implement optogenetic control over protein interactions

  • Create fusion proteins to force interactions and test functional hypotheses

• In situ studies:

  • Develop methods to study UbiB function directly in the native membrane environment

  • Apply correlative light and electron microscopy to localize complexes

  • Use genetically encoded sensors to monitor biosynthetic activity in living cells

These approaches, when combined with traditional biochemical and genetic methods, will provide unprecedented insights into the functional interactions of UbiB and advance our understanding of ubiquinone biosynthesis as an integrated cellular process.

How might understanding Sodalis-host interactions inform strategies for controlling other insect-borne diseases?

Insights from Sodalis-host interactions can inform novel strategies for controlling a range of insect-borne diseases through several mechanistic and conceptual advances:

• Translatable symbiont manipulation strategies:

  • Knowledge of how Sodalis establishes and maintains symbiosis could be applied to other insect-bacterial systems

  • Genetic manipulation techniques developed for Sodalis (conjugation, transduction) could be adapted for symbionts of other disease vectors

  • The O₂-independent ubiquinone biosynthesis pathway could be targeted to disrupt symbiont survival in microaerophilic insect tissues

• Comparative vector biology insights:

  • Understanding how the type III secretion system facilitates Sodalis invasion could reveal common mechanisms used by symbionts in mosquitoes, fleas, and other vectors

  • The interplay between social behavior and symbiont abundance observed in halictid bees could inform vector control strategies that target insect behavioral ecology

• Evolution-informed intervention design:

  • The parasitism-mutualism continuum hypothesis supported by Sodalis research suggests potential to redirect symbiont evolution in other vectors

  • Knowledge of how symbionts transition from pathogenic to mutualistic relationships could inform strategies to disrupt similar transitions in other systems

• Novel technological applications:

  • Paratransgenesis platforms similar to those designed for Sodalis could be developed for symbionts of mosquitoes (malaria, dengue), kissing bugs (Chagas disease), or fleas (plague)

  • Design of Experiments (DoE) approaches used to optimize Sodalis manipulation could improve efficiency of interventions for other vector systems

• Ecological impact assessment frameworks:

  • Models developed to predict the spread of engineered Sodalis in tsetse populations could be adapted for other vector-symbiont systems

  • Approaches to assess potential non-target effects of Sodalis manipulation could inform regulatory frameworks for other vector control methods