Recombinant Shewanella baltica Probable ubiquinone biosynthesis protein UbiB (ubiB)
Description
Molecular Identity and Classification
Shewanella baltica is a Gram-negative, facultative anaerobic bacterium frequently associated with seafood spoilage . Within this organism, the UbiB protein belongs to a class of enzymes involved in transferring phosphorus-containing groups (EC 2.7.-.-) . More specifically, UbiB is classified as a protein kinase that likely serves as a regulator of UbiI activity, which is directly involved in aerobic coenzyme Q (ubiquinone) biosynthesis . This regulatory function positions UbiB as a critical control element in bacterial energy production pathways.
The protein is known by several synonyms including "Probable protein kinase UbiB" and "Ubiquinone biosynthesis protein UbiB," reflecting both its enzymatic activity and biological role . In database classifications, the protein has been assigned various identifier codes including UniProt ID B8E6B4 (for the strain documented in the Cusabio datasheet) and A3D9F4 (for the Shewanella baltica OS155 strain) .
Protein Composition and Domains
While the full structural analysis of UbiB from Shewanella baltica is not comprehensively documented in the available research, related information from closely related species provides valuable insights. For instance, the UbiB protein from Shewanella pealeana consists of 549 amino acids with a specific sequence structure adapted for its enzymatic function . This related protein shares significant homology with the S. baltica version, suggesting similar structural organization.
The recombinant forms of this protein are often produced as partial sequences, with specific modifications to enhance production and purification efficiency. The commercially available recombinant product from Cusabio, for example, is described as a partial sequence of the native protein .
Post-translational Modifications and Tags
Recombinant versions of UbiB proteins are frequently produced with affinity tags to facilitate purification. The recombinant Shewanella pealeana UbiB, for instance, is produced with an N-terminal His-tag . Such modifications do not typically interfere with the protein's core functions but provide significant advantages for isolation and purification processes.
Table 1: Comparison of UbiB Protein Characteristics from Different Sources
| Characteristic | S. baltica UbiB | S. baltica OS155 UbiB | S. pealeana UbiB |
|---|---|---|---|
| UniProt ID | B8E6B4 | A3D9F4 | A8H968 |
| Function | Ubiquinone biosynthesis | Protein kinase regulator | Ubiquinone biosynthesis |
| Length | Partial | Not specified | Full (1-549 amino acids) |
| Expression System | Baculovirus | Not specified | E. coli |
| Purity | >85% (SDS-PAGE) | Not specified | >90% (SDS-PAGE) |
| Tag Information | Determined during manufacturing | Not specified | N-terminal His-tag |
Expression Systems
The commercial recombinant UbiB protein from Shewanella baltica is produced using a Baculovirus expression system according to the Cusabio datasheet . This expression platform is widely recognized for its ability to facilitate proper protein folding and post-translational modifications, making it suitable for the production of complex bacterial proteins.
Alternative expression systems have also proven effective, as demonstrated by the successful production of the related UbiB protein from Shewanella pealeana in Escherichia coli . The choice of expression system significantly impacts factors including yield, purity, and biological activity of the recombinant protein.
Purification and Quality Assessment
Recombinant UbiB proteins undergo rigorous purification procedures to ensure high quality for research applications. The purity of commercial preparations typically exceeds 85% as determined by SDS-PAGE analysis . This high level of purity is essential for ensuring reliable experimental results in biochemical and structural studies.
Quality control measures for these recombinant proteins include verification of molecular weight, purity assessment using gel electrophoresis, and in some cases, functional assays to confirm enzymatic activity. The specific tag systems used in recombinant production facilitate efficient purification through affinity chromatography techniques.
Enzymatic Function
The primary enzymatic function of UbiB appears to be as a protein kinase that regulates the activity of other enzymes involved in ubiquinone biosynthesis. According to the PubChem database, it "is probably a protein kinase regulator of UbiI activity which is involved in aerobic coenzyme Q (ubiquinone) biosynthesis" .
This regulatory relationship highlights the complex control mechanisms governing ubiquinone production in bacteria, which is essential for their aerobic respiration and energy generation. The specific substrates, kinetic parameters, and inhibitors of UbiB's kinase activity represent important areas for continued research.
Function in Ubiquinone Biosynthesis
The principal biological role of UbiB is in the biosynthetic pathway leading to the production of ubiquinone (coenzyme Q). Ubiquinone serves as a critical electron carrier in the respiratory chain of many bacteria, including Shewanella baltica. This lipid-soluble component is essential for aerobic energy production, making UbiB indirectly vital for bacterial survival and growth.
UbiB's specific contribution to this process appears to be regulatory in nature, controlling the activity of UbiI through phosphorylation mechanisms . This regulatory function positions UbiB as a potential control point for the entire ubiquinone biosynthetic pathway, suggesting its importance in bacterial energy metabolism regulation.
Contextual Role in Shewanella baltica Biology
While the direct function of UbiB centers on ubiquinone biosynthesis, the broader context of Shewanella baltica biology provides additional perspective on this protein's significance. S. baltica is recognized as a dominant spoilage bacterium in chilled fish and seafood , with complex regulatory networks controlling its metabolic activities.
The bacterium employs various regulatory systems including RpoS-mediated pathways that affect spoilage activity and biofilm formation , as well as quorum sensing mechanisms that influence its ecological behavior . Though not directly connected to UbiB in the available research, these regulatory networks collectively form the biological context in which UbiB functions.
Experimental Utility
Recombinant UbiB from Shewanella baltica serves as a valuable research tool for investigating ubiquinone biosynthesis pathways in bacteria. The availability of purified recombinant forms facilitates:
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Enzymatic assays to characterize kinase activity and substrate specificity
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Structural studies to determine three-dimensional organization and functional domains
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Interaction analyses to identify protein partners in the ubiquinone biosynthetic pathway
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Comparative biochemical studies across different bacterial species
Potential Biotechnological Applications
Understanding the function and regulation of UbiB presents several potential biotechnological applications:
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Development of targeted antibiotics that disrupt bacterial energy metabolism by inhibiting ubiquinone biosynthesis
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Engineering of bacterial strains with modified ubiquinone production for biofuel or bioremediation applications
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Creation of biosensors utilizing UbiB-based detection systems for environmental monitoring
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Production of ubiquinone and derivatives for pharmaceutical and nutritional applications
Future Research Directions
The current understanding of Recombinant Shewanella baltica Probable ubiquinone biosynthesis protein UbiB presents several promising avenues for future investigation:
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Detailed structural analyses through X-ray crystallography or cryo-electron microscopy to elucidate the three-dimensional organization and functional domains
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Comprehensive biochemical characterization of kinase activity, including identification of specific substrates and regulatory mechanisms
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In vivo studies to confirm the precise role in ubiquinone biosynthesis and explore connections to other metabolic pathways
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Investigation of potential relationships between UbiB function and the spoilage characteristics of Shewanella baltica in seafood preservation contexts
Such research directions would significantly enhance understanding of bacterial energy metabolism regulation and potentially lead to practical applications in antimicrobial development, biotechnology, and food preservation.
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, kindly include them in your order remarks. We will strive to fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. Should you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: sbl:Sbal_3897
STRING: 325240.Sbal_3897
Q&A
What is the probable ubiquinone biosynthesis protein UbiB in Shewanella baltica?
UbiB in Shewanella baltica is an atypical kinase-like protein involved in ubiquinone (UQ) biosynthesis. Similar to its homolog in Escherichia coli, it plays a crucial yet incompletely characterized role in the biosynthetic pathway of ubiquinone, which is an essential electron carrier in aerobic respiration. In E. coli, UbiB has been identified as an essential component for UQ8 synthesis, although its exact enzymatic function remains elusive . S. baltica, as a facultative anaerobe with versatile respiratory capabilities, likely relies on UbiB for similar functions, particularly when adapting to varying oxygen levels in its environment.
How does ubiquinone biosynthesis relate to S. baltica's ecological niche adaptation?
S. baltica was identified as the dominant culturable nitrate-reducing bacterium in the Baltic Sea during the 1980s, primarily inhabiting the oxic-anoxic transition zone . The core genome of S. baltica is enriched in anaerobic respiration-associated genes , suggesting that respiratory flexibility is key to its ecological success. Ubiquinone, synthesized through pathways involving UbiB, serves as a critical electron carrier during aerobic conditions, while under anaerobic conditions, S. baltica can employ alternative respiratory pathways. This respiratory versatility enables S. baltica to thrive in stratified water columns with varying redox conditions, contributing to its niche specialization.
What genomic evidence supports UbiB's role in S. baltica adaptation?
Comparative genomic hybridization studies of 46 S. baltica isolates revealed that genes associated with anaerobic respiration form part of the core genome . While UbiB is not specifically mentioned in these studies, the identification of genetic signatures related to redox-driven niche specialization suggests that ubiquinone biosynthesis pathways, including UbiB, may be critical for S. baltica's adaptation to specific water column positions. The genomic variations observed between different S. baltica clades, particularly those related to respiratory capabilities, indicate that proteins like UbiB may play roles in the species' remarkable adaptability to different electron acceptor availability in stratified environments.
What are the optimal expression systems for producing recombinant S. baltica UbiB protein?
For recombinant expression of S. baltica UbiB, E. coli-based expression systems typically offer the most practical approach for initial studies. Consider the following methodological recommendations:
Table 1: Recommended Expression Systems for S. baltica UbiB
| Expression System | Advantages | Considerations | Best For |
|---|---|---|---|
| E. coli BL21(DE3) | High yield, well-established protocols | May form inclusion bodies | Initial expression trials |
| E. coli Arctic Express | Better folding at low temperatures | Slower growth, lower yields | Improving solubility |
| E. coli Rosetta | Accommodates rare codons | More expensive | Optimizing translation |
| S. baltica native host | Natural chaperones, authentic folding | More complex genetics | Functional studies |
When expressing UbiB, it is critical to consider its potential membrane association and the possible requirement for specific lipid environments, given that ubiquinone biosynthesis involves a membrane-associated metabolon as observed in related systems .
What purification challenges are specific to S. baltica UbiB and how can they be addressed?
Purification of UbiB presents significant challenges due to its predicted membrane association and potential instability. A methodological approach should include:
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Initial membrane extraction: Use mild detergents like n-dodecyl-β-D-maltopyranoside (DDM) or digitonin to solubilize membrane-associated UbiB while preserving protein structure.
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Affinity purification: Employ a dual-tag strategy (e.g., His-tag for initial capture and a secondary tag like FLAG for additional purification) to achieve higher purity.
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Protein stabilization: Include glycerol (10-20%) and specific lipids in purification buffers to maintain the native environment for UbiB.
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Functional verification: Develop activity assays based on known functions of UbiB homologs, possibly involving ATP hydrolysis measurements since UbiB has kinase-like domains.
Drawing parallels from E. coli studies, it's important to note that UbiB may participate in protein complexes similar to the "Ubi metabolon" observed in E. coli, where UbiJ and UbiK bind lipids to facilitate ubiquinone biosynthesis in a hydrophilic environment .
How can researchers evaluate UbiB's contribution to ubiquinone biosynthesis experimentally?
To assess UbiB's role in ubiquinone biosynthesis, researchers should consider a multi-faceted approach:
In vivo approaches:
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Gene deletion studies: Generate ubiB knockout mutants and analyze ubiquinone levels using HPLC-MS/MS.
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Complementation assays: Test whether S. baltica UbiB can restore ubiquinone production in E. coli ubiB mutants.
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Oxygen-dependent expression analysis: Measure ubiB expression and ubiquinone production under varying oxygen concentrations to understand its regulation in response to environmental conditions.
In vitro approaches:
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Reconstitution experiments: Attempt to reconstitute ubiquinone biosynthesis using purified components including UbiB.
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Protein-protein interaction studies: Identify binding partners of UbiB using pull-down assays, co-immunoprecipitation, or bacterial two-hybrid systems.
What bioinformatic analyses can predict UbiB function in S. baltica?
Bioinformatic approaches provide valuable insights into UbiB function:
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Homology modeling: Construct structural models of S. baltica UbiB based on characterized homologs, focusing on the kinase-like domain.
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Conserved domain analysis: Compare UbiB sequences across Shewanella species and other bacteria to identify conserved regions that may be functionally important.
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Genomic context analysis: Examine genes located near ubiB in the S. baltica genome to identify potential functional relationships, similar to how UbiUVT genes in E. coli were found to enable O₂-independent ubiquinone biosynthesis .
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Phylogenetic analysis: Determine how UbiB evolved in S. baltica compared to other species, which may reveal adaptive signatures related to the Baltic Sea environment.
How does UbiB potentially interact with anaerobic ubiquinone biosynthesis pathways?
Recent research in E. coli has revealed an anaerobic O₂-independent ubiquinone biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes . For S. baltica, which thrives in oxic-anoxic transition zones, understanding potential interactions between UbiB and anaerobic ubiquinone synthesis is crucial:
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Investigate UbiUVT homologs: Determine if S. baltica possesses homologs of the UbiUVT system found in E. coli and examine their expression patterns relative to UbiB.
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Oxygen labeling experiments: Use ¹⁸O₂ labeling to distinguish between O₂-dependent and O₂-independent hydroxylation of ubiquinone precursors, similar to experiments in E. coli .
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Transcriptional regulation analysis: Investigate whether S. baltica has Fnr-like regulators (oxygen-sensing transcription factors) that control expression of ubiB and related genes, as Fnr controls ubiTUV in E. coli .
How does UbiB function relate to S. baltica's adaptation to redox gradients?
S. baltica thrives in the Baltic Sea's stratified water column, particularly in the oxic-anoxic transition zone . UbiB's role in ubiquinone biosynthesis likely contributes to this adaptation in several ways:
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Respiratory flexibility: Ubiquinone enables S. baltica to perform aerobic respiration when oxygen is available, while other electron acceptors (like nitrate) can be used when oxygen is scarce.
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Rapid metabolic switching: The presence of both aerobic and anaerobic ubiquinone synthesis pathways (potentially involving UbiB and UbiUVT homologs, respectively) would allow S. baltica to quickly adapt to fluctuating oxygen levels.
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Redox sensing: UbiB may participate in sensing redox conditions, potentially through its kinase-like activity, helping to coordinate appropriate metabolic responses.
Genomic analysis of S. baltica clades has revealed gene signatures related to specific redox-driven niches within the water column , suggesting that respiratory chain components, including those involved in ubiquinone biosynthesis, are important for niche specialization.
What role might UbiB play in biofilm formation and quorum sensing in S. baltica?
S. baltica has demonstrated biofilm formation capabilities regulated by quorum sensing (QS) systems . While direct evidence linking UbiB to these processes is lacking, several potential connections exist:
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Energy provision for biofilm processes: Ubiquinone-dependent respiration provides energy needed for biofilm formation, making UbiB indirectly important.
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Potential regulatory crosstalk: In S. baltica, the QS system employing cyclo-(L-Pro-L-Phe) (PP) as an autoinducer and LuxR-type proteins as receptors positively regulates biofilm formation . The energy status of the cell, influenced by respiratory efficiency and thus potentially by UbiB function, may affect QS signaling.
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Stress response coordination: Both biofilm formation and respiratory adaptation represent responses to environmental stresses, suggesting possible coordinated regulation involving UbiB.
How can CRISPR-Cas9 technology be applied to study UbiB function in S. baltica?
CRISPR-Cas9 genome editing offers powerful approaches for investigating UbiB:
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Precise gene modifications: Create point mutations in specific domains of UbiB to determine structure-function relationships without completely removing the protein.
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Conditional knockdowns: Implement CRISPRi (CRISPR interference) to achieve tunable repression of ubiB expression, allowing assessment of dose-dependent phenotypes.
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Fluorescent tagging: Insert fluorescent protein tags to monitor UbiB localization and dynamics in living cells under different growth conditions.
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Promoter replacements: Substitute the native ubiB promoter with inducible promoters to control expression timing and strength for functional studies.
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Multiplex editing: Simultaneously modify ubiB and related genes (potential UbiUVT homologs) to study genetic interactions and redundancies in ubiquinone biosynthesis pathways.
What proteomics approaches can elucidate UbiB interactions and modifications?
Advanced proteomics techniques can reveal critical insights about UbiB:
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Proximity-dependent biotin identification (BioID): Fuse BioID to UbiB to identify proximal proteins in the living bacterial cell, potentially revealing components of a S. baltica ubiquinone biosynthesis complex.
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Crosslinking mass spectrometry (XL-MS): Apply protein crosslinking followed by mass spectrometry to map interaction interfaces between UbiB and partner proteins.
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Thermal proteome profiling: Monitor thermal stability changes of UbiB and other proteins upon addition of potential substrates or inhibitors to infer functional interactions.
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Post-translational modification analysis: Investigate whether UbiB undergoes regulatory modifications such as phosphorylation, which might be expected given its kinase-like domains.
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Quantitative redox proteomics: Determine how UbiB's oxidation state changes under different oxygen conditions, potentially revealing mechanism of oxygen sensing.
How can metabolomic approaches track UbiB's impact on cellular metabolism?
Metabolomics offers powerful insights into UbiB's role in S. baltica metabolism:
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Targeted ubiquinone analysis: Employ UHPLC-MS/MS to quantify ubiquinone and biosynthetic intermediates in wild-type versus ubiB mutant strains under various oxygen conditions.
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Flux analysis with stable isotopes: Use 13C-labeled precursors to track carbon flow through the ubiquinone biosynthesis pathway and determine where UbiB functions.
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Global metabolite profiling: Compare metabolomes of wild-type and ubiB mutant strains to identify broader metabolic impacts beyond ubiquinone biosynthesis.
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Respiration-specific assays: Measure oxygen consumption rates, membrane potential, and ATP production in wild-type versus ubiB mutant strains to assess respiratory efficiency.
Table 2: Metabolite Changes in ubiB Mutants Under Different Oxygen Conditions
| Condition | Expected Ubiquinone Levels | Alternate Electron Carriers | Respiratory Capacity |
|---|---|---|---|
| Aerobic | Severely reduced | Increased menaquinone (compensatory) | Significantly impaired |
| Microaerobic | Moderately reduced | Mixed quinone pool | Moderately impaired |
| Anaerobic | Minimally affected | Primarily menaquinone | Minimally affected if UbiUVT homologs present |
How does S. baltica UbiB compare with homologs in other species?
Comparative analysis of UbiB across bacterial species reveals:
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Structural conservation: The kinase-like domain of UbiB is likely conserved across species, reflecting its fundamental role in ubiquinone biosynthesis.
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Specialization in facultative anaerobes: S. baltica and other facultative anaerobes may have evolved specialized regulatory features for UbiB to function optimally across oxygen gradients.
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Taxonomic distribution: UbiB homologs are widespread in proteobacteria but may have evolved different regulatory mechanisms in distinct ecological niches.
What can horizontal gene transfer patterns tell us about UbiB evolution in S. baltica?
Genomic island analysis in S. baltica has revealed extensive horizontal gene transfer (HGT) contributing to niche specialization . For UbiB research:
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Core vs. auxiliary gene status: Determine whether ubiB is part of the core genome or shows patterns consistent with horizontal acquisition.
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Clade-specific signatures: Examine whether different S. baltica clades show variations in ubiB and related genes that correlate with their environmental niches.
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Mobile genetic elements: Investigate the genomic context of ubiB for evidence of mobile genetic elements that might have facilitated its transfer or modification.
The study of S. baltica isolates has already revealed that gain/loss of functional genes drives gene content differences among less related strains , suggesting that genes involved in core metabolic functions like ubiquinone biosynthesis might show interesting evolutionary patterns.
