Recombinant UPF0145 protein YbjQ (ybjQ)

What is UPF0145 protein YbjQ and where is it found in bacterial species?

UPF0145 protein YbjQ is a bacterial protein that belongs to the UPF0145 family. It is found in various enterobacteria including Escherichia coli strains (O7:K1, O6:K15:H31, K12) and Shigella species (such as Shigella boydii) . The protein is relatively small, with 107 amino acid residues in E. coli strains, and has a molecular weight of approximately 11.4 kDa .

Structural studies have shown that YbjQ adopts a pentameric structure with elongated β-sheets, but lacks the small first β-strand and alpha helix that is characteristic of DUF1471 family proteins and RcsF . Unlike some other bacterial proteins that contain multiple domains, YbjQ consists of a single functional domain.

How is the ybjQ gene organized and regulated in bacteria?

The ybjQ gene is generally found as a single copy in bacterial genomes. In E. coli, it is identified by the gene symbol ybjQ with synonyms including ECK0857 and JW0850 . Unlike some other bacterial genes that are organized in operons with related functional genes, ybjQ appears to be independently regulated.

While specific transcriptional regulation of ybjQ has not been extensively characterized, some studies suggest it may be regulated as part of stress response systems. The protein has been detected in proteomics studies investigating bacterial responses to various environmental conditions , indicating that its expression may be modulated under specific stress conditions.

What is known about the three-dimensional structure of YbjQ protein?

Structural studies have revealed that YbjQ adopts a pentameric configuration, making it one of three proteins (along with a DUF74 domain protein and a selenium-binding protein from Methanococcus vannielii) that form pentameric structures in their family . The protein has more elongated β-sheets compared to related bacterial proteins but notably lacks the small first β-strand and alpha helix that characterize DUF1471 proteins and RcsF.

Structural analysis using tools like the Dali server has identified similar structures, with details presented in research studies . ModBase provides a predicted 3D structure for the P0A8C1 entry (E. coli K12 YbjQ) , which can serve as a model for further structural investigations.

The pentameric organization of YbjQ suggests it may function as part of a larger protein complex or may be involved in forming interfaces with other cellular components.

What are the functional roles of YbjQ in bacterial physiology?

While the precise function of YbjQ remains to be fully elucidated, several lines of evidence suggest potential roles:

• Stress Response: YbjQ may be involved in bacterial responses to environmental stresses, as it has been identified in proteomics studies examining stress conditions .

• Two-Component Signaling: There is evidence suggesting YbjQ may interact with or be regulated by two-component signaling systems in bacteria , potentially linking it to environmental sensing and response mechanisms.

• Possible Similarities to DUF1471 Proteins: While structurally distinct, YbjQ's functional role may have parallels with DUF1471 family proteins, which are involved in:

  • Biofilm formation

  • Response to oxidative and acid stress

  • Cell surface modification

  • Bacterial defense mechanisms

The structural similarity to RcsF is particularly noteworthy because RcsF is involved in regulating capsule synthesis and processes related to colanic acid production and biofilm formation in response to changes in the extracellular environment .

How does YbjQ compare structurally to proteins in the DUF1471 family?

Despite functional similarities, YbjQ shows distinct structural differences from the DUF1471 family proteins:

These structural differences suggest that YbjQ and DUF1471 proteins may have evolved to perform related but distinct functions in bacterial cells . The structural divergence may reflect adaptations to specific cellular processes or environments.

What expression systems are optimal for producing recombinant YbjQ protein?

Multiple expression systems have been successfully used to produce recombinant YbjQ protein:

• E. coli Expression System: The most commonly used system due to its simplicity and high yield. The protein can be expressed with various tags (N-terminal and/or C-terminal) to facilitate purification .

• Yeast Expression System: Offers post-translational modifications that might be important for specific applications, though typically with lower yields than E. coli systems .

• Baculovirus Expression System: Provides intermediate yields with eukaryotic post-translational modifications that may be relevant for certain functional studies .

• Mammalian Cell Expression System: Offers the most sophisticated post-translational modifications but with typically lower yields and higher costs .

For structural studies, the E. coli system has been successfully employed to produce properly folded YbjQ protein as demonstrated in crystallographic and NMR studies . When expressing the protein, consideration should be given to the inclusion of appropriate tags for purification while ensuring these do not interfere with protein folding or function.

What purification strategies are effective for recombinant YbjQ?

Effective purification of recombinant YbjQ typically involves:

• Affinity Chromatography: Using N-terminal or C-terminal tags such as His-tags for initial capture. The small size of YbjQ (11.4 kDa) makes it amenable to efficient binding to affinity resins .

• Size Exclusion Chromatography: Particularly important for separating the pentameric form from monomers or other oligomeric states.

• Ion Exchange Chromatography: Can be used as an additional purification step based on the protein's charge properties.

Researchers have achieved greater than 85% purity using SDS-PAGE analysis through these methods . For structural studies requiring higher purity, additional chromatographic steps may be necessary.

For NMR spectroscopy studies, isotopic labeling (15N, 13C) can be incorporated during expression in minimal media, followed by similar purification protocols .

How can researchers use YbjQ in functional and interaction studies?

To investigate YbjQ's function and interactions, researchers can employ several methodological approaches:

• Gene Deletion Studies: Constructing ybjQ knockout strains using techniques like λ red-mediated recombination and P1 transduction as described in protocols for similar bacterial genes . This allows assessment of phenotypic changes in the absence of YbjQ.

• Protein-Protein Interaction Assays:

  • Pull-down assays using tagged recombinant YbjQ

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation followed by mass spectrometry to identify interaction partners

• Structural Biology Approaches:

  • X-ray crystallography: Has been successfully used for related proteins

  • NMR spectroscopy: Effective for studying protein dynamics and interactions in solution

  • Cryo-electron microscopy: Particularly useful for examining YbjQ within larger complexes

• Functional Reconstitution: In vitro reconstitution of potential biochemical activities using purified components, which has been successful in studies of related bacterial proteins .

• Stress Response Assays: Monitoring changes in YbjQ expression or localization in response to various stress conditions, similar to studies performed with DUF1471 proteins .

What role might YbjQ play in bacterial stress responses and biofilm formation?

While direct evidence for YbjQ's role in stress responses is limited, structural similarities to proteins involved in these processes suggest potential functions:

• Stress Response:

  • The structural similarity to RcsF suggests YbjQ might be involved in sensing or responding to extracellular stresses .

  • Proteomics studies have identified YbjQ in stress response conditions, suggesting upregulation under specific environmental challenges .

• Biofilm Formation:

  • Related proteins like DUF1471 family members (YcfR/BhsA, YbiM/McbA, YjfO/BsmA, and YhcN) are involved in biofilm formation and modification of cell surface characteristics .

  • YbjQ might similarly influence bacterial adhesion, aggregation, or other processes related to biofilm development.

• Cell Surface Modification:

  • The pentameric structure of YbjQ could potentially function in modifying cell surface components, affecting bacterial interactions with their environment.

  • This could impact processes such as attachment to surfaces, cell-cell interactions, or responses to environmental stressors.

Experimental approaches to investigate these roles could include phenotypic characterization of ybjQ mutants under various stress conditions and biofilm formation assays comparing wild-type and mutant strains.

How does YbjQ interact with two-component signaling systems in bacteria?

Evidence suggests YbjQ may have connections to bacterial two-component signaling systems:

• Potential Regulatory Relationships:

  • Cross-talk between two-component systems can occur at different levels of signaling pathways, including between histidine kinases and non-cognate response regulators .

  • YbjQ may be regulated by these systems or modulate their activity, particularly in response to environmental stresses.

• Integration into Signaling Networks:

  • YbjQ could serve as an accessory protein that influences signal transduction in two-component systems.

  • It may function downstream of these systems as part of the cellular response mechanism.

• Experimental Evidence:

  • Studies have shown interactions between non-cognate components of two-component systems in E. coli using techniques like FRET microscopy .

  • Similar approaches could be used to investigate YbjQ's relationship with specific two-component systems.

To further investigate these interactions, researchers could employ techniques such as bacterial two-hybrid assays, co-immunoprecipitation studies, or phosphotransfer assays to identify specific two-component system components that interact with YbjQ.

What differentiates YbjQ proteins across various bacterial species and strains?

YbjQ proteins show both conservation and variation across bacterial species:

• Sequence Conservation:

  • The core functional domains of YbjQ are generally well-conserved across Enterobacteriaceae.

  • E. coli strains (K12, O7:K1, O6:K15:H31) contain highly similar YbjQ proteins .

  • Shigella boydii YbjQ shares significant sequence similarity with E. coli variants .

• Strain-Specific Variations:

  • Some differences are observed between pathogenic and non-pathogenic strains, potentially reflecting adaptation to different environmental niches.

  • These variations may influence protein function, stability, or interactions with other cellular components.

• Evolutionary Relationships:

  • Phylogenetic analysis of YbjQ proteins could provide insights into their evolutionary history and functional divergence.

  • Comparative genomics approaches can reveal patterns of conservation and variation that may correlate with specific bacterial phenotypes or ecological niches.

The differential distribution and sequence variation of YbjQ across bacterial species may reflect its adaptation to specific ecological contexts or functional roles within different bacterial lifestyles.

What are the major challenges in studying YbjQ function?

Researchers face several significant challenges when investigating YbjQ:

• Functional Redundancy: Like many bacterial proteins, functional redundancy may mask phenotypes in single gene deletion studies, necessitating multiple gene deletions to observe clear phenotypes.

• Contextual Function: YbjQ may only exhibit its physiological role under specific environmental conditions that might be difficult to reproduce in laboratory settings.

• Protein-Protein Interactions: Identifying interaction partners is challenging due to potentially transient interactions or requirements for specific conditions to trigger these interactions.

• Structural Heterogeneity: The pentameric structure of YbjQ may exist in different conformational states depending on cellular conditions, complicating structural analysis.

• Integration with Cellular Networks: Understanding how YbjQ integrates into broader cellular networks requires systems-level approaches that are technically challenging.

Addressing these challenges will require combining multiple experimental approaches, including genetic, biochemical, structural, and systems biology methods.

What technologies and approaches show promise for advancing YbjQ research?

Several cutting-edge technologies could significantly advance our understanding of YbjQ:

• CRISPR-Cas9 Gene Editing: For precise manipulation of ybjQ in various bacterial backgrounds to study function in different genetic contexts.

• Cryo-Electron Microscopy: To resolve high-resolution structures of YbjQ alone and in complex with interaction partners.

• Single-Cell Techniques: To examine heterogeneity in YbjQ expression and function within bacterial populations.

• Protein Crosslinking Mass Spectrometry: To identify interaction networks involving YbjQ under physiologically relevant conditions.

• RNA-Seq and Ribosome Profiling: To understand transcriptional and translational regulation of ybjQ under various conditions.

• Bacterial Cytological Profiling: To determine YbjQ's subcellular localization and potential changes in response to environmental stimuli.

• Computational Approaches: Machine learning and molecular dynamics simulations could predict functional sites and potential interactions of YbjQ.

These approaches, particularly when used in combination, have the potential to provide comprehensive insights into YbjQ's cellular functions and regulatory mechanisms.

How might understanding YbjQ function contribute to broader research in bacterial physiology?

Elucidating YbjQ function could impact multiple areas of bacterial research:

• Stress Response Mechanisms: Provide insights into how bacteria sense and respond to environmental stresses, particularly those affecting cell surface integrity.

• Biofilm Biology: Enhance understanding of molecular mechanisms underlying biofilm formation and maintenance, which has implications for both environmental and clinical microbiology.

• Evolution of Bacterial Signaling: Offer perspective on how specialized signaling proteins have evolved to coordinate responses to environmental changes.

• Bacterial Adaptation: Illuminate mechanisms of bacterial adaptation to specific ecological niches, including host environments for pathogenic strains.

• Antibiotic Resistance: Potentially reveal new targets for antimicrobial development if YbjQ is shown to play crucial roles in stress tolerance or biofilm formation, which are processes often linked to antibiotic resistance.

Understanding YbjQ's function represents one piece of the complex puzzle of bacterial physiology, with potential applications ranging from basic science to clinical microbiology and biotechnology.