Recombinant Rhizobium radiobacter Protein virB10 (virB10)

What is the basic structure of the VirB10 protein?

VirB10 is a multidomain protein that spans the entire bacterial cell envelope. Its structure consists of four distinct domains: (i) an N-terminal cytoplasmic region, (ii) a transmembrane (TM) α-helix anchoring the protein to the inner membrane, (iii) a proline-rich region (PRR) that extends across the periplasm, and (iv) a C-terminal β-barrel domain that interacts with the outer membrane components . The protein structurally integrates into the inner membrane and extends across the periplasmic space to interact with the VirB7-VirB9 complex at the outer membrane, forming a continuous channel through the bacterial cell envelope .

What is the functional role of VirB10 in the Type IV Secretion System?

VirB10 serves as an energy sensor and conformational coupling protein in the T4SS. It detects ATP consumption by the VirB/D4 ATPases in the inner membrane and undergoes a conformational change that facilitates DNA transfer across the outer membrane . This energy-dependent structural transition is essential for substrate translocation through the secretion channel. Additionally, VirB10 mediates the biogenesis of the virB-encoded T pilus, which is critical for bacterial attachment and subsequent DNA transfer to target cells .

How does VirB10 interact with other components of the T4SS?

VirB10 forms a "core complex" with VirB7 and VirB9 during the biogenesis of the VirB/VirD4 type IV secretion system . This core complex creates a structural scaffold spanning the periplasm and outer membrane. The β-barrel domain of VirB10 specifically interacts with the VirB7-VirB9 channel complex at the outer membrane, while its transmembrane domain anchors it to the inner membrane . These interactions create a continuous conduit through which secretion substrates can pass from the bacterial cytoplasm to the external environment or directly into target cells.

What are the recommended methods for purifying recombinant VirB10 protein?

For recombinant VirB10 purification, a methodological approach involving bacterial expression systems is recommended. Begin with cloning the virB10 gene from Rhizobium radiobacter into an expression vector containing an affinity tag (His6 or GST). Express in E. coli strains optimized for membrane protein expression (like C41/C43 or BL21-AI) at lower temperatures (16-20°C) to enhance proper folding. For extraction, use detergents like n-dodecyl-β-D-maltoside (DDM) or CHAPS that maintain protein integrity while solubilizing membrane components. Purify using affinity chromatography followed by size exclusion chromatography to ensure high purity. Verification should include SDS-PAGE, Western blotting, and mass spectrometry. Functional assays such as ATP binding or conformational change assays can confirm the biological activity of the purified protein.

How can researchers assess VirB10 conformational changes in response to ATP?

To assess ATP-dependent conformational changes in VirB10, researchers can employ protease accessibility assays as described in previous studies . This method involves comparing the protease susceptibility pattern of VirB10 in energized cells versus cells depleted of ATP by treatment with arsenate. When VirB10 undergoes conformational changes in response to ATP energy, certain regions become more or less accessible to proteases, resulting in different fragmentation patterns that can be visualized by immunoblotting . Additionally, researchers can utilize fluorescence resonance energy transfer (FRET) by introducing fluorescent labels at strategic positions in VirB10, allowing real-time monitoring of distance changes between domains during ATP binding and hydrolysis.

What approaches are most effective for studying VirB10 interactions with other T4SS components?

For studying VirB10 interactions with other T4SS components, a multi-faceted approach is most effective. Co-immunoprecipitation (Co-IP) can identify protein-protein interactions in vivo, while bacterial two-hybrid assays can validate direct protein interactions. For structural analysis, cryogenic electron microscopy (cryo-EM) has proven valuable in visualizing the entire T4SS complex architecture. Cross-linking experiments using formaldehyde or specific cross-linking agents, followed by mass spectrometry, can identify interaction interfaces. Pull-down assays using purified components can determine binding affinities and kinetics. Genetic complementation studies using chimeric proteins can define functional interaction domains. In vivo fluorescence microscopy with fluorescently tagged T4SS components can visualize complex assembly in real-time within bacterial cells.

How do mutations in different domains of VirB10 affect T4SS function and pilus biogenesis?

Different domains of VirB10 have distinct roles in T4SS function and pilus biogenesis, as evidenced by mutation studies. Mutations conferring a transfer- and pilus-minus (Tra-, Pil-) phenotype include PRR deletion and β-barrel substitution mutations that prevent VirB10 interaction with the outer membrane VirB7-VirB9 channel complex . Mutations in the cytoplasmic domain or TM domain insertions result in a transfer-positive but pilus-negative (Tra+, Pil-) phenotype, indicating these regions are specifically important for pilus biogenesis but not substrate transfer . Deletions of α-helical projections extending from the β-barrel domain cause a unique phenotype where substrate transfer occurs, but pilus assembly is disrupted, resulting in the release of pilin monomers to the milieu . These findings highlight the modular nature of VirB10 function, with distinct domains regulating either secretion channel formation or T pilus assembly.

What is the significance of the G272R mutation in VirB10 and how does it affect substrate regulation?

The G272R mutation in VirB10 represents a significant discovery in understanding channel gating mechanisms in the T4SS. This mutation has been found to allow unregulated release of the VirE2 secretion substrate to the cell surface independently of target cell contact . Importantly, G272R renders VirB10 conformationally insensitive to cellular ATP depletion, suggesting it "locks" the protein in an energy-activated, open conformation . While this mutation does not affect substrate transfer to target cells (maintaining a Tra+ phenotype), it completely blocks pilus production (Pil- phenotype) . This indicates that the G272 residue, located near the AP pore, is critical for proper channel gating. The mutation also enhances uptake of detergent and large antibiotics, further supporting the model that G272R creates a constitutively open channel state that bypasses the normal energy-dependent regulation mechanism.

How can site-directed mutagenesis approaches be optimized for studying VirB10 structure-function relationships?

For optimized site-directed mutagenesis of VirB10, researchers should follow a strategic approach based on structural insights and conservation analysis. Start by analyzing sequence alignments across bacterial species to identify highly conserved residues likely critical for function. Use available structural data to target specific domains: the N-terminal cytoplasmic domain, TM helix, PRR, and C-terminal β-barrel. Create point mutations, small deletions, and domain swaps to test functional hypotheses. For energy sensing, focus on residues near the inner membrane interface and ATP-binding regions. For conformational coupling, target flexible regions that might undergo structural changes. For channel gating, concentrate on the β-barrel domain, particularly residues facing the channel lumen. Design alanine scanning libraries of short segments to systematically map functional regions. Employ overlap extension PCR or commercial mutagenesis kits, and verify all constructs by sequencing. Test each mutant in complementation assays measuring both substrate transfer and pilus biogenesis to distinguish domain-specific functions.

How does VirB10 contribute to energy coupling in the T4SS?

VirB10 serves as a critical energy-coupling protein in the T4SS, transmitting energy from ATP hydrolysis at the inner membrane to facilitate substrate transfer across the bacterial cell envelope. Studies indicate that VirB10 undergoes a conformational change in response to ATP consumption by the VirB/D4 ATPases . This energy-dependent structural transition is required for DNA transfer across the outer membrane. The null mutation of virB10 blocks formaldehyde (FA) cross-linking of DNA with VirB2 and VirB9, suggesting that ATP energy at the inner membrane activates a structural transition in VirB10 required for outer membrane channel formation or gating . The G272R mutation, which renders VirB10 conformationally insensitive to cellular ATP levels, supports the model that energy sensing by VirB10 is a regulated process that normally prevents uncontrolled substrate release . This sophisticated energy coupling mechanism ensures that substrate translocation occurs only under appropriate conditions, likely when the secretion apparatus is properly engaged with a target cell.

What is the role of VirB10 in the assembly of the outer membrane complex?

VirB10 plays a crucial structural role in the assembly of the outer membrane complex of the T4SS. It forms a "core complex" with VirB7 and VirB9 that serves as a structural scaffold for the portion of the channel spanning the periplasm and outer membrane . The C-terminal β-barrel domain of VirB10 specifically interacts with the VirB7-VirB9 channel complex at the outer membrane . Mutation studies have shown that PRR deletion and β-barrel substitution mutations that prevent VirB10 interaction with the outer membrane VirB7-VirB9 channel complex result in a transfer- and pilus-minus phenotype . This indicates that proper assembly of this complex is essential for both substrate transfer and pilus biogenesis. The biochemical findings, together with structural data, support a model where VirB10 integrates into the inner membrane, extends via its PRR across the periplasm, and interacts via its β-barrel domain with the VirB7-VirB9 channel complex to form a continuous conduit through the bacterial cell envelope .

How does VirB10 regulate substrate specificity in the T4SS?

VirB10 contributes to substrate specificity in the T4SS through its C-terminal region, which plays a key role in regulating passage of secretion substrates across the outer membrane . This regulation involves controlled gating of the secretion channel in response to ATP energy consumption and possibly in response to signals associated with target cell contact. The G272R mutation, which allows unregulated release of the VirE2 secretion substrate, provides evidence for VirB10's role in substrate gating . Moreover, different domain mutations in VirB10 differentially affect substrate transfer versus pilus biogenesis, suggesting that the protein may participate in substrate discrimination . While the exact mechanism of substrate specificity is not fully elucidated, it likely involves coordinated action with other T4SS components, particularly the VirD4 coupling protein, which is known to recognize secretion substrates. In systems lacking VirD4, such as in some Sesbania-nodulating rhizobia, substrate transfer may occur through a VirD4-independent mechanism or via an unidentified gene with similar function , suggesting flexibility in the substrate recognition and transfer process.

How does VirB10 function differ between pathogenic and symbiotic interactions in Rhizobium species?

The function of VirB10 in Rhizobium species demonstrates important adaptations depending on whether the bacterium engages in pathogenic or symbiotic interactions. In pathogenic contexts, such as in Agrobacterium (reclassified as Rhizobium) causing Crown Gall disease, the VirB/D4 T4SS facilitates the transfer of oncogenic T-DNA into plant cells, altering the plant's DNA to synthesize opines that are metabolized by the pathogenic bacteria . In contrast, in symbiotic interactions, such as those between Rhizobium species and leguminous plants like Sesbania cannabina, the T4SS may function to establish nitrogen-fixing root nodules . Comparative genomic analyses of Sesbania-nodulating rhizobia have revealed that symbiotic strains contain transferrable plasmids (pSyms) with virB genes, but often lack virD4 . This suggests that symbiotic Rhizobium species may have evolved a virD4-independent mechanism for substrate transfer or utilize an unidentified gene with similar function . Additionally, the presence of highly similar nodA gene sequences among diverse Rhizobium and Sinorhizobium strains suggests that the legume host rigorously selects for specific Nod factor structures, potentially influencing T4SS function and VirB10 activity in symbiotic contexts .

What mechanisms allow for horizontal transfer of VirB10-containing genetic elements between bacterial species?

Horizontal transfer of VirB10-containing genetic elements between bacterial species is facilitated by several sophisticated mechanisms. In Rhizobium species, the virB genes, including virB10, are often located on transferrable symbiotic plasmids (pSyms) . These pSyms typically contain origin of transfer (oriT) sequences, conjugal transfer genes (traACDG), and quorum-sensing regulatory genes (cinIR), which together enable plasmid mobilization between bacteria . The complete virB1-11 gene cluster on these plasmids encodes the T4SS machinery necessary for DNA transfer . Interestingly, in Sesbania-nodulating rhizobia, the pSyms contain complete virB gene sets but often lack virD4, suggesting a virD4-independent transfer mechanism . The high similarity of nodulation genes across diverse Rhizobium species, despite their taxonomic differences, provides strong evidence for frequent horizontal gene transfer events . This genetic mobility explains the existence of diverse Sesbania-nodulating rhizobia and indicates that receptor strains adapted to local conditions can acquire the necessary symbiosis genes through horizontal transfer, potentially losing their original pSyms due to plasmid incompatibility .

How can VirB10 function be studied in the context of host immune responses to Rhizobium infection?

Studying VirB10 function in the context of host immune responses requires specialized methodologies addressing both plant and potential human host interactions. For plant hosts, researchers should employ Arabidopsis or tobacco with genetically modified immune receptors to track pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) responses to wild-type versus virB10 mutant strains. ROS burst assays, callose deposition measurements, and defense gene expression (PR1, PDF1.2) analyses can quantify immune activation. In human contexts, though Rhizobium radiobacter rarely infects humans, neutrophil extracellular trap formation, cytokine production assays using THP-1 or primary macrophages, and identification of human PRRs recognizing bacterial patterns are valuable approaches. To directly link immune responses to VirB10 function, localize VirB10 during infection using immunofluorescence microscopy with anti-VirB10 antibodies or tagged VirB10 constructs. Compare host transcriptomes upon exposure to wild-type versus VirB10-deficient bacteria to identify differentially regulated immune pathways. The unique case of R. radiobacter infection in an immunocompromised patient with sickle cell anemia demonstrates potential clinical relevance of this research.

What are the main challenges in expressing and purifying functional recombinant VirB10 protein?

The expression and purification of functional recombinant VirB10 protein presents several significant challenges. First, VirB10 is a membrane-spanning protein with multiple domains that must maintain proper folding and topology for functionality . Traditional expression systems often result in protein aggregation or improper folding. Second, the protein contains both hydrophobic transmembrane regions and hydrophilic periplasmic domains, making it difficult to solubilize and maintain in a native-like environment throughout purification. Third, VirB10 functions as part of a multi-protein complex with VirB7 and VirB9 , and isolation may disrupt critical protein-protein interactions necessary for proper conformation. Fourth, VirB10 undergoes ATP-dependent conformational changes , so maintaining this dynamic property during purification is challenging. Finally, the protein must be extracted from membranes using detergents, which can affect protein stability and activity. To address these challenges, researchers should consider using specialized expression systems for membrane proteins, mild detergents for extraction, and potentially co-expressing VirB10 with interacting partners like VirB7 and VirB9 to maintain native complex formation.

How can researchers effectively design experiments to study the dynamic conformational changes in VirB10?

To effectively study dynamic conformational changes in VirB10, researchers should implement a multi-technique approach. Begin with strategic cysteine substitutions at key positions throughout VirB10 to enable accessibility studies using membrane-permeable and -impermeable thiol-reactive reagents, which can identify regions that become exposed during conformational shifts. Complement this with protease susceptibility assays comparing VirB10 fragmentation patterns in ATP-energized versus ATP-depleted cells . For real-time monitoring, implement FRET using fluorescently labeled VirB10 with donor-acceptor pairs positioned to detect domain movements during activation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map regions with altered solvent accessibility during conformational changes. For structural visualization, employ cryo-electron microscopy of the T4SS complex under different energetic states. Validate findings using site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy to measure distances between domains. These approaches should be conducted in both in vitro reconstituted systems and, when possible, in the native membrane environment to capture the authentic conformational dynamics of VirB10.

What are the most appropriate model systems for studying VirB10 function in vivo?

The most appropriate model systems for studying VirB10 function in vivo require careful selection based on specific research questions. For fundamental mechanistic studies, Agrobacterium tumefaciens remains the gold standard due to its well-characterized VirB/D4 T4SS and established genetic tools . This system allows for direct assessment of DNA transfer efficiency and pilus formation through plant transformation assays and electron microscopy. For studying host-specific adaptations, researchers should use authentic Rhizobium radiobacter strains isolated from their natural environments . To examine symbiotic functions, Sesbania cannabina-nodulating Rhizobium strains provide valuable insights into how VirB10 contributes to plant-microbe symbiosis . For rare pathogenic interactions with humans, experimental systems using human cell lines can model interactions observed in immunocompromised patients . Essential experimental approaches include genetic complementation assays with virB10 mutants, fluorescent protein tagging for localization studies, and bacterial two-hybrid systems to map protein-protein interactions. For structural studies, bacterial minicells enriched for poles (where T4SS complexes often localize) can be used for cryo-electron tomography to visualize the T4SS machinery in a near-native state.

What can comparative analysis of VirB10 sequences tell us about T4SS evolution and specialization?

Comparative analysis of VirB10 sequences provides valuable insights into T4SS evolution and specialization across bacterial species. The conservation pattern of VirB10 reveals a core functional architecture that has been maintained throughout evolution, particularly in the transmembrane domain, PRR, and β-barrel domain . Variations in sequence and domain organization reflect adaptation to different ecological niches and functional requirements. For instance, the presence of similar nodA gene sequences (98.8-100% similarities) among diverse Rhizobium and Sinorhizobium strains that nodulate Sesbania cannabina suggests horizontal gene transfer events and convergent evolution driven by host selection pressure . The consistent presence of VirB10 in pSyms (symbiotic plasmids) across different rhizobial species, despite taxonomic diversity, indicates the crucial role of this protein in establishing symbiotic relationships . Interestingly, while virB10 is consistently preserved, some symbiotic strains lack virD4, suggesting evolutionary adaptation toward a virD4-independent mechanism for substrate transfer . This evolutionary plasticity in the T4SS machinery demonstrates how bacterial secretion systems can be modified and repurposed throughout evolution to serve different functions, from pathogenesis to symbiosis.

What structural domains of VirB10 show the highest conservation or variability across species?

Analysis of VirB10 across different bacterial species reveals distinct patterns of conservation and variability in its structural domains. The transmembrane (TM) α-helix and the proline-rich region (PRR) show high conservation, reflecting their essential roles in anchoring the protein to the inner membrane and spanning the periplasmic space, respectively . The C-terminal β-barrel domain, which interacts with the VirB7-VirB9 channel complex at the outer membrane, also displays significant sequence conservation, particularly in regions directly involved in protein-protein interactions and channel formation . In contrast, the N-terminal cytoplasmic region shows the highest variability across species, suggesting this domain may be involved in species-specific regulatory functions or interactions .

The table below summarizes the conservation patterns across VirB10 domains: