Recombinant Chromobacterium violaceum Flagellar P-ring protein 2 (flgI2)

What is the fundamental role of the P-ring in bacterial flagellar structures?

The P-ring, composed of FlgI protein, is located in the peptidoglycan layer of gram-negative bacteria like Chromobacterium violaceum . It functions primarily as a bushing that facilitates smooth rotation of the flagellum through the cell envelope . This structural component is essential for bacterial motility, as it creates a stable anchor point within the peptidoglycan layer while allowing the central rod to rotate freely . The P-ring works in concert with other basal body components to ensure proper flagellar function and efficient bacterial movement.

How does the molecular structure of FlgI contribute to P-ring assembly?

The FlgI protein contains highly conserved regions, particularly in the N-terminus (residues 1-120), that play critical roles in both stabilizing the protein's structure and mediating interactions between adjacent FlgI molecules during P-ring formation . Systematic cysteine mutagenesis studies have demonstrated that specific residues within this N-terminal region are essential for proper protein folding, stability, and functional assembly . The spatial arrangement of FlgI molecules creates a ring structure with precise dimensions that accommodates the flagellar rod while maintaining structural integrity within the periplasmic space .

What genomic characteristics are associated with C. violaceum flagellar genes?

Chromobacterium violaceum has a genome size of approximately 4,637,406 base pairs with a relatively high GC content of 64.89% . The genome contains 4,572 protein-coding sequences, including those for flagellar components . Flagellar genes in C. violaceum are typically arranged in operons that allow coordinated expression of multiple structural and regulatory components . While specific information about the FlgI gene locus is limited in the provided data, genomic analysis reveals complex regulatory networks that control flagellar assembly and function in response to environmental conditions.

What methodologies are most effective for studying FlgI protein interactions in the P-ring structure?

Systematic cysteine mutagenesis represents a powerful approach for investigating FlgI protein interactions and structure-function relationships . This methodology involves:

• Creation of cysteine substitutions at regular intervals throughout the protein sequence

• Expression of these variants in a FlgI-deficient background

• Assessment of protein stability via immunoblot analysis

• Functional evaluation through motility assays

• Probing of surface-exposed residues using thiol-specific reagents such as methoxypolyethylene glycol 5000 maleimide

This comprehensive strategy allows researchers to simultaneously evaluate the impact of mutations on protein stability, function, and surface accessibility, providing insights into which domains are involved in critical interactions versus those that are structurally protected .

How can researchers accurately assess the impact of FlgI mutations on flagellar function?

Quantitative assessment of flagellar function requires multiple complementary approaches:

Comprehensive evaluation should include both population-level measurements (swarm assays) and single-cell observations (microscopy) to distinguish between defects in flagellar assembly, structure, and motor function . Additionally, correlating these functional assessments with biochemical analyses provides mechanistic insights into how specific mutations impact flagellar performance.

What strategies can be employed to optimize recombinant expression of C. violaceum FlgI in heterologous systems?

Optimizing recombinant expression of C. violaceum FlgI requires addressing several challenges:

• Codon optimization based on C. violaceum's high GC content (64.89%) to enhance expression in common laboratory hosts like E. coli

• Design of expression constructs that preserve the N-terminal region (residues 1-120) critical for proper folding and stability

• Consideration of periplasmic targeting signals, as FlgI naturally localizes to the periplasmic space

• Potential co-expression with flagellar chaperones to enhance folding efficiency

• Temperature modulation during induction, as demonstrated in similar studies with recombinant violacein production systems

Expression systems utilizing the metabolic engineering principles successfully applied to other C. violaceum proteins (like those in the violacein pathway) may provide valuable strategies for FlgI production . Researchers should monitor both protein yield and proper folding, as improperly folded FlgI may form inclusion bodies or exhibit altered functional properties.

Which specific amino acid residues are critical for FlgI stability and function in C. violaceum?

Systematic mutagenesis studies have identified several key residues in the N-terminus of FlgI that are crucial for protein stability and flagellar function . Specifically:

• Residue G21: Substitution with cysteine (G21C) completely disrupts FlgI function, suggesting a critical role in protein folding or interactions

• Residues I3, G51, G81, and D111: Cysteine substitutions at these positions significantly reduce motility while maintaining some function

• N-terminal region (residues 1-120): Highly conserved across bacterial species and contains multiple residues essential for proper protein levels and function

Interestingly, these functionally critical residues do not overlap with surface-exposed residues identified through thiol-specific labeling experiments, suggesting they play structural roles rather than mediating surface interactions .

How does the quaternary structure of the P-ring facilitate flagellar rotation while maintaining structural integrity?

The P-ring forms a circular bushing structure in the peptidoglycan layer that must simultaneously provide structural support while allowing free rotation of the central rod . This dual function is achieved through:

• Precise spatial arrangement of FlgI monomers to form a ring with appropriate inner diameter

• Strategic interactions between conserved N-terminal domains (residues 1-120) that maintain ring stability

• Surface-exposed residues that potentially interact with the peptidoglycan layer to anchor the structure

• Non-overlapping functional domains that separate structural stability from interaction interfaces

This complex architecture creates a structure capable of withstanding mechanical stress while minimizing friction during flagellar rotation, essential for efficient bacterial motility under various environmental conditions.

What is the relationship between FlgI function and C. violaceum virulence in human infections?

Chromobacterium violaceum is primarily an environmental bacterium that rarely causes human infections, but when it does, these infections have a high fatality rate . While the direct relationship between FlgI and virulence hasn't been fully characterized, several connections can be inferred:

• Bacterial motility, dependent on proper flagellar function including the P-ring, enables tissue invasion and dissemination during infection

• Flagellar components including FlgI may trigger immune responses that contribute to the inflammatory cascade observed in severe C. violaceum infections

• The rapid progression from localized infection to systemic disease suggests efficient bacterial dissemination potentially facilitated by functional flagella

C. violaceum infections typically present with fever following localized wound infections, progressing to dark or purple vesicle abscesses with necrosis . Hematogenic spread, evidenced by positive blood cultures, leads to complications including sepsis, respiratory distress, and abscess formation in various organs . The potential role of flagellar motility in this dissemination process warrants further investigation.

How do flagellar mutations affect C. violaceum survival under antibiotic pressure?

C. violaceum exhibits intrinsic resistance to multiple antibiotics, a characteristic influenced by its purple pigment violacein and the presence of efflux pump systems . The potential interactions between flagellar mutations and antibiotic susceptibility may include:

• Altered energy allocation: Defects in flagellar assembly (including P-ring) may redirect cellular resources toward antibiotic resistance mechanisms

• Membrane permeability changes: Flagellar defects affecting membrane integrity could influence antibiotic penetration

• Regulatory cross-talk: Shared regulatory pathways between flagellar expression and antibiotic resistance genes

The genome of C. violaceum contains at least seven putative genes involved in efflux pump systems that confer multidrug resistance . Understanding how flagellar mutations interact with these resistance mechanisms could provide insights for developing more effective therapeutic approaches for the rare but often fatal C. violaceum infections.

What novel imaging techniques can provide insights into P-ring assembly dynamics in living cells?

Advanced imaging approaches that could elucidate P-ring assembly include:

• Fluorescence Recovery After Photobleaching (FRAP) using FlgI-fluorescent protein fusions to monitor assembly kinetics

• Single-molecule localization microscopy (SMLM) to visualize individual FlgI molecules during ring formation

• Cryo-electron tomography to capture three-dimensional structures of the flagellar basal body at different assembly stages

• Correlative light and electron microscopy (CLEM) combining fluorescence imaging of tagged FlgI with high-resolution ultrastructural analysis

These approaches would complement the biochemical data from cysteine labeling studies by providing spatial and temporal information about P-ring assembly in living bacteria. Such techniques could reveal intermediate steps in the assembly process and identify potential quality control mechanisms that ensure proper P-ring formation.

How might comparative genomics inform our understanding of FlgI evolution and specialization in C. violaceum?

Comparative genomic approaches could reveal evolutionary patterns in FlgI sequences across bacterial species, particularly focusing on:

• Identification of highly conserved domains beyond the known N-terminal region (residues 1-120)

• Detection of C. violaceum-specific adaptations that might reflect environmental niche specialization

• Correlation between FlgI sequence variations and differences in flagellar structure or function across species

• Analysis of selection pressures on different FlgI domains, potentially revealing regions under purifying versus diversifying selection

The high GC content (64.89%) of the C. violaceum genome might influence codon usage in the FlgI gene, potentially affecting translation efficiency and protein folding dynamics. Comparative analyses could identify whether this represents an adaptation to specific environmental conditions or a neutral evolutionary process.

What potential research applications exist for recombinant C. violaceum FlgI beyond structural studies?

Recombinant C. violaceum FlgI could serve diverse research applications:

• Development of structure-based inhibitors targeting bacterial motility as a novel antimicrobial approach

• Creation of self-assembling protein nanostructures based on the ring-forming properties of FlgI

• Design of biosensors utilizing FlgI structural changes upon binding to peptidoglycan components

• Investigation of evolutionary relationships between flagellar systems across bacterial species

• Development of vaccines targeting conserved epitopes in FlgI to protect against multiple flagellated pathogens

These applications would build upon the methodological approaches used in cysteine mutagenesis studies while extending the impact of FlgI research beyond basic structural biology to applied biotechnology and therapeutic development.