Recombinant Chromobacterium violaceum Flagellar L-ring protein 2 (flgH2)

What is the basic structure and function of the Flagellar L-ring protein 2 (flgH2) in Chromobacterium violaceum?

The FlgH2 protein in C. violaceum forms the L-ring component of the flagellar basal body, functioning as a molecular bushing anchored in the outer membrane. Structurally, FlgH proteins are highly extended with complex topology, typically featuring a β-barrel core from which complex loop insertions extend. In assembled flagella, the L-ring creates a passage for the flagellar rod through the outer membrane and provides structural stability to the entire flagellar complex.

Similar to other bacterial FlgH proteins (such as those from Salmonella), C. violaceum FlgH2 is likely synthesized as a precursor with a signal peptide and processed to its mature form. The mature protein would contain an N-terminal lipid modification (via a conserved cysteine residue) that anchors it to the outer membrane .

How does flgH2 differ from flgH1 in Chromobacterium violaceum, and what is the evolutionary significance of having multiple homologs?

C. violaceum contains multiple homologs of most flagellar genes, including flgH. The presence of flgH2 alongside flgH1 suggests specialized functions or regulatory mechanisms. Comparative analysis indicates that these paralogs likely arose through gene duplication events followed by functional divergence.

The evolutionary significance of maintaining multiple homologs may relate to:

• Adaptation to different environmental conditions

• Functional specialization for different motility requirements

• Redundancy to ensure flagellar assembly under various stress conditions

• Potential regulatory diversity in flagellar gene expression

This gene duplication pattern is consistent with C. violaceum's complex chemotaxis system, which features an unusually high number of chemosensory genes (67 genes in 10 gene clusters) .

What are the optimal conditions for expressing recombinant C. violaceum flgH2 in E. coli expression systems?

For optimal expression of recombinant C. violaceum flgH2 in E. coli, the following methodology is recommended:

Expression system optimization:

• Vector selection: pET-based vectors with T7 promoter systems provide high-level expression

• Host strain: BL21(DE3) or derivatives like Rosetta(DE3) to address potential codon bias

• Growth temperature: 16-18°C post-induction to minimize inclusion body formation

• Induction: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8

• Media supplementation: Addition of 0.2% glucose to minimize leaky expression

Key consideration: As flgH2 likely contains membrane-targeting sequences, expression strategies should account for potential toxicity and membrane localization. Using strains with reduced proteolytic activity (like BL21) and incorporating fusion tags (His6, MBP, or SUMO) can enhance solubility and facilitate purification .

What purification strategies are most effective for obtaining structurally intact recombinant flgH2 protein suitable for functional studies?

A multi-step purification approach is necessary to obtain structurally intact flgH2:

• Initial extraction:

  • For membrane-associated form: Solubilization using mild detergents (n-dodecyl β-D-maltoside or CHAPS at 0.5-1%)

  • For soluble constructs (if transmembrane regions are removed): Standard lysis buffers containing protease inhibitors

• Chromatography sequence:

  • IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA or Co-NTA resins for His-tagged constructs

  • Size exclusion chromatography to separate monomeric from oligomeric forms

  • Ion exchange chromatography as a polishing step

• Buffer optimization:

  • Inclusion of stabilizers: 5-10% glycerol and 1-5 mM β-mercaptoethanol

  • pH range: 7.0-8.0 (phosphate or Tris-based buffers)

  • Salt concentration: 150-300 mM NaCl to maintain protein stability

Storage in small aliquots at -80°C with flash-freezing in liquid nitrogen helps maintain structural integrity during long-term storage .

How do mutations in the N-terminal lipidation site of flgH2 affect protein localization and flagellar assembly in C. violaceum?

Mutations in the N-terminal lipidation site of flgH2 significantly impact protein localization and flagellar assembly. Research with homologous FlgH proteins in Salmonella provides insights applicable to C. violaceum:

• Cysteine substitution effects:

  • Replacement of the conserved N-terminal cysteine (lipidation site) with glycine or other amino acids prevents lipoylation

  • Without proper lipid modification, FlgH fails to anchor properly in the outer membrane

  • This results in unstable L-ring formation and compromised flagellar structure

• Experimental evidence:

  • In Salmonella studies, cells with FlgH lacking the lipidation site exhibited severely impaired motility

  • The mutant protein could still be processed to remove the signal peptide but failed to incorporate [³H]palmitate

  • Complementation experiments showed that high-copy expression of non-lipidated FlgH partially rescued motility, suggesting that proper localization can be partially achieved through mass action

• Localization defects:

  • Without lipidation, FlgH accumulates in the periplasm rather than inserting into the outer membrane

  • This mislocalization prevents proper L-ring assembly and disrupts the formation of a functional flagellar basal body

Researchers investigating C. violaceum flgH2 should consider site-directed mutagenesis approaches targeting the conserved cysteine residue to analyze the specific effects in this organism .

What structural differences have been identified between the L-ring structures in C. violaceum compared to other well-characterized bacterial flagellar systems?

Comparative structural analysis between C. violaceum L-ring and other bacterial systems reveals several noteworthy differences:

Architectural distinctions:

• Ring symmetry and subunit arrangement:

  • Salmonella FlgH forms a 26-fold symmetric ring with an inner diameter of ~140 Å

  • C. violaceum's L-ring likely exhibits similar symmetry but with unique species-specific features

  • The β-barrel structure is conserved, but loop insertions show considerable variation

• Membrane interactions:

  • C. violaceum FlgH2 likely forms specialized interactions with the unique lipopolysaccharide composition of its outer membrane

  • These interactions may provide enhanced stability in the diverse environments inhabited by C. violaceum

• Inter-ring connections:

  • The interaction between the L-ring (FlgH) and P-ring (FlgI) shows species-specific variations

  • C. violaceum may possess unique structural features that optimize flagellar function in soil and aquatic environments

These structural differences may relate to C. violaceum's adaptation to tropical and subtropical environments and its capacity for both free-living existence and host infection .

How does targeted mutagenesis of flgH2 affect flagellar assembly, motility, and virulence in C. violaceum?

Targeted mutagenesis of flgH2 produces cascading effects on flagellar assembly, motility, and virulence in C. violaceum:

Effects on flagellar assembly:

• Complete deletion of flgH2 likely results in compromised L-ring formation

• Depending on functional redundancy with flgH1, the phenotype may range from complete absence of flagella to reduced flagellar numbers

• Electron microscopy of basal bodies from flgH2 mutants would reveal structural defects in the L-ring region

Impacts on motility:

• Swimming motility assays would show reduced migration through semi-solid agar (0.3-0.4%)

• High-speed video microscopy would detect altered rotational patterns and reduced swimming speed

• Chemotaxis toward attractants would be compromised, affecting environmental adaptation

Virulence implications:

• Reduced invasion capability in cell culture models

• Diminished colonization in animal infection models

• Altered biofilm formation capacity, which is critical for C. violaceum pathogenicity

Complementation studies with wild-type flgH2 would restore these phenotypes, confirming the specific role of this protein. Data from similar mutations in Salmonella and Vibrio suggest that even point mutations in critical domains can significantly impair flagellar function .

What are the methodological approaches for studying protein-protein interactions between flgH2 and other flagellar components in C. violaceum?

Several complementary methodologies can elucidate protein-protein interactions between flgH2 and other flagellar components:

• Bacterial Two-Hybrid (B2H) assays:

  • Fusion of flgH2 and potential interaction partners to DNA-binding and activation domains

  • Measurement of reporter gene expression as indicator of protein interaction

  • This approach successfully identified interactions between FlhF and FliG in P. aeruginosa

• Co-immunoprecipitation assays:

  • Generation of specific antibodies against flgH2 or epitope-tagged versions

  • Pull-down experiments followed by immunoblotting for potential interacting partners

  • Mass spectrometry analysis of co-precipitated proteins to identify novel interactions

• Cross-linking coupled with mass spectrometry:

  • Application of membrane-permeable cross-linkers to intact cells

  • Isolation of flgH2-containing complexes

  • Mass spectrometric identification of cross-linked peptides to map interaction interfaces

• FRET-based approaches:

  • Fusion of flgH2 and potential partners with fluorescent proteins

  • Measurement of energy transfer as indication of protein proximity

  • Live-cell imaging to monitor dynamics of interactions during flagellar assembly

These approaches have been successfully applied to other flagellar components and can be adapted for studying C. violaceum flgH2 interactions with neighboring proteins like FlgI (P-ring) and other outer membrane components .

How does flgH2 expression correlate with quorum sensing and violacein production in C. violaceum?

The relationship between flgH2 expression, quorum sensing (QS), and violacein production in C. violaceum represents a complex regulatory network:

Regulatory connections:

• Quorum sensing control:

  • C. violaceum uses N-hexanoyl-L-homoserine lactone (C6-HSL) as its primary QS signal

  • The CviI/CviR QS system regulates multiple phenotypes, including biofilm formation and violacein production

  • Flagellar gene expression, including flgH2, is likely modulated by QS signals at specific cell densities

• Correlation with violacein production:

  • High cell density activates QS, triggering both violacein biosynthesis genes (vioABCDE) and potentially altering flagellar gene expression

  • Temporal expression analysis shows that flagellar and violacein genes exhibit distinct but potentially coordinated expression patterns

  • The Air two-component regulatory system, which responds to antibiotic stress, regulates both violacein production and likely influences flagellar gene expression

• Environmental response integration:

  • Translation-inhibiting antibiotics induce both violacein production and biofilm formation

  • These stress responses may coincide with changes in flagellar gene expression, including flgH2

  • The physiological state of cells transitioning between motile and sessile lifestyles involves coordinated regulation of both systems

Experimental methods like qRT-PCR and reporter gene assays (using promoter fusions to GFP or luciferase) have demonstrated these correlations in C. violaceum under various growth conditions .

What methodological approaches can distinguish the specific roles of flgH1 and flgH2 in C. violaceum motility?

Distinguishing the specific roles of flgH1 and flgH2 requires a multifaceted experimental approach:

Genetic approaches:

• Individual and double gene knockout studies:

  • Generation of ΔflgH1, ΔflgH2, and ΔflgH1ΔflgH2 mutants using allelic exchange

  • Phenotypic characterization of swimming, swarming, and twitching motility

  • Microscopic examination of flagellar number and morphology

• Complementation analysis:

  • Cross-complementation testing (flgH1 in ΔflgH2 and vice versa)

  • Domain-swapping experiments to identify functional specificity regions

  • Heterologous expression of each gene in model organisms like E. coli

Expression analysis:

• Transcriptional profiling:

  • RNA-seq or microarray analysis of wild-type and mutant strains

  • Quantitative RT-PCR with gene-specific primers to detect differential expression

  • Promoter-reporter fusions to monitor expression under various conditions

• Protein localization:

  • Fluorescent protein fusions to determine subcellular localization

  • Immunogold electron microscopy to visualize protein incorporation into flagellar structures

  • Fractionation studies to determine membrane association patterns

These approaches would reveal whether the two homologs have redundant, complementary, or distinct functions in C. violaceum motility, potentially relating to different environmental conditions or growth phases .

What are the challenges and solutions for obtaining high-resolution structural data of C. violaceum flgH2 using cryo-electron microscopy?

Obtaining high-resolution structural data of C. violaceum flgH2 via cryo-electron microscopy (cryo-EM) presents several challenges and corresponding solutions:

Challenges and technical solutions:

Workflow optimization:

• Express recombinant flgH2 with minimal modifications (small affinity tags)

• Purify intact basal bodies or reconstitute flgH2 rings in vitro

• Apply vitrification conditions optimized for membrane protein complexes

• Collect data with energy filters and direct electron detectors

• Process using motion correction, CTF estimation, and particle picking

• Apply symmetry-based reconstruction (likely C26 symmetry based on homologous structures)

These approaches have successfully revealed the structure of flagellar components from other bacteria at resolutions sufficient to build atomic models .

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) be applied to study the dynamic properties of flgH2 during flagellar assembly?

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers powerful insights into the dynamic properties of flgH2 during flagellar assembly:

Methodological approach:

• Sample preparation stages:

  • Purify recombinant flgH2 in various states:

    • Monomeric form

    • Assembled into rings in vitro

    • In complex with interaction partners (e.g., FlgI)

  • Perform deuterium labeling at controlled time points (10 sec to 24 hr)

  • Quench the exchange reaction and digest with protease (typically pepsin)

  • Analyze resulting peptides by LC-MS/MS

• Data interpretation framework:

  • Regions with rapid exchange: Exposed/flexible domains

  • Regions with slow exchange: Core structural elements or protected interfaces

  • Differential exchange patterns between states: Conformational changes during assembly

• Application to specific research questions:

  • Map interaction surfaces between flgH2 and flgI (P-ring protein)

  • Identify conformational changes upon membrane association

  • Characterize the dynamic properties of the β-barrel and loop regions

  • Monitor structural changes induced by lipidation

• Technical considerations:

  • Temperature control (0-4°C during quenching and digestion)

  • pH control (pH 2.5 during quenching)

  • Use of protease inhibitors to prevent non-specific degradation

  • Optimization of peptide coverage through multiple protease approaches

This approach has successfully revealed assembly mechanisms of other macromolecular complexes and would provide valuable insights into the dynamic properties of flgH2 during flagellar morphogenesis .

How does C. violaceum flgH2 compare to homologous proteins in other pathogenic bacteria, and what does this reveal about flagellar evolution?

Comparative analysis of C. violaceum flgH2 with homologs from other pathogenic bacteria reveals important evolutionary and functional insights:

Phylogenetic relationships and structural conservation:

Evolutionary insights:

• Domain architecture conservation:

  • The core β-barrel structure is highly conserved across species

  • Species-specific adaptations occur primarily in surface-exposed loops

  • The N-terminal lipidation motif shows high conservation, underscoring its functional importance

• Gene duplication patterns:

  • C. violaceum's possession of flgH1 and flgH2 represents a duplication event not seen in many other species

  • This duplication likely provided adaptive advantages in the diverse environments C. violaceum inhabits

  • Similar duplications are observed in other flagellar genes of C. violaceum (41 MCP genes compared to 5 in E. coli)

• Functional divergence:

  • Vibrio species possess additional flagellar ring components (H-ring) that may reflect adaptation to specific environmental pressures

  • The existence of multiple homologs in C. violaceum suggests functional specialization for different environmental conditions

These comparative analyses provide insights into the evolutionary forces shaping bacterial motility systems and the specific adaptations of C. violaceum .

What genomic analysis approaches can identify conserved and variable domains in flgH proteins across the Chromobacterium genus?

Several genomic analysis approaches can effectively identify conserved and variable domains in flgH proteins across the Chromobacterium genus:

Comprehensive analytical framework:

• Sequence-based approaches:

  • Multiple sequence alignment (MSA) using MUSCLE or MAFFT algorithms

  • Conservation scoring using methods like Jensen-Shannon divergence

  • Sliding window analysis of nucleotide and amino acid diversity (π, θ)

  • Selection pressure analysis using dN/dS ratio to identify positions under positive selection

• Structure-informed analysis:

  • Homology modeling based on solved structures (e.g., Salmonella FlgH)

  • Mapping of conservation scores onto structural models

  • Prediction of transmembrane regions and signal peptides

  • Identification of lipidation motifs and other post-translational modification sites

• Comparative genomics approaches:

  • Synteny analysis of flagellar gene clusters across Chromobacterium species

  • Identification of species-specific insertions/deletions

  • Analysis of operon structure and promoter regions

  • Investigation of horizontal gene transfer events using codon usage and GC content analysis

• Functional domain prediction:

  • Conserved domain search using CDD, PFAM, and InterPro databases

  • Secondary structure prediction using PSIPRED

  • Disorder prediction using PONDR or IUPred

  • Coevolution analysis to identify functionally linked residues

These approaches would reveal domains essential for core FlgH function versus regions that may confer species-specific adaptations in different Chromobacterium strains, providing valuable information for targeted mutagenesis studies .

How does flgH2 function contribute to the formation and structural integrity of C. violaceum biofilms?

The contribution of flgH2 to C. violaceum biofilm formation involves several interconnected mechanisms:

Structural and functional contributions:

• Initial surface attachment:

  • Functional flagella are essential for initial surface sensing and attachment

  • FlgH2, as part of the L-ring, ensures proper flagellar rotation needed for this process

  • Defects in flgH2 would impair the ability of cells to approach and interact with surfaces

• Biofilm matrix architecture:

  • AFM studies reveal that C. violaceum undergoes morphological differentiation during biofilm development

  • This differentiation involves membrane invaginations that later form polymer matrix extrusions

  • Proper flagellar function, dependent on intact flgH2, is required for this differentiation process

• Quorum sensing integration:

  • C. violaceum biofilm development is regulated by N-hexanoyl-L-homoserine lactone (C6-HSL)

  • Flagellar function and biofilm formation are coordinated through quorum sensing networks

  • Experimental evidence from atomic force microscopy shows that wild-type cells form organized matrix structures, while QS mutants produce diffuse extracellular substances

• Virulence connection:

  • Translation-inhibiting antibiotics induce both biofilm formation and virulence in C. violaceum

  • This induction requires functional flagellar components, including flgH2

  • The antibiotic-induced response (air) two-component regulatory system links these phenotypes

Disruption of flgH2 would therefore affect not only motility but also biofilm development and potentially virulence, highlighting the multifunctional nature of flagellar components in bacterial adaptation .

What experimental designs can elucidate the role of flgH2 in C. violaceum interactions with other microorganisms in mixed-species biofilms?

Several experimental designs can effectively elucidate flgH2's role in interspecies interactions:

Experimental approaches for mixed-species biofilm studies:

• Confocal microscopy-based co-culture systems:

  • Fluorescently label wild-type and flgH2 mutant C. violaceum (GFP, mCherry)

  • Establish mixed biofilms with relevant partner species (e.g., Bacillus cereus, Saccharomyces cerevisiae)

  • Analyze spatial organization, biovolume ratios, and species distribution

  • Time-lapse imaging to capture dynamic interactions during biofilm development

• Transcriptomic profiling of interspecies interactions:

  • RNA-seq analysis of C. violaceum wild-type and ΔflgH2 in mono- versus mixed-species biofilms

  • Identification of differentially expressed genes in response to partner species

  • Focus on quorum sensing, virulence, and antimicrobial production pathways

  • Validation of key findings with reporter gene constructs

• Metabolomic analysis of chemical communication:

  • LC-MS/MS profiling of metabolites in mixed biofilm supernatants

  • Targeted analysis of signaling molecules (AHLs, violacein, hydrogen cyanide)

  • Comparison between wild-type and flgH2 mutant interactions

  • Correlation of metabolite profiles with biofilm structural properties

• Competition and predation assays:

  • Quantitative assessment of competitive fitness in mixed biofilms

  • Prey-predator interactions with protozoan grazers

  • Resistance to antimicrobial compounds produced by competing species

  • Long-term evolutionary experiments to track adaptation in mixed communities

These experimental designs would provide comprehensive insights into how flagellar function, mediated by flgH2, influences the complex social interactions of C. violaceum in polymicrobial communities .

What are the implications of targeting flgH2 for developing novel antimicrobial strategies against C. violaceum infections?

Targeting flgH2 offers promising avenues for antimicrobial development against C. violaceum infections:

Therapeutic potential and considerations:

• Rationale for targeting flgH2:

  • C. violaceum infections are rare but often fatal with mortality rates >50%

  • Conventional antibiotics face resistance challenges

  • Targeting virulence factors like flagella may reduce selective pressure for resistance

  • FlgH2 is essential for flagellar function and likely plays roles in host colonization

• Potential inhibition strategies:

  • Small-molecule inhibitors of FlgH2 assembly or function

  • Peptide-based inhibitors targeting the FlgH2-FlgI interaction interface

  • Antibodies or nanobodies against surface-exposed FlgH2 epitopes

  • Inhibitors of lipidation to prevent proper L-ring anchoring

• Therapeutic applications:

  • Prevention of initial host colonization

  • Reduction of bacterial dissemination within the host

  • Combination therapy with conventional antibiotics

  • Potential for broad-spectrum activity against multiple flagellated pathogens

• Experimental validation approaches:

  • High-throughput screening assays for inhibitor identification

  • In vitro motility inhibition assays

  • Cell culture infection models

  • Mouse infection models, particularly in chronic granulomatous disease (CGD) models where C. violaceum is especially virulent

The therapeutic potential is particularly relevant for immunocompromised patients, such as those with CGD, who are especially susceptible to C. violaceum infections .

How can recombinant flgH2 be utilized as a research tool for studying bacterial motility and host-pathogen interactions?

Recombinant flgH2 offers versatile applications as a research tool:

Research applications:

• Structural biology platforms:

  • Template for computational drug design targeting flagellar assembly

  • Model system for studying membrane protein assembly

  • Reference for comparative studies of flagellar evolution

• Immunological research:

  • Development of antibodies for flagellar localization studies

  • Investigation of innate immune recognition of flagellar components

  • Study of host-pathogen interactions in C. violaceum infections

• Synthetic biology applications:

  • Design of chimeric flagellar systems with novel properties

  • Engineering of bacteria with controlled motility

  • Development of biosensors using flagellar components

• Biotechnological tools:

  • Protein scaffolds for nanobiotechnology applications

  • Templates for self-assembling protein nanotubes

  • Building blocks for bottom-up synthetic flagella

Methodological approaches:

• Site-directed mutagenesis to generate protein variants

• Fluorescent protein fusions for live-cell imaging

• Creation of affinity-tagged versions for protein-protein interaction studies

• Development of in vitro assembly systems to study flagellar morphogenesis

These applications leverage the structural and functional properties of flgH2 to address fundamental questions in bacterial physiology and pathogenesis while also enabling biotechnological innovations .

What recent technological advances have revolutionized the study of flagellar L-ring proteins in C. violaceum and related bacteria?

Recent technological advances have transformed our understanding of flagellar L-ring proteins:

Breakthrough methodologies:

• Structural biology innovations:

  • Cryo-electron tomography of intact bacterial flagella in situ

  • High-resolution cryo-EM of isolated basal bodies reaching 2.2Å resolution

  • Advanced image processing algorithms for asymmetric reconstruction

  • Integrative structural biology combining multiple data sources

• Genetic tool development:

  • CRISPR-Cas9 genome editing in non-model bacteria including Chromobacterium

  • Inducible gene expression systems for temporal control

  • Single-cell tracking of protein dynamics using photoactivatable fluorescent proteins

  • Transposon sequencing (Tn-seq) for high-throughput functional genomics

• Biochemical and biophysical approaches:

  • Native mass spectrometry of membrane protein complexes

  • Single-molecule force spectroscopy of flagellar components

  • In vitro reconstitution of flagellar substructures

  • Microfluidic platforms for precise control of bacterial microenvironments

• Computational advances:

  • Molecular dynamics simulations of membrane-embedded flagellar rings

  • Deep learning approaches for protein structure prediction

  • Systems biology models of flagellar gene regulation

  • Coevolutionary analysis to predict protein-protein interactions

These advances have enabled unprecedented insights into flagellar structure and function, revealing details of protein-protein interactions, assembly pathways, and evolutionary relationships that were previously inaccessible .

What are the most pressing unanswered questions regarding flgH2 function in C. violaceum, and what experimental approaches might address these gaps?

Several critical knowledge gaps remain regarding flgH2 function in C. violaceum:

Unanswered questions and experimental approaches: