Recombinant Photorhabdus luminescens subsp. laumondii Cell division protein ZipA homolog (zipA), partial

What is the Cell Division Protein ZipA homolog in Photorhabdus luminescens?

The Cell Division Protein ZipA homolog in Photorhabdus luminescens is a membrane-anchored protein that plays a crucial role in bacterial cell division. It functions by interacting with the bacterial tubulin homolog FtsZ, promoting and stabilizing the assembly of FtsZ protofilaments into the Z-ring structure that defines the division plane in bacterial cells . The N-terminal cytoplasmic domain of ZipA contains sequence elements resembling microtubule-binding signature motifs found in eukaryotic Tau, MAP2, and MAP4 proteins, suggesting an evolutionary conservation of protein-protein interactions involved in cytoskeletal organization . In P. luminescens, ZipA is part of the complex cellular machinery responsible for cell division, contributing to the bacterium's ability to proliferate during its lifecycle, which includes both symbiotic and pathogenic phases .

How does ZipA function in bacterial cell division?

ZipA functions in bacterial cell division through multiple mechanisms:

• Protofilament Stabilization: Purified ZipA promotes and stabilizes protofilament assembly of FtsZ in vitro and cosediments with these protofilaments .

• Structural Organization: ZipA organizes FtsZ protofilaments into arrays of long bundles or sheets that likely represent the physiological organization of the FtsZ ring in bacterial cells .

• Membrane Tethering: As a membrane-anchored protein, ZipA helps tether the FtsZ ring to the cytoplasmic membrane, providing structural support for the division apparatus .

• pH-Dependent Assembly Regulation: The effects of ZipA on FtsZ assembly vary with pH. In acidic conditions (pH 6.5), ZipA induces FtsZ to form large bundles, while at neutral pH (7.5), FtsZ-ZipA protofilaments do not bundle but maintain structural integrity .

The MAP-Tau-homologous motifs in ZipA likely mediate its binding to FtsZ, representing an ancient prototype of protein-protein interactions that suppress microtubule catastrophe and/or promote rescue in more complex systems .

What are the storage and handling recommendations for recombinant ZipA?

The recombinant Cell Division Protein ZipA homolog requires specific storage and handling conditions to maintain its structural and functional integrity:

• Storage Temperature: Store at -20°C/-80°C. The shelf life of liquid form is approximately 6 months, while the lyophilized form maintains stability for up to 12 months at these temperatures .

• Reconstitution Protocol: Before opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add 5-50% glycerol (final concentration) and aliquot before storing at -20°C/-80°C .

• Working Conditions: Store working aliquots at 4°C for up to one week. Repeated freezing and thawing is not recommended as it can compromise protein stability and function .

• Quality Assessment: The commercially available recombinant protein typically has a purity of >85% as determined by SDS-PAGE .

What is the relationship between ZipA and the quorum-sensing system in P. luminescens?

The relationship between ZipA and the quorum-sensing system in P. luminescens reveals complex regulatory networks in bacterial cell physiology:

• Quorum-Sensing Control of Cell Division: In P. luminescens, the quorum-sensing autoinducer AI-2 regulates more than 300 targets across diverse cellular compartments and metabolic pathways . While direct regulation of zipA gene expression by AI-2 is not explicitly documented in the available research, AI-2 controls several transcriptional regulators that could potentially influence cell division protein expression, including ZipA .

• Biofilm Formation Connection: AI-2 positively regulates biofilm formation in P. luminescens, with luxS-deficient strains exhibiting decreased biofilm formation . ZipA, as a cell division protein that interacts with the bacterial cytoskeleton, may contribute to the cellular architecture necessary for biofilm development. This suggests a potential indirect relationship between quorum sensing and ZipA function in community structure formation.

• Coordinated Regulation During Host Infection: Both quorum sensing and cell division must be precisely regulated during the infection cycle of P. luminescens in insect hosts. AI-2 is involved mainly in early steps of insect invasion , a phase that likely requires coordinated cell division to establish bacterial populations within the host.

• Metabolic Integration: AI-2 activates glutamate transport and arginine biosynthesis in P. luminescens , potentially providing metabolic precursors necessary for cell growth and division, which would indirectly influence ZipA function in dividing cells.

How does pH affect the interaction between ZipA and FtsZ in experimental systems?

The pH-dependent behavior of ZipA-FtsZ interactions provides critical insights for experimental design and physiological understanding:

• Bundling Behavior: At pH 6.5, ZipA induces FtsZ to form large bundles in vitro, while at pH 7.5, FtsZ-ZipA protofilaments do not bundle but maintain structural associations . This pH-dependent behavior has been observed in studies with Pseudomonas aeruginosa FtsZ (PaFtsZ) and likely extends to P. luminescens ZipA-FtsZ interactions.

• Experimental Implications: This pH sensitivity has significant implications for in vitro assays designed to study ZipA-FtsZ interactions. Researchers must carefully control and report pH conditions when conducting protein-protein interaction studies, polymerization assays, or structural analyses .

• Physiological Relevance: The pH dependency may reflect adaptation to microenvironmental changes during the P. luminescens lifecycle. During insect infection, the bacterium encounters varying pH environments, from the neutral hemolymph to potentially more acidic conditions in damaged tissues or within specialized nematode structures during the symbiotic phase .

• Mechanism of pH Sensitivity: The pH effect likely involves changes in protein surface charge distribution that alter electrostatic interactions between ZipA and FtsZ molecules. At lower pH, protonation of key amino acid residues may facilitate lateral interactions between protofilaments, promoting bundle formation .

What are the best approaches for expressing and purifying recombinant ZipA from P. luminescens?

Successful expression and purification of recombinant ZipA from P. luminescens requires careful consideration of several factors:

• Expression System Selection:

  • E. coli-based expression systems are typically preferred due to their well-established protocols and high yield potential . BL21(DE3) or its derivatives are commonly used for membrane-associated proteins like ZipA.

  • Consider using pET series vectors with inducible T7 promoters for controlled expression.

  • For challenging membrane-associated proteins, specialized E. coli strains like C41(DE3) or C43(DE3) may improve yield and reduce toxicity.

• Solubility Enhancement Strategies:

  • Express ZipA as fusion proteins with solubility-enhancing tags such as MBP (maltose-binding protein), GST (glutathione S-transferase), or SUMO.

  • Consider expressing only the cytoplasmic domain if full-length protein proves difficult, as this domain contains the FtsZ-interacting region .

  • Optimize induction conditions: lower temperature (16-20°C), reduced IPTG concentration, and longer induction times often improve soluble protein yield.

• Purification Protocol:

  • Implement a multi-step purification strategy beginning with affinity chromatography (Ni-NTA for His-tagged constructs).

  • Follow with ion-exchange chromatography to separate charged variants.

  • Complete with size-exclusion chromatography to achieve high purity and remove aggregates.

  • Throughout purification, maintain buffer conditions that stabilize the protein, typically including 50-150 mM NaCl, 20-50 mM Tris-HCl or phosphate buffer, and potentially glycerol or reducing agents.

• Quality Control:

  • Verify purity using SDS-PAGE (>85% is typically acceptable for most applications) .

  • Confirm identity by Western blotting and/or mass spectrometry.

  • Assess functionality through FtsZ binding assays or protofilament formation assays.

What in vitro assays can be used to study ZipA-FtsZ interactions?

Several complementary assays can effectively characterize ZipA-FtsZ interactions:

• Co-sedimentation Assays:

  • Mix purified ZipA and FtsZ in the presence of GTP, incubate to allow polymerization, then centrifuge at high speed (≥100,000 × g).

  • Analyze supernatant and pellet fractions by SDS-PAGE to quantify the amount of ZipA that co-sediments with FtsZ polymers .

  • This assay can determine binding stoichiometry and affinity when performed with varying protein concentrations.

• Light Scattering Assays:

  • Monitor FtsZ polymerization kinetics in real-time by measuring light scattering at 350-400 nm in the presence and absence of ZipA.

  • This approach provides information about the rate and extent of polymer formation and how ZipA affects these parameters.

  • Critical for comparing wild-type and mutant ZipA variants or examining pH-dependent effects .

• Electron Microscopy:

  • Negative staining electron microscopy allows direct visualization of FtsZ protofilaments and how ZipA affects their organization (e.g., bundling, sheet formation) .

  • Sample preparation typically involves adsorption to carbon-coated grids followed by staining with uranyl acetate or phosphotungstic acid.

  • Quantify filament length, width, and morphology to assess ZipA's effects on FtsZ polymer structure.

• Fluorescence-Based Interaction Assays:

  • Fluorescence anisotropy or FRET (Förster Resonance Energy Transfer) with fluorescently labeled proteins can measure direct interactions.

  • These approaches allow determination of binding constants and kinetics under various conditions.

  • Particularly useful for testing how pH, salt concentration, or mutations affect binding .

How can researchers distinguish between direct and indirect effects of ZipA on cellular processes?

Distinguishing direct from indirect effects of ZipA requires multiple complementary approaches:

• Genetic Approaches:

  • Generate conditional zipA mutants or depletion strains in P. luminescens to observe immediate versus delayed phenotypic changes.

  • Immediate effects (within one cell cycle) are more likely to be direct consequences of ZipA function.

  • Complement with wild-type or domain-specific mutant variants to map functional regions.

  • Construct double mutants with known interacting partners (e.g., ftsZ variants) to identify genetic interactions.

• Biochemical Approaches:

  • Perform in vitro reconstitution experiments with purified components to establish direct molecular interactions.

  • Use chemical crosslinking coupled with mass spectrometry to identify direct protein binding partners of ZipA in vivo.

  • Apply proximity-based labeling methods (BioID or APEX) to identify proteins in close proximity to ZipA in living cells.

• Temporal Analysis:

  • Implement time-resolved studies following ZipA depletion or inhibition to establish the sequence of cellular events.

  • Early events (minutes to hours) are more likely direct consequences, while later events (multiple hours to days) may represent indirect effects or compensatory responses.

  • Combine with transcriptomics or proteomics to identify rapid changes in gene or protein expression.

• Domain-Specific Perturbations:

  • Design mutations that specifically disrupt individual ZipA domains or functions.

  • The N-terminal cytoplasmic domain with MAP-Tau-homologous motifs is particularly important for FtsZ interaction .

  • Mutations disrupting ZipA-FtsZ interaction but preserving protein stability can help isolate direct effects on cell division.

What strategies can be used to study ZipA function in the context of P. luminescens pathogenicity?

Investigating ZipA's role in P. luminescens pathogenicity requires integrating molecular, cellular, and infection model approaches:

• Genetic Manipulation Strategies:

  • Generate zipA conditional expression strains using inducible promoters to control protein levels during infection.

  • Create domain-specific mutants that maintain cell viability but alter specific ZipA functions.

  • Develop fluorescently tagged ZipA variants (ensuring tag does not disrupt function) to track localization during infection.

  • Implement CRISPR interference (CRISPRi) for transient, tunable reduction in zipA expression during specific infection phases.

• Insect Infection Models:

  • Compare the virulence of wild-type and zipA-modified P. luminescens strains in susceptible insect larvae (e.g., Spodoptera littoralis) .

  • Measure key virulence parameters including time to death, bacterial load, and insect immune response markers.

  • Assess whether zipA mutants show altered abilities to overcome insect immune defenses, particularly oxidative stress responses that are known to be modulated by quorum sensing .

  • Examine bacterial morphology and division patterns within infected insect tissues using fluorescence microscopy.

• Nematode Symbiosis Analysis:

  • Evaluate the capacity of zipA-modified strains to establish and maintain symbiotic relationships with nematode partners.

  • Assess bacterial colonization efficiency, persistence, and transmission to new insect hosts.

  • Determine if altered cell division affects the bacterium's ability to transition between pathogenic and symbiotic lifestyles .

• Integration with Other Pathogenicity Systems:

  • Investigate potential interactions between ZipA-mediated cell division and the quorum-sensing system, which is known to regulate virulence factors .

  • Examine whether zipA expression or ZipA localization changes in response to AI-2 levels or in luxS-deficient backgrounds.

  • Determine if disruptions in ZipA function affect bioluminescence, which is both a characteristic feature of P. luminescens and regulated in part by spermidine levels controlled by quorum sensing .

How can researchers accurately assess the effects of pH on ZipA-FtsZ interactions in experimental settings?

Accurately assessing pH effects on ZipA-FtsZ interactions requires careful experimental design and multiple complementary approaches:

• Buffer Selection and pH Control:

  • Use buffers with appropriate pKa values for the pH range of interest (e.g., MES for pH 6.0-6.5, PIPES for pH 6.5-7.0, HEPES for pH 7.0-7.5).

  • Ensure buffers don't interfere with protein interactions or GTP hydrolysis.

  • Maintain consistent ionic strength across different pH conditions by adjusting salt concentrations.

  • Verify pH before and after protein addition and at experimental temperature.

• Complementary Assay Approaches:

  • Implement multiple experimental techniques to comprehensively characterize pH effects:

  Technique	Measurement	pH-Related Information
  Light Scattering	Polymerization kinetics	Rate and extent of assembly at different pH
  Electron Microscopy	Polymer morphology	Bundle formation vs. single filaments
  GTPase Activity	FtsZ functional state	How pH affects catalytic activity with/without ZipA
  FRET/Anisotropy	Direct binding	Binding affinity changes with pH
  Co-sedimentation	Complex formation	Quantitative assessment of interaction strength

• Critical Controls and Comparisons:

  • Include FtsZ-only controls at each pH to distinguish ZipA-specific effects from pH effects on FtsZ itself.

  • Test pH effects on ZipA and FtsZ separately to identify individual protein responses.

  • Compare results with known pH-responsive domains from other proteins as positive controls.

  • Include pH-insensitive proteins as negative controls to rule out non-specific effects.

• Physiological Relevance Considerations:

  • Correlate in vitro findings with pH measurements from relevant microenvironments in P. luminescens lifecycle.

  • During insect infection, measure pH in hemolymph and infected tissues.

  • During nematode symbiosis, determine pH in relevant compartments.

  • Design experiments that mimic pH fluctuations rather than static conditions, which may better reflect natural environments.

What are the key considerations for developing and validating ZipA-targeting compounds for antimicrobial applications?

Developing ZipA-targeting antimicrobial compounds requires systematic evaluation of several critical factors:

• Target Validation and Assay Development:

  • Confirm ZipA essentiality in P. luminescens through genetic approaches.

  • Develop robust primary screening assays based on ZipA-FtsZ interactions that are amenable to high-throughput screening.

  • Establish clear criteria for hit identification, including Z' factors >0.5 for statistical reliability.

  • Design orthogonal secondary assays to eliminate false positives and confirm mechanism of action.

• Structure-Based Approaches:

  • Determine the three-dimensional structure of P. luminescens ZipA, particularly the FtsZ-binding domain, through X-ray crystallography or cryo-EM.

  • Identify druggable pockets that, when occupied, would disrupt ZipA-FtsZ interactions.

  • Consider differences between ZipA from P. luminescens and human pathogens to potentially develop broad-spectrum agents.

  • Implement in silico screening to identify initial chemical matter for experimental validation.

• Selectivity and Specificity Evaluation:

  • Test compound effects against human proteins containing MAP-Tau domains to assess potential off-target interactions .

  • Compare efficacy across ZipA homologs from different bacterial species to determine spectrum of activity.

  • Evaluate activity in the presence of biological matrices (serum, tissue homogenates) to assess non-specific binding.

  • Conduct proteomic profiling to identify unintended protein targets.

• Efficacy Validation in Complex Systems:

  • Progress from biochemical to cellular assays, measuring effects on:

    • Bacterial growth (MIC determination)

    • Cell division (microscopy for filamentous phenotypes)

    • FtsZ ring formation (fluorescence microscopy)

    • Biofilm formation (crystal violet staining)

  • Validate in insect infection models, measuring bacterial load reduction and survival improvements.

  • Assess impacts on beneficial microbiota if considering agricultural applications.