Recombinant Photobacterium profundum Cell division protein ZapC (zapC)

What is the genomic context of zapC in Photobacterium profundum?

The zapC gene in Photobacterium profundum is part of the cell division gene cluster. In P. profundum 3TCK, which has been fully sequenced, the genome consists of 11 scaffolds totaling 6,186,725 bp with an average 41.3% GC content encoding 5,549 ORFs. The genome appears to be organized into two chromosomes, similar to other members of the Vibrionaceae family . The zapC gene is conserved across various strains of P. profundum, including both the deep-sea piezopsychrophilic strain SS9 and the shallow-water non-piezophilic strain 3TCK, suggesting its fundamental importance to cell division regardless of pressure adaptation .

How does P. profundum ZapC contribute to bacterial cell division?

Based on studies of ZapC homologs in other bacterial species, P. profundum ZapC likely functions as a Z-ring stabilizer during bacterial cell division. The Z-ring, composed primarily of the tubulin-like protein FtsZ, is essential for bacterial cytokinesis. ZapC directly interacts with FtsZ to promote lateral interactions between FtsZ protofilaments, enhancing Z-ring stability . In the context of P. profundum, which can grow under various pressure conditions, ZapC may play a particularly important role in maintaining proper Z-ring formation under environmental stresses. Experimental evidence from other bacterial systems indicates that ZapC acts as a positive regulator of Z-ring assembly, and its deletion often results in elongated cells due to delayed or defective cell division .

What is the predicted structure and functional domains of P. profundum ZapC?

P. profundum ZapC belongs to the ZapC family of cell division proteins. While the specific crystal structure of P. profundum ZapC has not been reported in the provided research, comparative analysis with ZapC proteins from other bacterial species suggests it likely contains:

• An N-terminal domain that mediates FtsZ binding

• A central core domain with structural elements for protein stability

• Potential C-terminal interaction regions that may interact with other divisome components

The protein likely forms oligomers that enhance its function in Z-ring stabilization. Similar to ZapT described in other bacteria, ZapC may have domains that specifically interact with DNA or other Z-ring associated proteins (Zap proteins) to coordinate chromosome organization with cell division .

What are the optimal conditions for expressing recombinant P. profundum ZapC in E. coli?

For optimal heterologous expression of recombinant P. profundum ZapC in E. coli, the following protocol is recommended:

Expression System:

• Host strain: BL21(DE3) or Rosetta(DE3) for rare codon optimization

• Vector: pET-based expression vectors (pET28a for N-terminal His-tag)

• Induction: 0.5 mM IPTG at OD600 = 0.6-0.8

Culture Conditions:

• Growth temperature: 18-25°C post-induction (lower temperatures reduce inclusion body formation)

• Growth duration: 16-18 hours post-induction

• Media: LB or 2xYT supplemented with appropriate antibiotics

Purification Strategy:

• Cell lysis using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography for final purification

The purified protein should achieve ≥85% purity as determined by SDS-PAGE . For applications requiring higher purity, additional purification steps may be necessary.

How can researchers assess the functional activity of recombinant P. profundum ZapC?

Several complementary approaches can be used to assess the functional activity of recombinant P. profundum ZapC:

In vitro FtsZ Bundling Assay:

• Purify FtsZ from E. coli or P. profundum

• Incubate 5 μM FtsZ with varying concentrations of purified ZapC (0-5 μM)

• Add GTP to initiate FtsZ polymerization

• Monitor polymer formation through 90° angle light scattering at 350 nm

• Visualize FtsZ bundles by negative-stain electron microscopy

FtsZ GTPase Activity Assay:

• Incubate FtsZ with ZapC at various molar ratios

• Measure GTP hydrolysis rates using a malachite green phosphate assay

• Calculate the effect of ZapC on FtsZ's GTPase activity

Complementation Assays:

• Transform zapC deletion strains with plasmids expressing P. profundum ZapC

• Evaluate restoration of normal cell morphology and division

• Quantify cell length distribution and division frequency

Protein-Protein Interaction Studies:

• Use bacterial two-hybrid assays to verify ZapC-FtsZ interaction

• Perform co-immunoprecipitation experiments to identify other interaction partners

• Conduct fluorescence microscopy with tagged proteins to visualize subcellular localization

These assays collectively provide a comprehensive assessment of ZapC functionality in both in vitro and in vivo contexts.

What methodologies are suitable for studying ZapC localization in P. profundum cells?

Studying ZapC localization in P. profundum cells presents unique challenges due to the organism's adaptation to various pressure environments. The following methodologies are recommended:

Fluorescent Protein Fusion Approaches:

• Construct C-terminal or N-terminal fluorescent protein fusions (GFP, mCherry) with ZapC

• Verify fusion protein functionality through complementation assays

• Express the fusion protein from native promoter for physiological expression levels

• Use high-resolution fluorescence microscopy to visualize localization patterns

• For pressure-adapted studies, utilize pressure-resistant fluorescent proteins

Immunofluorescence Microscopy:

• Generate specific antibodies against P. profundum ZapC

• Fix P. profundum cells while preserving cellular architecture

• Permeabilize cells and perform immunostaining with anti-ZapC antibodies

• Use fluorophore-conjugated secondary antibodies for visualization

• Perform co-staining with DNA and FtsZ to correlate localization with cell cycle stages

Pressure-Adapted Visualization Systems:

• Employ specialized high-pressure microscopy chambers for live-cell imaging

• Compare ZapC localization patterns under various pressure conditions (atmospheric vs. high pressure)

• Analyze dynamics using time-lapse microscopy where possible

Transmission Electron Microscopy with Immunogold Labeling:

• Process P. profundum cells for electron microscopy

• Perform immunogold labeling using anti-ZapC antibodies

• Analyze ultrathin sections to determine precise subcellular localization at nanometer resolution

These approaches should be optimized for P. profundum's specific growth requirements, including salinity, temperature, and pressure conditions as relevant to the experimental questions being addressed.

How does P. profundum ZapC compare structurally and functionally with ZapC homologs from other bacterial species?

P. profundum ZapC shares conserved features with ZapC homologs from other bacterial species while exhibiting adaptations potentially related to its marine and pressure-varied environment:

Functionally, all ZapC homologs appear to stabilize the Z-ring during cell division, but P. profundum ZapC may have unique adaptations for function under various pressure conditions. The Z-ring binding and stabilization mechanism is likely conserved, though the regulation of ZapC activity might differ between species to accommodate their specific environmental niches .

What insights can be gained from studying ZapC in the context of pressure adaptation in P. profundum?

Studying ZapC in the context of pressure adaptation in P. profundum offers unique insights into how fundamental cell division processes adapt to extreme environments:

• Structural Adaptations: Comparing ZapC from piezophilic (pressure-loving) strain SS9 with non-piezophilic strain 3TCK may reveal amino acid substitutions that enhance protein stability and function under pressure . These adaptations could involve changes in hydrophobic core packing, surface charge distribution, or flexibility of key functional regions.

• Expression Regulation: Pressure may influence zapC expression levels or timing during the cell cycle. Transcriptomic analysis under different pressure conditions could reveal pressure-responsive elements in the zapC promoter region.

• Protein-Protein Interaction Dynamics: High pressure can alter protein-protein interaction affinities. Studies comparing ZapC-FtsZ interactions under various pressure conditions might reveal pressure-adapted interaction interfaces.

• Cell Division Kinetics: Different P. profundum strains show remarkable differences in physiological responses to pressure . Comparing the role of ZapC in cell division timing and efficiency between strains could illuminate adaptation mechanisms.

• Evolutionary Conservation: Genomic comparison between P. profundum strains reveals variations that correlate to environmental differences and define the Hutchinsonian niche of each strain . Analyzing selection pressure on zapC across strains can identify critical adaptive features.

These studies would contribute not only to understanding P. profundum biology but also to broader questions about protein evolution and adaptation to extreme environments.

How do Z-ring associated proteins interact with ZapC to regulate bacterial cell division?

Z-ring associated proteins (Zap proteins) form a complex network of interactions that collectively regulate FtsZ assembly and Z-ring dynamics. Based on studies in model organisms and the limited information available for P. profundum, the following interactions are likely relevant:

Direct Interactions with ZapC:

• FtsZ-ZapC: Primary interaction where ZapC directly binds FtsZ protofilaments to promote lateral interactions and bundling.

• ZapC-ZapC: Self-interaction forming dimers or higher-order oligomers that enhance Z-ring stabilization.

• ZapC-ZapA: Potential cooperative interaction that enhances Z-ring stability, similar to the ZapA-ZauP-ZapT complex described in C. crescentus .

Functional Interaction Network:

ZapC functions within a broader network of Z-ring regulators that includes both positive and negative regulators:

In C. crescentus, the ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring . Similar mechanisms may exist in P. profundum, with ZapC potentially participating in complexes that coordinate chromosome organization with cell division.

How can CRISPR-Cas9 gene editing be applied to study zapC function in P. profundum?

CRISPR-Cas9 gene editing offers powerful approaches for investigating zapC function in P. profundum through targeted genetic modifications:

Knockout Strategy:

• Design guide RNAs targeting the zapC gene with minimal off-target effects

• Construct a CRISPR-Cas9 system compatible with P. profundum (consider codon optimization)

• Include homology-directed repair templates to replace zapC with an antibiotic resistance marker

• Transform P. profundum using electroporation optimized for marine bacteria

• Select transformants and verify gene disruption by sequencing

• Analyze phenotypic consequences on cell morphology, division, and pressure adaptation

Tagged Variant Generation:

• Design repair templates containing zapC fused to epitope tags or fluorescent proteins

• Include homology arms flanking the modification site

• Co-transform with CRISPR components targeting the C-terminus of zapC

• Select and verify correct integration

• Use tagged variants for localization studies and protein interaction analyses

Point Mutation Analysis:

• Identify conserved residues likely critical for ZapC function through comparative sequence analysis

• Generate repair templates with specific codon changes

• Introduce mutations and assess functional consequences

• Create a panel of mutations affecting different predicted functional domains

Pressure-Responsive Promoter Replacement:

• Design CRISPR strategy to replace the native zapC promoter

• Introduce conditional or inducible promoters

• Study zapC expression under different pressure conditions

• Analyze consequences of altered expression timing or levels

This approach would need to be optimized for P. profundum's specific genetic characteristics and transformation efficiency. The pressure-adapted characteristics of different P. profundum strains (SS9 vs. 3TCK) provide an excellent comparative system for understanding ZapC function under different environmental conditions .

What are the methodological challenges in resolving protein-protein interaction networks involving ZapC under high-pressure conditions?

Studying protein-protein interactions involving ZapC under high-pressure conditions presents several methodological challenges that require specialized approaches:

In vitro Interaction Analysis Challenges:

• Pressure-Compatible Biophysical Instruments: Standard protein interaction analysis tools (SPR, ITC, MST) must be modified for high-pressure compatibility.

• Maintaining Protein Stability: Proteins may denature or aggregate under pressure, requiring careful buffer optimization.

• Real-Time Measurements: Capturing dynamic interactions while maintaining pressure conditions requires specialized equipment.

Recommended Approaches:

• High-Pressure NMR Spectroscopy:

  • Use pressure-resistant NMR tubes and specialized equipment

  • Label proteins with 15N/13C for structural studies under pressure

  • Monitor chemical shift perturbations to map interaction interfaces

• Fluorescence-Based Interaction Assays:

  • Employ FRET pairs with pressure-stable fluorophores

  • Utilize pressure-resistant cuvettes and modified spectrofluorimeters

  • Measure interactions at incrementally increasing pressures

• Crosslinking Mass Spectrometry:

  • Perform protein crosslinking under pressure conditions

  • Release pressure before sample processing

  • Identify interaction sites through mass spectrometry analysis of crosslinked peptides

In vivo Interaction Analysis Challenges:

• Pressure Chamber Compatibility: Microscopy and other cell-based assays must function within pressure chambers.

• Cell Viability: Maintaining cell health during extended high-pressure experiments.

• Signal Detection: Reduced signal strength through pressure chamber windows.

Recommended Approaches:

• Split Fluorescent Protein Complementation:

  • Design ZapC fusions with split fluorescent protein fragments

  • Monitor interaction-dependent fluorescence recovery under pressure

  • Use pressure-resistant microscopy setups

• Modified Bacterial Two-Hybrid Systems:

  • Adapt bacterial two-hybrid assays to function in pressure-tolerant strains

  • Develop reporter systems stable under pressure conditions

  • Compare interaction patterns across pressure gradients

• In situ Proximity Ligation Assays:

  • Fix cells under pressure before performing the assay

  • Detect proximity of interacting proteins through antibody-based detection

  • Quantify interaction frequency under different pressure conditions

These approaches would provide complementary data on how pressure affects ZapC interactions within the cell division machinery of P. profundum.

How can structural biology approaches be utilized to understand pressure adaptation mechanisms in P. profundum ZapC?

Structural biology offers powerful tools for understanding how P. profundum ZapC has adapted to function under varying pressure conditions:

X-ray Crystallography Under Pressure:

• Purify recombinant P. profundum ZapC to high homogeneity (>95%)

• Perform crystallization screening to identify optimal conditions

• Utilize specialized pressure cells for crystallization under various pressures

• Collect diffraction data and solve structures at atmospheric and elevated pressures

• Compare structural elements, focusing on cavities, ion pairs, and hydration layers that may change under pressure

High-Pressure NMR Spectroscopy:

• Produce 15N/13C-labeled ZapC for NMR studies

• Collect spectra at varying pressures (1-1000 bar)

• Track chemical shift perturbations to identify pressure-sensitive regions

• Model conformational changes induced by pressure

• Identify structural elements that maintain function under pressure

Comparative Molecular Dynamics Simulations:

• Generate molecular models of ZapC from both piezophilic (SS9) and non-piezophilic (3TCK) strains

• Perform extended MD simulations under varying pressure conditions

• Analyze protein flexibility, water penetration, and structural stability

• Identify pressure-adaptive mutations by comparing simulations

• Calculate free energy landscapes to understand pressure effects on conformational equilibria

These structural approaches, combined with functional assays, would provide mechanistic insights into how ZapC maintains its cell division functions across the range of pressures encountered by different P. profundum strains in their natural habitats .

What evolutionary adaptations are observed in ZapC proteins from bacteria living in different pressure environments?

Evolutionary adaptations in ZapC proteins from bacteria inhabiting different pressure environments reveal important mechanisms for maintaining protein function under extreme conditions:

Amino Acid Composition Shifts:
Piezophilic (pressure-adapted) ZapC variants typically show:

• Increased proportion of small amino acids (Ala, Gly) that provide conformational flexibility

• Reduced frequency of bulky hydrophobic residues to decrease internal cavities

• Enhanced charged residue content (especially negatively charged) that maintains hydration under pressure

• Strategic placement of proline residues that restrict conformational changes induced by pressure

Structural Adaptations:

• Reduced internal void volumes that would collapse under pressure

• Enhanced electrostatic interactions (salt bridges) to stabilize tertiary structure

• Increased surface hydrophilicity to maintain hydration layer

• Modified flexibility in functional regions to preserve activity under pressure

Comparative analysis between ZapC from P. profundum strains SS9 (deep-sea, piezophilic) and 3TCK (shallow-water, non-piezophilic) would likely reveal specific substitutions responsible for pressure adaptation . These adaptations reflect evolutionary responses to the selection pressures of their respective ecological niches.

How does ZapC contribute to maintaining genomic stability during bacterial cell division?

ZapC contributes to genomic stability during bacterial cell division through several mechanisms that ensure proper chromosome segregation and cell division timing:

Z-ring Stabilization and Positioning:

• By promoting proper Z-ring formation and stability, ZapC ensures that cell division occurs at the correct cellular location (midcell)

• This precise positioning is critical for equal distribution of chromosomal DNA to daughter cells

Coordination with Chromosome Organization:
Studies of Z-ring associated proteins in C. crescentus indicate that these proteins help organize the chromosome terminus into a compact structure at midcell through interactions with DNA-binding proteins . While direct evidence for P. profundum ZapC is limited, it likely participates in similar mechanisms:

• Potential interaction with terminus-binding proteins analogous to ZapT

• Formation of higher-order complexes that physically link the divisome to the chromosome

• Contribution to the timing mechanism that ensures chromosome replication completion before cell division initiation

Prevention of Premature Division:

• ZapC's role in Z-ring dynamics may contribute to checkpoint mechanisms that prevent premature constriction

• This temporal regulation ensures complete chromosome segregation before cytokinesis

Response to Environmental Stresses:
In P. profundum, which experiences varying pressures in its marine environment, ZapC may be particularly important for maintaining genomic stability under stress conditions:

• Ensuring proper division under pressure challenges

• Potentially responding to pressure-induced changes in nucleoid organization

• Maintaining division site selection despite physical stresses on cellular structures

These functions collectively contribute to the high fidelity of chromosome transmission during bacterial cell division, essential for maintaining genomic stability across generations.

What is the relationship between ZapC function and adaptation to the deep-sea environment in P. profundum?

The relationship between ZapC function and adaptation to the deep-sea environment in P. profundum represents a fascinating example of how fundamental cellular processes adjust to extreme conditions:

Pressure-Adapted Cell Division:
Deep-sea environments subject bacteria to hydrostatic pressures up to 1,000 atmospheres, which can significantly impact protein structure and interactions. P. profundum strain SS9, isolated from deep ocean sediments, displays remarkable adaptation to high pressure conditions compared to shallow-water strain 3TCK . ZapC's role in cell division likely includes:

• Maintaining proper Z-ring formation under pressure conditions that might otherwise destabilize FtsZ polymers

• Compensating for pressure-induced changes in cytoskeletal protein dynamics

• Ensuring divisome assembly proceeds efficiently despite physical constraints

Cold Adaptation Synergy:
Deep-sea environments are also characterized by constant cold temperatures (2-4°C), which affects:

• Protein folding kinetics and stability

• Membrane fluidity relevant to divisome assembly

• Enzymatic reaction rates in division processes

ZapC from deep-sea P. profundum strains likely exhibits dual adaptation to both pressure and cold, with structural features that maintain flexibility and function under these combined stresses.

Genomic Evidence of Adaptation:
Comparative genome analysis between P. profundum strains reveals "remarkable differences in their physiological responses to pressure" . These differences likely extend to cell division proteins including ZapC, with specific genetic variations that:

• Alter amino acid composition to maintain structural integrity under pressure

• Modify regulatory elements controlling zapC expression under different conditions

• Adjust interaction interfaces with partner proteins to maintain functional complexes under pressure

Ecological Significance:
ZapC's adaptation in deep-sea P. profundum reflects the broader ecological strategy of this organism:

• Enabling colonization of deep-sea niches with few competitors

• Supporting growth and reproduction under constant high-pressure conditions

• Contributing to the remarkable ability of P. profundum to thrive across a wide range of depths and pressures

This adaptation represents an excellent model for studying how essential cellular processes evolve to function in extreme environments, with potential applications in biotechnology and astrobiology.

What are the most promising research directions for understanding ZapC function in P. profundum?

The most promising research directions for understanding ZapC function in P. profundum include:

• Comparative Functional Genomics: Systematic comparison of zapC gene sequences, expression patterns, and phenotypic consequences between piezophilic (SS9) and non-piezophilic (3TCK) strains to identify pressure-adaptive features .

• High-Resolution Structural Studies: Determination of ZapC structure under varying pressure conditions to elucidate molecular mechanisms of pressure adaptation.

• Protein Interaction Networks: Comprehensive mapping of ZapC interaction partners across different pressure conditions to understand how the divisome network adapts to environmental challenges.

• In vivo Dynamics: Development of advanced imaging techniques to visualize ZapC localization and dynamics during cell division under native pressure conditions.

• Synthetic Biology Approaches: Creation of chimeric ZapC proteins combining domains from pressure-adapted and non-adapted strains to identify critical regions for pressure tolerance.

• Systems Biology Integration: Computational modeling of how ZapC contributes to the entire cell division process across varying pressure conditions.

• Evolutionary Studies: Broad phylogenetic analysis of ZapC across marine bacteria from different depths to trace the evolutionary history of pressure adaptation.

These approaches would significantly advance our understanding of bacterial adaptation to extreme environments while providing insights into fundamental aspects of bacterial cell division.

How might insights from P. profundum ZapC research be applied in biotechnology and synthetic biology?

Insights from P. profundum ZapC research have several potential applications in biotechnology and synthetic biology:

Pressure-Stable Protein Engineering:

• Identifying structural elements that confer pressure stability could inform the design of pressure-resistant enzymes for industrial bioprocessing

• Development of pressure-stable protein scaffolds for biotechnological applications

• Creation of biosensors that function reliably under varying pressure conditions

Extremozyme Development:

• Engineering pressure-adapted variants of industrially relevant enzymes using design principles from ZapC

• Development of biocatalysts that function efficiently in high-pressure bioprocessing

• Creation of cold-adapted, pressure-stable enzyme variants for food processing applications

Synthetic Cell Division Control:

• Utilizing modified ZapC proteins as modulators of bacterial cell size and division timing

• Engineering growth rate controls for biotechnology applications

• Development of synthetic bacterial chassis with enhanced growth characteristics under specific conditions

Biomaterials Development:

• Leveraging insights from pressure-adapted protein structures to design novel biomaterials

• Creating self-assembling protein structures based on ZapC oligomerization properties

• Developing pressure-responsive biomaterials for specialized applications

Deep-Sea Bioprospecting Tools:

• Using knowledge of pressure adaptation to develop improved cultivation methods for currently uncultivable deep-sea microorganisms

• Creating genetic tools optimized for genetic manipulation of piezophilic bacteria

• Developing sampling technologies that maintain native pressure during collection and processing