Recombinant Photobacterium profundum Chromosomal replication initiator protein DnaA (dnaA)

What is Photobacterium profundum and why is it significant for studying DnaA?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It has significant research value as a model organism for studying adaptations to extreme environments, particularly high-pressure conditions. The most studied strain, P. profundum SS9, was isolated from the Sulu Sea and exhibits optimal growth at 15°C and 28 MPa (approximately 280 atmospheres), making it both a psychrophile and a piezophile .

P. profundum is particularly valuable for studying DnaA because its initiation of DNA replication is significantly affected by pressure, with DnaA-related proteins playing crucial roles in high-pressure adaptation. Studies have shown that mutations in DnaA-associated proteins like DiaA (positive regulator) and SeqA (negative regulator) directly affect the organism's ability to grow under high-pressure conditions .

How does the DnaA protein function in bacterial DNA replication initiation?

DnaA protein serves as the master regulator of bacterial DNA replication initiation through a multi-step process:

• DnaA binds to specific "DnaA-boxes" in the origin of replication (oriC)

• This binding facilitates strand opening at an AT-rich "DNA unwinding element" (DUE) region

• The opened DNA allows for replisome assembly and subsequent bidirectional replication

The E. coli DnaA protein contains four functional domains:

• Domain I: N-terminal protein interaction domain

• Domain II: A linker domain

• Domain III: AAA+ ATPase domain responsible for ATP/ADP binding and ATP hydrolysis

• Domain IV: Double-stranded DNA binding domain

What are the optimal methods for culturing P. profundum under different pressure conditions?

For successful cultivation of P. profundum strains, particularly for pressure-related experiments:

Standard Culture Conditions:

• Medium: 2216 marine medium (Difco) supplemented with 20 mM glucose and 10 mM HEPES (pH 7.5)

• Temperature: 15°C for most experiments with SS9 strain

• Pressure conditions:

  • Low pressure (0.1 MPa/atmospheric pressure)

  • High pressure (45 MPa for most experiments)

  • Can be cultured at pressures up to 70 MPa depending on strain

Pressure Application Methods:

• Custom-designed stainless steel pressure vessels (e.g., 2-liter vessel from Autoclave Engineers)

• Pressurization using water and a hydraulic pump

• For microtiter plate experiments, seal plates with PCR Axymat, ensuring no air bubbles are trapped

Strain Selection:

• SS9R (rifampin-resistant derivative) is commonly used for genetic manipulation studies

• Different strains have different optimal conditions:

  • SS9: optimal at 15°C and 28 MPa

  • 3TCK: optimal at 9°C and 0.1 MPa

  • DSJ4: optimal at 10°C and 10 MPa

How can genomic DNA be efficiently extracted from P. profundum for DnaA gene cloning?

Two effective methods for extracting high-quality genomic DNA from P. profundum are:

Method 1: EDTA-Lysozyme-Proteinase K Method

• Harvest approximately 1 liter of mid-exponential culture by centrifugation (15 minutes at 5,000×g)

• Resuspend pellet in 5 ml buffer A (50 mM Tris, 50 mM EDTA, pH 8.0)

• Incubate suspension overnight at -20°C

• Thaw at room temperature and add 500 µl buffer B (250 mM Tris, pH 8.0, 10 mg/ml lysozyme)

• Incubate on ice for 45 minutes

• Add 1 ml buffer C (0.5% SDS, 50 mM Tris, 400 mM EDTA, pH 7.5, 1 mg/ml Proteinase K)

• Incubate at 50°C for 60 minutes

• Add additional 750 µl buffer C and incubate at 50°C for 30 minutes

• Extract twice with 5 ml phenol:chloroform:isoamyl alcohol (24:24:1)

• Precipitate with 0.8 volumes of isopropanol

Method 2: Commercial Kit Method

• Harvest 3 ml of 48-hour-old P. profundum cultures

• Extract genomic DNA using a Wizard genomic kit (Promega)

The extracted DNA is suitable for PCR amplification, cloning, and can be used for transposon mutagenesis studies of the DnaA gene and associated factors.

What are the effective strategies for cloning and expressing the P. profundum dnaA gene?

Cloning Strategies:

• PCR-Based Cloning:

  • Design primers based on the P. profundum dnaA sequence

  • Suggested primers should include restriction sites compatible with expression vectors

  • Use high-fidelity polymerase (e.g., Expand Long Template PCR system) for amplification

  • Thermal cycling conditions: 92-95°C for 1 min, 48-52°C for 30-60 sec, 72°C for 1 min per kb, 25-30 cycles

• Genomic Library Screening:

  • Create a genomic library in a vector like pUC18 or broad-host-range plasmids

  • Screen using dnaA-specific probes or by complementation in E. coli dnaA mutants

Expression Systems:

• E. coli Expression:

  • Clone into pET29a with a C-terminal hexa-histidine tag

  • Transform into E. coli expression strains (BL21 or derivatives)

  • Expression can be verified by Western blotting

• Functional Complementation:

  • The P. profundum dnaA gene can be tested for function by complementing E. coli dnaA temperature-sensitive mutants

  • Use broad-host-range plasmids like pFL190 with appropriate promoters (e.g., arabinose-inducible)

Verification Methods:

• Flow cytometry with DNA binding dyes (e.g., SYTOX Green) to assess DNA replication patterns

• Western blotting to verify protein expression

• Functional assays to confirm DnaA activity

How do DiaA and SeqA proteins interact with DnaA to regulate DNA replication under high pressure?

Research on P. profundum reveals a sophisticated regulatory network involving DnaA, DiaA, and SeqA that is particularly important under high-pressure conditions:

DiaA (Positive Regulator):

• The PBPRA3229 gene in P. profundum encodes a DiaA homolog with 75% identity to E. coli DiaA

• Functions as a positive regulator of DNA replication initiation

• Disruption of this gene in P. profundum (strain FL23) results in high-pressure sensitivity

• The ratio of growth at 45 MPa to growth at 0.1 MPa is substantially reduced in the DiaA mutant

• DiaA forms tetramers and facilitates:

  • DnaA binding to the origin of replication (oriC)

  • ATP-DnaA-specific conformational changes

  • Unwinding of oriC DNA

SeqA (Negative Regulator):

• The PBPRA1039 gene in P. profundum encodes a SeqA homolog

• Functions as a negative regulator of DNA replication initiation

• In E. coli, SeqA binds to hemimethylated oriC and prevents binding of ATP-DnaA

• Disruption of this gene in P. profundum (strain FL28) results in enhanced growth at high pressure

• This suggests that removing a replication inhibitor enhances high-pressure growth

Regulatory Model Under High Pressure:

• High pressure appears to negatively affect the initiation of DNA replication in P. profundum

• Either the presence of the positive regulator (DiaA) or the removal of the negative regulator (SeqA) promotes growth under high-pressure conditions

• The effect of DiaA on timing of DNA replication initiation appears to be SeqA-independent

This research indicates that the regulation of DNA replication initiation is particularly sensitive to pressure, and P. profundum has evolved specific adaptations in these regulatory pathways to maintain proper replication under deep-sea conditions.

What approaches can be used to study the pressure-adaptive features of recombinant P. profundum DnaA?

Several sophisticated approaches can be employed to investigate the unique pressure-adaptive features of P. profundum DnaA:

Comparative Structural Analysis:

• Express recombinant DnaA proteins from P. profundum and mesophilic bacteria

• Use X-ray crystallography or cryo-electron microscopy to determine protein structures

• Compare structures at different pressures using high-pressure X-ray crystallography

• Analyze domains that might contribute to pressure adaptation, particularly focusing on protein hydration and conformational states

Biophysical Characterization:

• Pressure-perturbation studies to examine:

  • Changes in protein hydration

  • Ligand binding affinities

  • Spin transitions

• Pressure-temperature phase diagrams to determine stability ranges

• Thermal and pressure denaturation profiles using circular dichroism or fluorescence spectroscopy

Functional Assays Under Pressure:

• ATP binding and hydrolysis assays at varying pressures

• DNA binding assays to determine pressure effects on DnaA-DNA interactions

• Helicase loading and activation studies at different pressures

• In vitro replication initiation assays under pressure

Genetic Approaches:

• Create chimeric DnaA proteins with domains from piezophilic and mesophilic organisms

• Site-directed mutagenesis targeting residues predicted to be involved in pressure adaptation

• Suppressor screens to identify compensatory mutations

• Flow cytometry and microscopy to examine replication patterns in vivo under pressure

These approaches would provide insights into how P. profundum DnaA has adapted to function optimally in the deep-sea environment with high hydrostatic pressure.

How can functional complementation assays be designed to test P. profundum DnaA activity?

Functional complementation assays provide powerful tools for assessing P. profundum DnaA activity:

E. coli dnaA Mutant Complementation:

• Using Temperature-Sensitive Mutants:

  • Transform E. coli dnaA(Cs) temperature-sensitive mutants with P. profundum dnaA constructs

  • Assess growth restoration at non-permissive temperatures

  • Analyze the effect of P. profundum DnaA on the cold sensitivity phenotype of E. coli dnaA(Cs) mutants

• Flow Cytometry Analysis:

  • Transform E. coli dnaA mutants with P. profundum dnaA under inducible promoters

  • Stain cells with DNA binding dyes like SYTOX Green

  • Analyze chromosome copy number and replication synchrony

  • Compare fluorescence intensity patterns between:

    • Wild-type E. coli (typically showing 2, 4, and 8 chromosome copies)

    • dnaA mutants (showing asynchronous replication with odd chromosome numbers)

    • Complemented strains (should restore normal patterns if functional)

Replication Initiator Screening System:

• Use a dual reporter system like that described for E. coli:

  • A mini-chromosome (e.g., pRNK6) carrying a repressor gene

  • A chromosomal fluorescent reporter (e.g., GFPmut2) under repressor control

• When functional DnaA is present, the mini-chromosome is maintained and reporter expression is repressed

• Loss of DnaA function leads to mini-chromosome loss and reporter expression

• Measure relative fluorescence to quantify DnaA activity

Combined Genetic Approach:

• Construct strains with combinations of mutations in dnaA, diaA, and seqA

• Test the ability of P. profundum genes to restore normal growth and replication patterns

• This approach can reveal interactions between these regulatory components

Controls and Validation:

• Include both positive controls (E. coli dnaA) and negative controls (empty vector)

• Verify protein expression by Western blotting

• Use multiple E. coli genetic backgrounds to ensure consistent results

• Test complementation under different temperature and pressure conditions

These complementation assays have successfully demonstrated that P. profundum DnaA-related proteins are functional and can restore proper replication patterns in E. coli mutants, suggesting conserved mechanisms despite adaptation to high-pressure environments.

What are the common challenges in expressing recombinant P. profundum DnaA and how can they be addressed?

Expressing recombinant proteins from extremophilic organisms like P. profundum presents several challenges:

Challenge 1: Codon Usage Bias

• P. profundum, as a deep-sea bacterium, may have codon preferences different from common expression hosts

• Solution:

  • Use codon-optimized synthetic genes

  • Express in Rosetta or CodonPlus E. coli strains that supply rare tRNAs

  • Consider expression in P. profundum itself using compatible expression vectors

Challenge 2: Protein Solubility

• Proteins adapted to high pressure may fold incorrectly at atmospheric pressure

• Solution:

  • Use solubility enhancing tags (MBP, SUMO, thioredoxin)

  • Express at lower temperatures (15-20°C)

  • Include osmolytes or pressure-mimicking agents in buffer systems

  • Consider high-pressure expression systems if available

Challenge 3: Protein Stability

• DnaA proteins involve multiple domains with complex interactions

• Solution:

  • Include stabilizing agents like glycerol or specific ions in purification buffers

  • Optimize purification to minimize time at atmospheric pressure

  • Consider expressing functional domains separately

  • Use site-directed mutagenesis to enhance stability without affecting function

Challenge 4: Host Toxicity

• Overexpression of DnaA can disrupt host cell replication

• Solution:

  • Use tightly regulated expression systems (e.g., arabinose-inducible promoters)

  • Express in specialized strains with replication controls

  • Consider cell-free expression systems for toxic constructs

Challenge 5: Verification of Proper Folding

• Ensuring the recombinant protein adopts the correct conformation

• Solution:

  • Perform activity assays (ATP binding/hydrolysis)

  • Compare circular dichroism spectra with known DnaA proteins

  • Test functional complementation in suitable genetic backgrounds

These strategies can help overcome the unique challenges associated with expressing and purifying functional recombinant DnaA from a high-pressure-adapted organism.

How can researchers optimize PCR amplification of the dnaA gene from P. profundum genomic DNA?

Optimizing PCR amplification of the dnaA gene from P. profundum requires addressing several technical considerations:

Template Preparation:

• Extract high-quality genomic DNA using either:

  • The EDTA-Lysozyme-Proteinase K method described in search result

  • Commercial kits like Wizard genomic kit (Promega) as used in search result

• Measure DNA concentration and purity (A260/A280 ratio)

• Perform a gel electrophoresis to verify DNA integrity

• Use 10-100 ng of high-quality genomic DNA per reaction

Primer Design Considerations:

• Design primers based on the P. profundum genome sequence

• Include appropriate restriction sites for subsequent cloning

• Optimal primers should:

  • Be 18-30 nucleotides in length

  • Have GC content between 40-60%

  • End with G or C bases (GC clamp)

  • Have melting temperatures within 5°C of each other

• For challenging regions, consider adding GC clamps or using nested PCR approach

PCR Reaction Optimization:

• Use high-fidelity polymerases like Expand Long Template PCR system (Roche)

• Optimize Mg²⁺ concentration (typically 1.5-3 mM)

• Test different DMSO concentrations (2-10%) to reduce secondary structure

• Try various annealing temperatures (gradient PCR)

• Consider touchdown PCR protocols for difficult templates

Recommended PCR Conditions:

• Initial denaturation: 95°C for 5 minutes

• Cycling conditions:

  • Denaturation: 94-95°C for 30 seconds

  • Annealing: 48-52.5°C for 30 seconds

  • Extension: 72°C for 1 minute per kb

  • 30 cycles

• Final extension: 72°C for 7 minutes

Troubleshooting Strategies:

• For difficult templates, try nested PCR approaches as described in arbitrary PCR protocols

• For templates with high GC content, add DMSO or betaine

• For non-specific amplification, increase annealing temperature and reduce cycle number

• For no amplification, check template quality and try new primer pairs

These optimized PCR conditions have been successfully used to amplify genes from P. profundum for subsequent cloning and functional studies.

How can the study of P. profundum DnaA contribute to understanding bacterial adaptation to extreme environments?

Research on P. profundum DnaA provides unique insights into bacterial adaptation to extreme environments:

Pressure-Adaptive Mechanisms:

• DnaA and its regulatory proteins (DiaA, SeqA) show significant adaptation to high-pressure environments

• Mutations in these proteins directly affect high-pressure growth capabilities

• Understanding these adaptations reveals how essential cellular processes like DNA replication can be modified to function in extreme conditions

Comparative Genomics Applications:

• Comparison of DnaA sequences between piezophilic (e.g., P. profundum SS9) and piezosensitive (e.g., P. profundum 3TCK) strains

• Analysis of selection pressures on different domains of the protein

• Identification of key residues that confer pressure adaptation

• These comparisons help identify convergent evolution patterns across deep-sea bacteria

Evolutionary Implications:

• P. profundum strains show different degrees of adaptation to high pressure

• Closely related strains (SS9 and 3TCK) have 97% identical putative P450 proteins but very different pressure optima

• This suggests that relatively few genetic changes can facilitate adaptation to extreme conditions

• Similar principles may apply to DnaA and its regulatory proteins

Broader Applications:

• Insights gained can be applied to other extremophiles (thermophiles, acidophiles, etc.)

• Understanding how essential proteins function under extreme conditions informs synthetic biology approaches

• Identification of common strategies for protein adaptation to physical stress

• Development of stress-resistant organisms for biotechnological applications

The study of P. profundum DnaA thus serves as a model for understanding how fundamental biological processes can be maintained in extreme environments through protein adaptation.

What potential biotechnological applications exist for recombinant P. profundum DnaA?

Recombinant P. profundum DnaA offers several promising biotechnological applications:

Pressure-Stable Protein Engineering:

• The pressure-adaptive features of P. profundum DnaA can serve as a model for engineering pressure-resistant proteins

• Domains or motifs conferring pressure stability could be incorporated into other proteins

• This would be valuable for developing biocatalysts for high-pressure industrial processes

Novel Antimicrobial Development:

• DnaA is essential for bacterial replication and represents a potential antibiotic target

• Studies with anti-DnaA peptide nucleic acids (PNAs) have shown efficacy in inhibiting bacterial growth

• P. profundum DnaA could be used to screen for compounds effective against atypical bacteria

• Understanding differences between DnaA proteins could lead to species-specific inhibitors

High-Pressure Biotechnology Tools:

• Recombinant DnaA proteins adapted to high pressure could be used in high-pressure PCR and other molecular biology techniques

• Potential applications in high-pressure enzyme catalysis and bioreactors

• Development of pressure-resistant expression systems for industrial enzyme production

Bionanotechnology Applications:

• DnaA's ability to bind specific DNA sequences and initiate controlled unwinding could be harnessed for DNA nanotechnology

• Pressure-resistant variants could offer unique capabilities for controlled assembly/disassembly of DNA nanostructures

• Potential applications in biosensing and molecular machines

Synthetic Biology Chassis Development:

• Understanding DnaA function in P. profundum could facilitate the development of synthetic biology platforms functioning under high pressure

• Such platforms would be valuable for deep-sea bioremediation and resource recovery

• Could enable in situ biotechnology applications in deep ocean environments

These applications leverage the unique adaptations of P. profundum DnaA to extreme environments, potentially opening new avenues for biotechnology in previously inaccessible conditions.

How can genetic modification techniques be optimized for studying P. profundum DnaA in its native context?

Studying P. profundum DnaA in its native context requires specialized genetic modification techniques adapted for this piezophilic organism:

Optimized Conjugation Protocols:

• Triparental mating using:

  • E. coli donor strain (e.g., BW20767 containing donor plasmid)

  • Helper strain (e.g., E. coli with pRK2073)

  • P. profundum recipient (typically strain SS9R, a rifampin-resistant derivative)

• Conjugation procedure:

  • Harvest donor, helper, and recipient strains by centrifugation

  • Resuspend cells in 2216 medium

  • Spot 100 μl of mixture onto 0.4-μm-pore-size polycarbonate membrane filters

  • Incubate at room temperature for 12-16 hours

  • Wash cells from filters and plate on selective medium

  • Incubate plates at 15°C for 3-5 days

Transposon Mutagenesis Systems:

• Mini-Tn5 shows more random insertion than mini-Tn10 (which exhibits hotspots)

• Successful systems include:

  • Mini-Tn10 delivery for initial studies

  • Mini-Tn5 using pRL27 donor plasmid for more comprehensive mutagenesis

• Screening for pressure-related phenotypes using replica plating at different pressures (0.1 MPa vs. 45 MPa)

• Calculate pressure sensitivity ratio (growth at 45 MPa / growth at 0.1 MPa) to identify mutants

Gene Deletion and Replacement Strategies:

• Suicide plasmid-based methods:

  • Construct deletion using PCR to amplify flanking regions

  • Clone into suicide vectors (e.g., pRL271) with selectable markers

  • Add counter-selectable markers (e.g., sacB) for second recombination

  • Select for double crossover events using sucrose resistance

• Specific example for flagellin gene deletion:

  • Amplify upstream and downstream regions

  • Join fragments with NotI digestion and ligation

  • Clone into suicide plasmid with kanamycin resistance

  • Select sucrose-resistant colonies for deletion mutants

Expression Analysis Methods:

• RT-PCR protocols optimized for P. profundum

• Protein tagging systems compatible with high-pressure growth conditions

• Reporter gene systems functional at high pressure

• Flow cytometry methods for analyzing DNA content and replication patterns

Verification Techniques:

• Arbitrary PCR to identify transposon insertion sites

• Southern blotting to confirm gene modifications

• Whole genome sequencing for comprehensive mutation verification

• Phenotypic assays under varying pressure conditions

These optimized genetic techniques enable detailed study of DnaA function directly in P. profundum, providing insights that may not be obtainable through heterologous expression systems.

How does P. profundum DnaA compare structurally and functionally to DnaA from mesophilic bacteria?

A detailed comparison of P. profundum DnaA with mesophilic counterparts reveals important structural and functional differences:

Sequence and Domain Similarities:

Functional Adaptations:

• ATP Binding and Hydrolysis:

  • P. profundum DnaA likely retains essential ATP binding capability

  • May have altered ATP hydrolysis rates optimized for high-pressure environments

  • ATP-DnaA is the active form for initiation in both organisms

• Regulation by Associated Proteins:

  • PBPRA3229 (DiaA homolog) and PBPRA1039 (SeqA homolog) in P. profundum

  • Both can functionally complement E. coli mutants

  • Suggests conserved regulatory mechanisms despite pressure adaptation

  • Mutations in these regulators have more pronounced effects under high pressure

• Pressure Sensitivity:

  • Initiation of DNA replication is pressure-sensitive in P. profundum

  • Presence of positive regulators (DiaA) or absence of negative regulators (SeqA) promotes growth under high pressure

  • Suggests DnaA function may be rate-limiting under high pressure

• Conformational Transitions:

  • Research on P. profundum cytochrome P450 suggests a general mechanism where pressure affects protein hydration and conformational equilibrium

  • Similar principles may apply to DnaA, with a potential shift in conformational equilibrium to accommodate high pressure

  • May involve adaptation to enhanced protein hydration at high pressure

These comparisons indicate that while P. profundum DnaA maintains the core functional domains of mesophilic DnaA proteins, it likely possesses specific adaptations that allow it to function optimally under high hydrostatic pressure conditions characteristic of the deep sea.

What is the current understanding of how piezophilic bacteria like P. profundum adapt their DNA replication machinery to high pressure?

The adaptation of DNA replication machinery to high pressure in piezophilic bacteria reveals sophisticated evolutionary strategies:

DNA Replication Initiation Adaptations:

• DnaA-Mediated Regulatory Networks:

  • P. profundum has evolved a specialized regulatory network involving DnaA, DiaA, and SeqA

  • Disruption of DiaA (positive regulator) causes high-pressure sensitivity

  • Disruption of SeqA (negative regulator) enhances growth at high pressure

  • This suggests a fine-tuned balance of positive and negative regulation is critical under pressure

• Pressure Effects on Initiation Complexes:

  • High pressure may affect the stability of protein-DNA complexes at the origin

  • These effects may be mitigated through specific protein modifications

  • The ATP-bound form of DnaA may have different stability characteristics under pressure

Genome Organization Adaptations:

• Chromosome Structure:

  • P. profundum has two circular chromosomes, which may provide advantages for replication under pressure

  • Changes in DNA topology under pressure may require specialized replication machinery

• Origin Recognition:

  • DnaA boxes and DNA unwinding elements may have sequence adaptations that maintain function under pressure

  • The interaction between DnaA and origin DNA may be modified to function optimally at high pressure

Cellular Adaptations Related to Replication:

• Membrane Composition:

  • P. profundum alters membrane fatty acid composition in response to pressure

  • This may affect membrane-associated replication processes

  • DNA replication is often associated with the cell membrane in bacteria

• Stress Response Integration:

  • Several stress response genes (htpG, dnaK, dnaJ, groEL) are upregulated in response to pressure changes

  • These chaperones may help maintain proper folding of replication proteins under pressure

• Pressure-Specific DNA Repair:

  • RecD function is required for high-pressure growth

  • DNA recombination and repair pathways may be critical for managing replication errors under pressure

  • Suggests pressure introduces unique challenges to DNA integrity during replication