Recombinant Photobacterium profundum Negative modulator of initiation of replication (seqA)

What is Photobacterium profundum and why is it important for studying pressure adaptation?

Photobacterium profundum is a deep-sea bacterium that has developed significant adaptations for growth under high hydrostatic pressure conditions. P. profundum strain SS9 is particularly valued as a model organism for studying pressure adaptation (piezophily) because it demonstrates optimal growth under elevated pressure conditions, reflecting its deep-sea origin. This bacterium can be cultured in laboratory settings, making it amenable to genetic manipulation and experimental study. Unlike mesophilic organisms such as Escherichia coli, which experience stress at high pressures, P. profundum SS9 experiences greater stress at atmospheric pressure than at elevated pressure, providing a valuable contrast for comparative studies of pressure adaptation mechanisms .

What is SeqA and how does it function in DNA replication?

SeqA (encoded by PBPRA1039 in P. profundum) is a negative regulator of the cell cycle and specifically of DNA replication initiation. In E. coli, SeqA binds to hemimethylated oriC (origin of replication) and prevents the binding of ATP-DnaA, thereby inhibiting the initiation of DNA replication. The protein plays a crucial role in ensuring proper timing and synchrony of chromosome replication. In P. profundum, the SeqA homolog appears to maintain similar fundamental functions while having evolved specialized adaptations for high-pressure environments .

What phenotypes are associated with SeqA mutations in P. profundum?

The most notable phenotype associated with SeqA mutation in P. profundum SS9 is a pressure-enhanced growth pattern. When the seqA gene (PBPRA1039) is disrupted, the mutant strain grows better at high hydrostatic pressure than at atmospheric pressure. This contrasts with the wild-type strain, which experiences some stress at high pressure. Additionally, similar to E. coli SeqA mutants, P. profundum SeqA mutants demonstrate irregular growth caused by asynchronous patterns of replication. This asynchrony might be exacerbated at low pressure in P. profundum .

What are the optimal culture conditions for working with P. profundum in laboratory settings?

For laboratory cultivation of P. profundum strains:

• Media: P. profundum strains are routinely cultured in 2216 medium (28 g/liter; Difco Laboratories) or 0.75× 2216 medium

• Temperature: Optimal growth occurs at 16°C or 15°C

• Pressure conditions:

  • Atmospheric pressure (0.1 MPa) for routine cultivation

  • High-pressure growth (typically 30 MPa) for pressure studies

• For high-pressure growth experiments:

  • Culture media should be supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5)

  • Late-exponential-phase cultures are diluted 500-fold into fresh medium

  • Cultures are sealed in polyethylene transfer pipettes and incubated in stainless-steel pressure vessels

• Antibiotic concentrations when needed:

  • Chloramphenicol: 30 μg/ml

  • Kanamycin: 75 μg/ml for E. coli and 200 μg/ml for P. profundum

  • Streptomycin: 50 μg/ml for E. coli and 150 μg/ml for P. profundum

How can researchers generate and identify SeqA mutants in P. profundum?

Researchers can generate SeqA mutants in P. profundum using several approaches:

• Transposon mutagenesis:

  • Mini-Tn10 or mini-Tn5 transposable elements can be used

  • Conjugal delivery of transposon donor plasmids (e.g., pLOF for mini-Tn10) via triparental conjugation

  • Using E. coli strains containing helper plasmids such as pRK2073 or pRK2013

  • Note: Mini-Tn10 has shown insertion bias in P. profundum, while mini-Tn5 appears to have more random insertion

• Targeted gene disruption:

  • Construction of in-frame deletions using suicide vectors like pRL271 containing the sacB gene

  • Designing primers to amplify upstream and downstream regions of the target gene

  • Cloning these regions into the suicide vector

  • Conjugal transfer of the resulting plasmid into P. profundum

  • Selection for double-crossover events using sucrose counter-selection

Identifying SeqA mutants involves:

• Screening for pressure-enhanced growth phenotypes

• Testing for asynchronous DNA replication patterns

• Molecular confirmation through PCR and sequencing to verify the mutation location

What methods can be used to evaluate DNA replication synchrony in P. profundum?

To evaluate DNA replication synchrony in P. profundum, researchers can employ several techniques:

• Flow cytometry analysis of DNA content:

  • Cells are fixed and stained with DNA-binding fluorescent dyes

  • Distribution of DNA content in a population is measured

  • Asynchronous replication results in abnormal DNA content distributions

• Marker frequency analysis:

  • Quantitative PCR targeting sequences near oriC and terminus regions

  • Ratio of oriC to terminus reveals information about replication initiation frequency

  • Higher variability in this ratio indicates asynchronous replication

• Complementation studies:

  • Introduction of the wild-type seqA gene into mutant strains

  • Measuring restoration of synchronous replication patterns

  • As demonstrated in the research, P. profundum PBPRA1039 (SeqA homolog) can restore synchrony to the initiation of DNA replication in E. coli mutants lacking SeqA

• Temperature and pressure modulation:

  • Evaluating replication patterns under different pressure and temperature conditions

  • Particularly informative since SeqA function appears to be both pressure and temperature dependent in P. profundum

What is the molecular relationship between SeqA and pressure adaptation in P. profundum?

The relationship between SeqA and pressure adaptation in P. profundum reflects a complex evolutionary specialization. Several key aspects of this relationship have been identified:

• Differential regulation: SeqA appears to have evolved piezo-specific traits in P. profundum that differ from its role in temperature adaptation. While H-NS-deficient mutants of both E. coli and P. profundum are cold sensitive, P. profundum SeqA mutants exhibit a pressure-enhanced phenotype, indicating that SeqA has a specialized role in pressure response .

• DNA replication control: The importance of SeqA in regulating DNA replication timing appears critical under different pressure conditions. Since the initiation of DNA replication is one of the most pressure-sensitive cellular processes, SeqA's negative regulatory role likely helps coordinate proper timing of replication under high pressure .

• Suppression of pressure sensitivity: PBPRA1039 (SeqA) can suppress cold sensitivity phenotypes in E. coli dnaA(Cs) mutants, suggesting that SeqA's interaction with the replication machinery has evolved to function optimally under pressure and low-temperature conditions that characterize the deep-sea environment .

• Cellular homeostasis: SeqA may contribute to pressure adaptation by influencing membrane structure. Research in E. coli has shown that SeqA can affect membrane structure, and this function may be particularly important for maintaining cellular integrity under high-pressure conditions in P. profundum .

How does SeqA interact with other DNA replication proteins under high pressure?

The interaction between SeqA and other DNA replication proteins under high pressure involves several key relationships:

• DnaA interaction: SeqA primarily functions by preventing the binding of ATP-DnaA to oriC. Under high pressure, this regulatory mechanism appears modified in P. profundum to allow proper DNA replication initiation despite the physical stress of pressure .

• Relationship with DiaA: A notable relationship exists between SeqA and DiaA (encoded by PBPRA3229) in P. profundum. While SeqA acts as a negative regulator of replication initiation, DiaA functions as a positive regulator. Disruption of diaA results in a pressure-sensitive phenotype, contrasting with the pressure-enhanced phenotype of seqA mutants. This opposing relationship suggests a balanced regulatory system for DNA replication under pressure .

• Coordination with DNA repair machinery: The interplay between SeqA and DNA repair proteins becomes particularly important under high pressure. The observation that RecD function is required for high-pressure growth in P. profundum suggests a potential regulatory network involving both replication initiation control (SeqA) and DNA repair mechanisms (RecD) to maintain genomic integrity under pressure .

• Pressure-specific adaptations: Both DiaA and SeqA from P. profundum can functionally complement their respective E. coli homologs, but they appear to have evolved specific features that optimize their function under high pressure. This suggests structural or regulatory adaptations in their interactions with the replication machinery .

What is known about SeqA's role in asynchronous replication under different pressure conditions?

SeqA's role in managing asynchronous replication under varying pressure conditions reveals important insights:

How does P. profundum SeqA compare functionally to E. coli SeqA?

The functional comparison between P. profundum SeqA and E. coli SeqA reveals both conservation and adaptation:

This comparison demonstrates that while the core function of SeqA as a negative regulator of DNA replication initiation is conserved between the species, P. profundum SeqA has evolved specific adaptations for functioning optimally under high pressure conditions that characterize its deep-sea habitat .

What unique structural features might P. profundum SeqA possess for pressure adaptation?

Although the search results don't provide direct structural analysis of P. profundum SeqA, several key features can be inferred based on its functional properties and evolutionary adaptations:

• Pressure-stable binding domains: P. profundum SeqA likely possesses structural modifications that maintain proper DNA binding under high pressure conditions, which would typically disrupt protein-nucleic acid interactions in non-adapted proteins.

• Altered oligomerization interfaces: SeqA proteins typically form dimers and higher-order structures. The interfaces mediating these interactions in P. profundum SeqA may contain adaptations that prevent pressure-induced dissociation or improper assembly.

• Modified hemimethylated DNA recognition: Since SeqA binds specifically to hemimethylated DNA at the origin of replication, the DNA-binding domain of P. profundum SeqA may contain adaptations that optimize this recognition under pressure.

• Pressure-resistant conformational changes: The protein likely has evolved structural elements that resist pressure-induced conformational changes that would otherwise disrupt its regulatory function.

• Enhanced stability in membrane interactions: Given SeqA's effect on membrane structure, P. profundum SeqA may possess unique structural features that facilitate membrane interactions under high pressure.

The ability of P. profundum SeqA to functionally complement E. coli seqA mutants suggests that these structural adaptations maintain the core functional domains while optimizing them for high-pressure environments .

What are the current challenges in studying SeqA function in piezophilic bacteria?

Several significant challenges exist in studying SeqA function in piezophilic bacteria:

• Technical limitations in high-pressure experimentation:

  • Specialized equipment requirements for cultivating and observing bacteria under high pressure

  • Difficulties in real-time monitoring of cellular processes under pressure

  • Challenges in adapting standard molecular biology techniques to high-pressure conditions

• Genetic manipulation constraints:

  • Limited genetic tools optimized for piezophilic bacteria

  • Challenges in creating precise genetic modifications without pleiotropic effects

  • The need for appropriate control strains that account for pressure variables

• Complex environmental adaptation:

  • Difficulty in isolating pressure effects from temperature adaptation

  • Multiple regulatory systems responding simultaneously to environmental stressors

  • Challenges in recreating authentic deep-sea conditions in laboratory settings

• Structural analysis limitations:

  • Difficulties in obtaining high-resolution structural data under pressure conditions

  • Technical challenges in protein crystallization from piezophilic organisms

  • Need for specialized equipment to analyze protein-DNA interactions under pressure

• Functional redundancy:

  • Potential overlap between regulatory systems controlling DNA replication under pressure

  • Difficulty in isolating SeqA-specific effects from other pressure response mechanisms

How might advanced genomic approaches expand our understanding of SeqA in P. profundum?

Advanced genomic approaches offer promising avenues for expanding our understanding of SeqA in P. profundum:

• Comparative genomics across piezophilic bacteria:

  • Systematic comparison of seqA sequences from bacteria adapted to different depths

  • Identification of conserved pressure-adaptive features across diverse piezophilic species

  • Correlation of genetic variations with depth adaptation

• Whole-genome sequencing of evolved strains:

  • Experimental evolution of P. profundum under various pressure conditions

  • Identification of compensatory mutations in seqA mutants

  • Analysis of genetic adaptations that emerge during pressure adaptation

• ChIP-seq analysis under different pressure conditions:

  • Genome-wide mapping of SeqA binding sites under various pressure conditions

  • Identification of pressure-dependent changes in binding patterns

  • Correlation with changes in gene expression and replication timing

• Transcriptome analysis of seqA mutants:

  • RNA-seq of wild-type and seqA mutant strains under varying pressure

  • Identification of genes and pathways affected by SeqA disruption

  • Elucidation of the broader regulatory network influenced by SeqA

• CRISPR-based approaches:

  • Development of CRISPR-Cas systems optimized for P. profundum

  • Creation of precise gene modifications to study specific SeqA domains

  • Generation of conditional seqA mutants to study essential functions