Recombinant Photobacterium profundum Probable transcriptional regulatory protein PBPRA1113 (PBPRA1113)

What is Photobacterium profundum and why is it significant for studying transcriptional regulation under extreme conditions?

Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains have been isolated from different ocean depths, displaying remarkable differences in their physiological responses to pressure. The genome sequence of the deep-sea piezopsychrophilic strain Photobacterium profundum SS9 has provided insights into genetic features required for growth in the deep sea, while the genome of strain 3TCK (a non-piezophilic strain from shallow water) offers comparative data on environmental adaptations .

The study of transcriptional regulatory proteins like PBPRA1113 is particularly significant because they likely control gene expression patterns that enable adaptation to extreme pressure and temperature conditions. Understanding these regulatory mechanisms helps elucidate how bacteria rapidly adapt to specific environmental niches.

How is the genome of Photobacterium profundum organized, and where is PBPRA1113 located?

Photobacterium profundum's genome is organized into two chromosomes, similar to other members of the Vibrionaceae family. This organization is evident from gene synteny plots and the existence of two different origins of replication. The draft genome of strain 3TCK contains 11 scaffolds totaling 6,186,725 bp with an average 41.3% GC content, encoding 5,549 ORFs . This structure is comparable to the deep bathytype strain SS9, though 3TCK lacks an 80 kb dispensable plasmid specific to SS9 .

PBPRA1113 is specifically designated by its locus tag in the SS9 strain, indicating its location in the genome. Comparative genomic analyses between bathytypes can help identify whether this regulatory protein is conserved across different Photobacterium profundum strains or represents a specialization for deep-sea environments.

What is the hypothesized function of PBPRA1113 based on sequence homology?

Based on genomic characterization, PBPRA1113 is classified as a probable transcriptional regulatory protein. Transcriptional regulators typically function by binding to specific DNA sequences to either activate or repress gene expression. In the context of Photobacterium profundum, such proteins likely help coordinate the expression of genes involved in adaptation to environmental stressors such as high pressure, low temperature, and nutrient availability.

Sequence homology analyses would typically reveal conserved domains characteristic of specific families of transcriptional regulators, such as helix-turn-helix motifs for DNA binding. The "probable" designation indicates that while the protein's function has been predicted through bioinformatic analyses, experimental validation is still needed to confirm its precise role.

What expression systems are most effective for producing recombinant PBPRA1113?

For recombinant expression of PBPRA1113, researchers should consider several systems optimized for bacterial transcription factors:

• E. coli-based expression systems: The BL21(DE3) strain with pET vector systems offers a robust platform for initial expression attempts, particularly when tagged with 6xHis for purification.

• Cold-adapted expression systems: Since Photobacterium profundum is psychrophilic, expression at lower temperatures (15-20°C) may improve protein folding and solubility, even in mesophilic hosts.

• Pressure-adapted expression: For proteins potentially affected by pressure, expression under moderate pressure conditions may be necessary to obtain properly folded protein.

• Homologous expression: Expression within Photobacterium itself may be advantageous if specific chaperones or post-translational modifications are required.

A systematic comparison of expression conditions should include:

How can multiple-probe experimental designs be implemented to study PBPRA1113 function?

Multiple-probe experimental designs can systematically evaluate PBPRA1113 function across various conditions. Based on the PEAK Relational Training System approach, researchers could:

This approach is particularly valuable for characterizing transcriptional regulators under multiple conditions, as it allows for systematic assessment of functional parameters while maintaining experimental rigor.

What methodological approaches can identify DNA binding sites for PBPRA1113?

To identify PBPRA1113 DNA binding sites, researchers should employ a multi-tiered experimental strategy:

• In vitro approaches:

  • Electrophoretic Mobility Shift Assays (EMSA) with purified recombinant PBPRA1113 and target DNA fragments

  • DNase footprinting to precisely map protected regions

  • Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to identify consensus binding sequences

• In vivo approaches:

  • Chromatin Immunoprecipitation (ChIP) followed by sequencing (ChIP-seq)

  • DNA adenine methyltransferase identification (DamID) as an alternative to ChIP

  • Reporter gene assays to validate putative binding sites

• Computational approaches:

  • Motif discovery in promoter regions of co-regulated genes

  • Comparative genomics to identify conserved regulatory elements

  • Machine learning algorithms trained on known binding sites

These methods should be applied under various pressure conditions to determine whether PBPRA1113 binding specificity is pressure-dependent.

How can high-pressure experimental systems be designed to study PBPRA1113 function in vitro?

Designing high-pressure experimental systems for studying PBPRA1113 requires specialized equipment and methodological considerations:

• Pressure vessels and bioreactors:

  • Stainless steel pressure chambers capable of maintaining 0.1-100 MPa

  • Temperature control systems (4-15°C) integrated with pressure chambers

  • Real-time monitoring capabilities for pH, oxygen, and other parameters

• Biochemical assays under pressure:

  • Modified binding assays using fluorescence polarization suitable for high-pressure cuvettes

  • Pressure-resistant optical cells for spectroscopic measurements

  • Rapid depressurization protocols for sample retrieval with minimal disruption

• Data collection considerations:

  • Experimental controls at atmospheric pressure

  • Stepwise pressure increases to determine threshold effects

  • Time-series measurements to capture adaptation responses

The rapid development of mutants tolerant to pressure inactivation might aid specific taxa in rapidly adapting to new environmental conditions , making it essential to study PBPRA1113 function under varying pressure conditions relevant to its natural environment.

How can the interaction between pressure and temperature adaptation be disentangled in PBPRA1113 functional studies?

Disentangling pressure and temperature effects requires factorial experimental designs:

• Factorial experimental matrix:

• Statistical approaches:

  • Two-way ANOVA to assess main effects and interactions

  • Principal Component Analysis to identify dominant factors

  • Response surface methodology to model complex interactions

• Molecular probes:

  • Site-directed mutagenesis to identify pressure vs. temperature-sensitive domains

  • Isothermal titration calorimetry under varying conditions

  • Hydrogen-deuterium exchange mass spectrometry to monitor structural changes

These approaches enable researchers to distinguish between pressure-specific and temperature-specific effects on PBPRA1113 function, crucial for understanding its role in environmental adaptation.

How can comparative genomics approaches identify the evolutionary history of PBPRA1113?

Comparative genomics can illuminate the evolutionary history of PBPRA1113 through several sophisticated approaches:

• Phylogenetic analysis:

  • Construct maximum likelihood trees using homologous proteins from related species

  • Calculate selection pressures (dN/dS ratios) to identify signatures of positive selection

  • Date divergence events using molecular clock analyses

• Genomic context analysis:

  • Examine synteny conservation around PBPRA1113 across species

  • Identify horizontally transferred genomic regions through GC content and codon usage analysis

  • Map gene neighborhood networks to understand functional associations

• Domain architecture analysis:

  • Compare domain organization with other transcriptional regulators

  • Identify lineage-specific domain acquisitions or losses

  • Reconstruct ancestral sequences to trace functional evolution

The genome plasticity between bathytypes of P. profundum suggests that horizontal gene transfer (HGT) may be one possible mechanism for the rapid evolution of new bathytypes . This could be particularly relevant for understanding how PBPRA1113 may have been acquired or modified to enable adaptation to specific depth-related stresses.

What transcriptomic approaches can identify the PBPRA1113 regulon under different pressure conditions?

To identify the PBPRA1113 regulon under varying pressure conditions, researchers should implement:

• Differential expression analysis:

  • RNA-seq comparing wild-type and PBPRA1113 knockout strains

  • Time-course analysis during pressure adaptation

  • Comparison across multiple Photobacterium profundum strains

• Network inference:

  • Co-expression network analysis to identify genes with similar expression patterns

  • Bayesian network modeling to infer causal relationships

  • Integration with ChIP-seq data to distinguish direct vs. indirect regulation

• Validation experiments:

  • qRT-PCR validation of key differentially expressed genes

  • Reporter assays for predicted target promoters

  • Complementation studies with PBPRA1113 variants

When analyzing differential gene expression data, appropriate statistical approaches must be selected, possibly adapting methods from the PRISMA 2020 guidelines for systematic reviews to ensure methodological rigor in data synthesis and meta-analysis .

What are the most effective genetic manipulation systems for studying PBPRA1113 in vivo?

Effective genetic manipulation of Photobacterium profundum to study PBPRA1113 function requires:

• Gene knockout strategies:

  • Homologous recombination-based approaches using suicide vectors

  • CRISPR-Cas9 systems optimized for marine bacteria

  • Transposon mutagenesis for random insertional inactivation

• Complementation and expression systems:

  • Plasmid vectors with inducible promoters functional in Photobacterium

  • Integration of constructs at neutral genomic sites

  • Expression of PBPRA1113 variants with domain mutations

• Reporter systems:

  • Luciferase or fluorescent protein fusions for activity monitoring

  • Transcriptional fusions to putative target promoters

  • Translational fusions to study protein localization

For precise genetic manipulations, researchers can adapt approaches similar to those used in the deletion construction Δ22 described in the literature, where specific genes were removed by cutting plasmid constructs with restriction enzymes and re-ligating .

How can protein-protein interactions of PBPRA1113 be characterized under various pressure conditions?

Characterizing PBPRA1113 protein-protein interactions under pressure requires specialized approaches:

• In vitro interaction studies:

  • Surface Plasmon Resonance (SPR) in pressure-resistant flow cells

  • Isothermal Titration Calorimetry (ITC) with pressure modifications

  • Cross-linking mass spectrometry under varying pressure conditions

• In vivo interaction studies:

  • Bacterial two-hybrid systems with pressure treatment

  • Co-immunoprecipitation followed by mass spectrometry

  • Fluorescence Resonance Energy Transfer (FRET) microscopy in pressure chambers

• Computational predictions:

  • Molecular dynamics simulations under varying pressure

  • Protein-protein docking with pressure-induced conformational changes

  • Coevolution analysis to predict interaction interfaces

These approaches can reveal whether PBPRA1113 forms different protein complexes under varying pressure conditions, potentially explaining pressure-specific transcriptional responses.

How should researchers approach systematic reviews of transcriptional regulation in piezophilic bacteria?

Systematic reviews of transcriptional regulation in piezophilic bacteria should follow the PRISMA 2020 guidelines with domain-specific considerations:

• Search strategy:

  • Develop comprehensive search terms covering piezophily, transcriptional regulation, and marine adaptation

  • Search multiple databases including specialized marine microbiology repositories

  • Include grey literature from oceanographic expeditions and environmental sampling

• Study selection and data extraction:

  • Define clear inclusion criteria based on experimental pressure conditions

  • Extract metadata on bacterial strains, depth of isolation, and experimental methods

  • Assess risk of bias in pressure measurement and adaptation assessment

• Synthesis methods:

  • Apply appropriate effect measures for transcriptional responses

  • Explore heterogeneity based on phylogenetic relationships

  • Conduct sensitivity analyses to assess robustness of synthesized results

The PRISMA 2020 checklist provides a structured approach including rationale, objectives, eligibility criteria, information sources, search strategy, and synthesis methods , which can be adapted specifically for the field of piezophilic transcriptional regulation.

What statistical approaches are most appropriate for analyzing differential binding of PBPRA1113 under varying pressure conditions?

Appropriate statistical approaches for analyzing PBPRA1113 differential binding include:

• Peak calling and differential binding analysis:

  • Utilize peak calling algorithms (MACS2, GEM) adapted for different pressure conditions

  • Apply statistical frameworks that account for biological replicates

  • Implement differential binding analysis (DiffBind, MAnorm) with appropriate normalization

• Motif enrichment analysis:

  • Compare motif enrichment across pressure conditions

  • Test for motif strength correlation with pressure levels

  • Identify pressure-specific co-occurring motifs

• Integration with expression data:

  • Correlation analysis between binding strength and target gene expression

  • Gene set enrichment analysis of differentially bound targets

  • Bayesian integration of binding and expression data

How can single-molecule approaches advance our understanding of PBPRA1113 dynamics under pressure?

Single-molecule approaches offer unprecedented insights into PBPRA1113 behavior under pressure:

• Single-molecule techniques:

  • Pressure-adapted atomic force microscopy (AFM) to observe conformational changes

  • Single-molecule FRET to measure intramolecular distance changes

  • Optical tweezers combined with microfluidic pressure systems

• Experimental considerations:

  • Time-resolved measurements to capture transient conformational states

  • Force-distance curves under varying pressure

  • Correlation of single-molecule behavior with bulk biochemical activity

• Data analysis:

  • Hidden Markov modeling to identify discrete conformational states

  • Transition path analysis to characterize pressure-dependent state changes

  • Energy landscape reconstruction under different pressure regimes

These approaches can reveal how pressure affects the dynamic behavior of PBPRA1113, potentially explaining its role in pressure adaptation at the molecular level.

What are the methodological considerations for applying cryoelectron microscopy to study PBPRA1113 structure under pressure?

Applying cryoelectron microscopy (cryo-EM) to study PBPRA1113 structure under pressure involves several methodological considerations:

• Sample preparation:

  • High-pressure freezing to capture pressure-induced conformational states

  • Vitrification under controlled pressure conditions

  • Grid preparation techniques compatible with pressure treatment

• Data collection:

  • Specialized holders to maintain or simulate pressure effects

  • Tilt series acquisition strategies for pressure-treated samples

  • Beam-induced damage assessment under pressure conditions

• Computational analysis:

  • 3D reconstruction algorithms robust to pressure-induced heterogeneity

  • Classification methods to identify pressure-specific conformations

  • Molecular dynamics flexible fitting to interpret pressure effects

Researchers must carefully document these methodological details when reporting results, similar to the systematic approach recommended in the PRISMA 2020 guidelines .