Recombinant Photobacterium profundum UPF0301 protein PBPRA3139 (PBPRA3139)

What is Photobacterium profundum and why is it significant for protein research?

Photobacterium profundum is a gram-negative rod-shaped marine bacterium belonging to the family Vibrionaceae. It is particularly significant to research due to its remarkable adaptation to deep-sea environments, with the ability to grow at temperatures from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa depending on the strain . Four well-characterized wild-type strains exist: SS9 (isolated from the Sulu Sea), 3TCK (from San Diego Bay), DSJ4 (from Ryukyu Trench), and 1230 .

The significance for protein research lies in P. profundum's adaptation mechanisms to extreme pressure and cold. Strain SS9, for instance, exhibits optimal growth at 15°C and 28 MPa, making it both a psychrophile and piezophile . These adaptations involve specialized proteins that function under high pressure, providing unique research opportunities to understand protein structure-function relationships in extreme environments. Studying recombinant proteins from this organism can reveal novel molecular mechanisms of pressure adaptation, which has implications for biotechnology, evolutionary biology, and astrobiology.

What genomic features distinguish different strains of P. profundum?

P. profundum strains show remarkable diversity in their genome content, reflecting their adaptation to different ocean depths. The genome sequence of strain SS9 (piezopsychrophilic, deep-sea) differs significantly from strain 3TCK (non-piezophilic, shallow-water), illustrating how genomic adaptations define their respective Hutchinsonian niches .

Key genomic distinctions include:

• Gene content variations between strains

• Specific gene sequences under positive selection pressure

• Genome plasticity that reflects environmental adaptation

Additionally, P. profundum possesses two circular chromosomes, a feature shared with other members of the Photobacterium genus . Comparative genomic analysis of these strains provides insights into the genetic basis of pressure adaptation, which informs research on proteins like PBPRA3139 whose functions may be pressure-sensitive.

How do researchers experimentally study pressure effects on P. profundum proteins?

Studying pressure effects on proteins from P. profundum involves specialized methodologies:

• Genetic manipulation systems: Researchers create in-frame deletions for gene function analysis, as demonstrated with flagellar genes (flaA, flaC, flaB, motA2, motA1) in strain SS9 .

• Comparative expression analysis: Analyzing differential gene expression under varying pressure conditions. For example, several stress response genes (htpG, dnaK, dnaJ, groEL) are upregulated in strain SS9 in response to atmospheric pressure .

• Membrane adaptation studies: Examining changes in fatty acid chains in the cell membrane that respond to pressure and temperature variations .

• Motility assays: Comparing motility between piezophilic (SS9) and piezosensitive (3TCK) strains under different pressure conditions to understand protein functionality .

For specific proteins like PBPRA3139, researchers typically express the recombinant protein in mesophilic hosts, then study its structure and function under simulated high-pressure conditions using specialized equipment such as pressure chambers for enzyme assays or high-pressure NMR for structural studies.

What is known about UPF0301 family proteins in bacterial systems?

The UPF0301 protein family (which includes PBPRA3139) belongs to a group of proteins with uncharacterized function (hence the UPF designation). While specific information about PBPRA3139 is limited, several general characteristics of UPF0301 family proteins can be outlined:

• Conservation: UPF0301 proteins are conserved across various bacterial species, suggesting an important functional role.

• Structure: These proteins typically have a conserved domain architecture, though structural data for the specific PBPRA3139 protein remains limited.

• Potential functions: Based on context analysis and comparative genomics, UPF0301 proteins may be involved in:

  • Stress response mechanisms

  • Cell envelope biogenesis

  • Metabolic adaptation to environmental changes

Research approaches to characterize UPF0301 proteins typically involve comparative genomics, structural biology techniques, and phenotypic analysis of deletion mutants. For PBPRA3139 specifically, studies would need to account for the pressure-adaptive characteristics of P. profundum.

What expression systems are suitable for recombinant production of P. profundum proteins?

Expressing recombinant proteins from extremophiles like P. profundum presents unique challenges. Based on documented approaches with similar organisms, the following expression systems can be considered:

• E. coli-based systems: Most commonly used, but may require optimization:

  • Cold-inducible promoters (pCold series) for psychrophilic proteins

  • Codon optimization for mesophilic expression hosts

  • Fusion partners (MBP, SUMO) to enhance solubility

• Psychrophilic expression hosts: Systems based on psychrophilic bacteria that can express proteins at lower temperatures (10-15°C).

• Cell-free expression systems: These bypass cellular toxicity issues and can be conducted under various pressure conditions.

For PBPRA3139 specifically, a methodological approach would include:

• Pilot expression trials in multiple E. coli strains (BL21, Arctic Express, Rosetta)

• Temperature optimization (15-25°C for expression)

• Testing native vs. codon-optimized gene sequences

• Evaluating both N- and C-terminal purification tags

Expression success can be validated through techniques like confocal imaging of fluorescent protein fusions, similar to methods used for visualizing P. profundum SS9 .

How do researchers investigate functional differences between homologous proteins from piezophilic and non-piezophilic strains?

Investigating functional differences between homologous proteins from piezophilic (e.g., SS9) and non-piezophilic (e.g., 3TCK) strains requires sophisticated comparative approaches:

• Comparative biochemistry: Recombinant proteins from both strains are purified and characterized under identical conditions, with key parameters measured across a pressure range (0.1-70 MPa). For enzymes, this includes:

  • Kinetic parameters (Km, Vmax, kcat)

  • Thermodynamic stability (ΔG, melting temperature)

  • Conformational dynamics using pressure-resistant spectroscopic methods

• Structure determination under pressure: Using specialized equipment like:

  • High-pressure crystallography

  • Pressure-adapted NMR spectroscopy

  • High-pressure small-angle X-ray scattering (HP-SAXS)

• Genetic complementation experiments: Creating chimeric constructs by:

  • Swapping domains between homologs

  • Site-directed mutagenesis targeting key residues

  • Expression of each variant in both strain backgrounds

A particularly informative approach is the implementation of in vivo assays under varying pressure conditions. For example, similar to how photoreactivation studies were conducted with P. profundum strains , functional assays for PBPRA3139 could be performed under controlled pressure conditions to directly compare activity between homologs.

What structural adaptations enable proteins like PBPRA3139 to function under high hydrostatic pressure?

Proteins from piezophilic organisms like P. profundum SS9 exhibit specific structural adaptations that maintain functionality under high hydrostatic pressure. While direct structural data for PBPRA3139 is not available, research on piezophilic proteins reveals several common adaptations:

• Increased flexibility in certain regions: Strategic increases in backbone and side chain flexibility that counteract pressure-induced rigidification

• Modified core packing: Often featuring:

  • Reduced hydrophobic core volume

  • Increased number of small residues (Ala, Gly) in core positions

  • Strategic placement of charged residues

• Altered surface properties:

  • Increased negative surface charge

  • Modified ion pair networks

  • Reduced surface hydrophobicity

• Pressure-sensing domains: Some piezophilic proteins contain specialized domains that undergo conformational changes in response to pressure, acting as molecular switches

Methodological approaches to investigate these features in PBPRA3139 would include:

• Comparative modeling with homologs from non-piezophilic strains

• Molecular dynamics simulations under varying pressure conditions

• Hydrogen-deuterium exchange mass spectrometry to map flexibility differences

• Site-directed mutagenesis to test the importance of specific residues

What omics approaches are valuable for contextualizing PBPRA3139 function within P. profundum biology?

Multi-omics approaches provide powerful frameworks for understanding the biological context of PBPRA3139:

• Comparative genomics:

  • Analysis of gene neighborhoods across multiple Photobacterium strains

  • Identification of genetic linkage patterns that suggest functional associations

  • Phylogenetic profiling to identify co-evolving genes

• Transcriptomics:

  • RNA-seq analysis under varying pressure, temperature, and nutritional conditions

  • Identification of co-expressed gene clusters that include PBPRA3139

  • Comparison of expression patterns between SS9 and 3TCK strains

• Proteomics:

  • Quantitative proteomic analysis at different pressures

  • Protein-protein interaction studies using proximity labeling approaches

  • Post-translational modification mapping

• Metabolomics:

  • Identification of metabolic changes in PBPRA3139 knockout strains

  • Flux analysis to determine if metabolic pathways are affected

An integrated approach combining these methods would be especially powerful. For example, researchers could correlate pressure-dependent changes in PBPRA3139 expression with specific metabolic shifts, protein interaction changes, and phenotypic outcomes.

How can researchers investigate the role of PBPRA3139 in pressure adaptation using genetic approaches?

Genetic investigation of PBPRA3139's role in pressure adaptation can employ several sophisticated strategies:

• Precise gene deletion and complementation:

  • Generation of PBPRA3139 knockout strains using techniques similar to those employed for flagellar gene deletions in SS9

  • Complementation with native and mutated versions of the gene

  • Cross-complementation with homologs from non-piezophilic strains

• Conditional expression systems:

  • Pressure-responsive promoter systems

  • Inducible expression constructs to control protein levels

• Growth phenotype characterization:

  • Pressure-dependent growth curve analysis

  • Competitive growth assays with wild-type strain

  • Survival assays under pressure shock conditions

• Reporter fusion systems:

  • Transcriptional and translational fusions to monitor expression

  • Protein localization studies using fluorescent tags, similar to GFP visualization methods used with P. profundum SS9

• Suppressor screens:

  • Identification of mutations that suppress phenotypes of PBPRA3139 deletion

  • Chemical genomics to identify compounds that specifically affect mutant strains

These approaches could be combined with detailed phenotypic analysis, examining how various cellular processes (particularly those known to be pressure-sensitive, such as motility ) are affected by PBPRA3139 manipulation under different pressure conditions.

What are the current challenges in differentiating between pressure-specific and general stress responses in P. profundum?

Distinguishing pressure-specific from general stress responses represents a significant challenge in piezophile research:

• Overlapping stress response systems:

  • Many stress response genes (htpG, dnaK, dnaJ, groEL) upregulated under pressure stress are also induced by other stressors

  • Difficulty in isolating pressure-specific pathways from general stress circuits

• Methodological challenges:

  • High-pressure cultivation requires specialized equipment

  • Maintaining consistent conditions during sampling from pressure vessels

  • Time-course experiments under pressure are technically demanding

• Signal specificity issues:

  • Pressure may act as a secondary stressor by affecting membrane fluidity, similar to temperature

  • Effects on protein conformation may trigger general unfolded protein responses

• Data interpretation complexities:

  • Changes in gene expression may represent adaptive responses or damage responses

  • Distinguishing between direct pressure sensing and indirect effects

Experimental approaches to address these challenges include:

• Comparative transcriptomics/proteomics with careful stress controls

• Development of specific biosensors for pressure responses

• Genetic screens for pressure-specific versus general stress mutants

• Time-resolved studies to separate immediate from adaptive responses

For PBPRA3139 specifically, determining whether it functions in a pressure-specific manner would require careful experimental design controlling for other stressors while manipulating pressure conditions.

What protein purification strategies are effective for UPF0301 family proteins from P. profundum?

Purifying UPF0301 family proteins from piezophiles requires specialized approaches to maintain native structure and function:

• Optimized buffer systems:

  • Higher ionic strength buffers (400-500 mM NaCl) to mimic marine conditions

  • Kosmotropic agents (glycerol, trehalose) to stabilize protein structure

  • Careful pH optimization, typically 7.5-8.0 for marine proteins

• Affinity chromatography options:

  • His-tag purification under native conditions, optimized for high salt

  • Fusion protein approaches (MBP, GST, SUMO) with cleavable linkers

  • Specialized affinity resins stable under high salt conditions

• Multi-step purification protocols:

  Purification Step	Purpose	Typical Conditions
  Affinity chromatography	Initial capture	50 mM Tris pH 8.0, 400 mM NaCl, 10% glycerol
  Ion exchange	Remove nucleic acids	Salt gradient in 50 mM HEPES pH 7.5
  Size exclusion	Final polishing	25 mM PIPES pH 7.0, 300 mM NaCl, 5% glycerol

• Cold-adapted purification protocols:

  • Maintaining 4-10°C throughout purification

  • Pre-chilled buffers and collection vessels

  • Minimal exposure to room temperature

• Quality control considerations:

  • Multiple biophysical characterization steps (SEC-MALS, DLS, CD)

  • Activity assays at varying pressures

  • Stability testing under storage conditions

These strategies should be systematically optimized for PBPRA3139, with particular attention to salt concentration and temperature during purification to maintain the native structure of this deep-sea bacterial protein.

How can researchers design functional assays for proteins of unknown function like PBPRA3139?

Designing functional assays for hypothetical proteins like PBPRA3139 requires a systematic approach:

• Computational prediction-based assays:

  • Structural homology modeling to identify potential active sites

  • Domain analysis to guide assay design

  • Molecular docking with potential substrates

• Activity-guided screening approaches:

  • Substrate profiling using compound libraries

  • Activity-based protein profiling with reactive probes

  • Metabolite profiling of knockout vs. wild-type strains

• Physical interaction studies:

  • Pull-down assays with cellular extracts

  • Yeast two-hybrid or bacterial two-hybrid screening

  • Proximity labeling in vivo to identify interacting partners

• Phenotypic assays based on genetic manipulation:

  • Growth phenotypes under varying pressure conditions

  • Stress resistance profiling of deletion mutants

  • Quantitative fitness measurements using barcoded mutant libraries

• Targeted biochemical assays:

  Potential Function	Assay Approach	Detection Method
  Enzymatic activity	Substrate conversion	HPLC, spectrophotometry
  Binding function	Interaction with nucleic acids/proteins	EMSA, SPR, MST
  Structural role	Membrane association	Fractionation, microscopy
  Signaling function	Second messenger levels	LC-MS/MS

For PBPRA3139 specifically, a key consideration would be performing these assays across a range of pressure conditions, given P. profundum's piezophilic nature. This would reveal whether the protein's function is pressure-dependent or pressure-modulated.

What high-pressure experimental systems are available for studying recombinant PBPRA3139 function?

High-pressure experimental systems for studying proteins from piezophiles like P. profundum include:

• High-pressure bioreactors:

  • Continuous cultivation systems operating at 0.1-70 MPa

  • Temperature-controlled pressure vessels for batch cultures

  • Systems with sampling ports for time-course experiments

• Spectroscopic high-pressure cells:

  • UV-visible spectrophotometry cells (0.1-200 MPa)

  • Fluorescence spectroscopy pressure cells

  • FTIR-compatible high-pressure systems

• Enzyme activity measurement systems:

  • Stopped-flow systems coupled to pressure cells

  • Real-time pressure-variable enzyme assay equipment

  • Microfluidic devices with pressure control

• Structural biology under pressure:

  • High-pressure NMR probes (up to 200 MPa)

  • Diamond anvil cells for high-pressure crystallography

  • Small-angle X-ray scattering pressure cells

• Specialized equipment configurations:

  Technique	Pressure Range	Key Applications
  HP-SAXS	0.1-400 MPa	Solution structure determination
  HP-NMR	0.1-200 MPa	Dynamic structural changes
  HP Enzyme Kinetics	0.1-100 MPa	Catalytic parameter determination
  HP-CD	0.1-200 MPa	Secondary structure stability

For PBPRA3139, these systems would allow researchers to characterize structural changes, activity profiles, and interaction dynamics across the relevant pressure range for P. profundum (0.1-70 MPa), providing insight into how this protein functions in its native deep-sea environment.

How can structural biology approaches inform PBPRA3139 function?

Structural biology offers powerful approaches to elucidate the function of uncharacterized proteins like PBPRA3139:

For PBPRA3139 specifically, combining these approaches could reveal structural adaptations that enable function under high pressure, potentially identifying it as a model system for understanding pressure adaptation at the molecular level.

What in silico approaches can predict the function of PBPRA3139?

Computational prediction of protein function is particularly valuable for uncharacterized proteins like PBPRA3139:

• Advanced sequence analysis methods:

  • Profile Hidden Markov Models for remote homology detection

  • Coevolution analysis to identify functionally coupled residues

  • Deep learning-based function prediction (DeepFRI, ESM-1b)

• Structural bioinformatics approaches:

  • Structural classification database searches (CATH, SCOP)

  • Local structural motif matching for active site identification

  • Molecular dynamics simulations under varying pressure conditions

• Systems biology integration:

  • Gene neighborhood analysis across multiple genomes

  • Protein-protein interaction network inference

  • Pathway enrichment analysis

• Machine learning prediction pipelines:

  Approach	Key Information	Example Tools
  GO term prediction	Molecular function	DeepGOPlus, PANNZER2
  Enzyme classification	Potential catalytic activity	ECPred, DeepEC
  Ligand binding prediction	Potential substrates	COACH-D, ILbind
  Subcellular localization	Cellular context	DeepLoc, BUSCA

• Comparative genomics approaches:

  • Phylogenetic profiling across diverse bacteria

  • Genomic context analysis in multiple Photobacterium species

  • Comparison between piezophilic and non-piezophilic strains to identify pressure-adaptive features

For PBPRA3139, a comprehensive computational analysis would focus on identifying features conserved among UPF0301 proteins in piezophiles versus non-piezophiles, potentially revealing pressure-specific adaptations or functions.