Recombinant Photobacterium profundum Ornithine carbamoyltransferase, catabolic (arcB)

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

Photobacterium profundum SS9 is a deep-sea Gram-negative bacterium belonging to the Vibrionaceae family. It serves as a model organism for studying piezophily (adaptation to high pressure) because it can grow over a wide range of pressures from atmospheric pressure (0.1 MPa) to nearly 90 MPa, with optimal growth at 28 MPa and 15°C . Unlike many other piezophiles, P. profundum can grow at atmospheric pressure, which allows for easy genetic manipulation and culturing, making it an ideal system for studying deep-sea adaptations . Its genome has been fully sequenced and consists of two chromosomes and an 80 kb plasmid .

What is ArcB in Photobacterium profundum and what function does it serve?

ArcB in P. profundum is a catabolic ornithine carbamoyltransferase that catalyzes the third step of the arginine deiminase (ADI) pathway . This enzyme is responsible for converting citrulline into carbamoyl phosphate (CP) and ornithine . The gene encoding ArcB is located in an operon (arcABDC) on the chromosome, with arcB positioned between arcA and arcC . The ADI pathway allows bacteria to generate ATP from arginine under anaerobic conditions, which may be important for energy production in deep-sea environments .

How does the catabolic ArcB differ from the anabolic ornithine carbamoyltransferase?

While both enzymes catalyze reactions involving ornithine, carbamoyl phosphate, and citrulline, they function in opposite directions and have distinct characteristics:

While the anabolic OTC (ArgF) primarily functions in arginine biosynthesis, catabolic OTC (ArcB) can sometimes function in both directions under certain conditions, as demonstrated in studies with Staphylococcus aureus .

How can I isolate and clone the arcB gene from P. profundum?

To isolate the arcB gene from P. profundum, researchers have successfully employed PCR-based methods. The following approach has proven effective:

• Design appropriate primers: You can design primers based on conserved regions from alignment of related organisms. For example, researchers have successfully amplified arcB using primers designed from conserved regions from alignment of E. coli and Vibrio harveyi fabF sequences .

• PCR amplification: Use genomic DNA from P. profundum as a template. The following PCR conditions have been effective for similar genes in P. profundum:

  • First round: 5 cycles of 94°C for 30s, 30°C for 30s, 72°C for 60s, followed by 30 cycles of 94°C for 30s, 45°C for 30s, and 72°C for 60s .

  • Second round: 30 cycles of 95°C for 30s, 55°C for 30s, and 72°C for 45s with nested primers .

• Cloning and verification:

  • Clone the PCR product into a suitable vector like pCR2.1

  • Verify the identity by sequencing using standard primers (e.g., M13R, T7)

  • For further studies, subclone into an appropriate expression vector or a mobilizable vector for complementation studies .

Genomic DNA can be purchased from repositories such as ATCC (catalog BAA-1253D-5) if you don't have access to the organism .

What expression systems work best for recombinant production of P. profundum ArcB?

Based on research with similar P. profundum proteins, the following expression systems have been effective:

• E. coli-based expression systems:

  • BL21(DE3) strains have been successfully used for expression of proteins from piezophilic organisms

  • Expression at reduced temperatures (15-20°C) often improves folding of psychrophilic proteins

  • Vectors with T7 promoters have shown good results for similar deep-sea bacterial proteins

• Optimization considerations:

  • Codon optimization may be necessary since P. profundum has different codon usage than E. coli

  • Addition of solubility tags (MBP, SUMO, etc.) can improve protein solubility

  • Growth at atmospheric pressure is sufficient for expression, but some researchers have explored expression under pressure for improved folding

• Culture conditions:

  • For studying pressure effects, cultures can be grown in sealed vessels (like Pasteur pipettes) and incubated at different pressures

  • Example protocol: Grow cultures at 17°C anaerobically in marine broth supplemented with 20 mM glucose and 100 mM HEPES buffer (pH 7.5)

What methods can I use to measure ArcB enzyme activity?

Several methods have been successfully employed to measure ornithine carbamoyltransferase activity:

• Colorimetric assays:

  • The most common approach measures citrulline or carbamoyl phosphate formation/consumption

  • For catabolic direction (citrulline → ornithine + CP): measure CP formation using the colorimetric reaction with specific reagents

• Coupled enzyme assays:

  • ArcB activity can be coupled with carbamate kinase (the next enzyme in the ADI pathway) to monitor ATP formation

  • This can be measured using luciferase or coupled to other ATP-dependent enzymes

• Experimental conditions:

  • Buffer: Typically Tris-HCl (50-100 mM, pH 7.5-8.0)

  • Temperature: Compare activity at different temperatures (4°C, 15°C, 28°C, 37°C)

  • Pressure: For pressure effects, specialized equipment is needed to maintain reaction mixtures under pressure during the assay

• Data analysis:

  • Calculate specific activity (μmol product formed per minute per mg protein)

  • Determine kinetic parameters (Km, kcat) under different conditions

  • For CP cooperativity analysis, create Hill plots of activity vs. CP concentration

How do mutations in arcB affect P. profundum growth under different pressure and temperature conditions?

Research on P. profundum mutants has revealed important insights about the role of various genes in pressure and temperature adaptation. While specific arcB mutant data is limited, the general approach and findings regarding similar genes provide a framework:

• Generation of arcB mutants:

  • Transposon mutagenesis has been successfully used to generate P. profundum mutants

  • Two transposable elements, mini-Tn10 and mini-Tn5, have been effective, though mini-Tn5 showed less insertion bias

  • For targeted mutations, plasmid-based methods involving homologous recombination can be used

• Phenotypic characterization:

  • Growth rate comparison at different pressures (0.1 MPa vs. 28 MPa) and temperatures (4°C vs. 15°C vs. 20°C)

  • Calculate cold sensitivity ratio (growth rate at 15°C/growth rate at 4°C) and pressure sensitivity ratio (growth rate at 28 MPa/growth rate at 0.1 MPa)

• Complementation analysis:

  • To verify gene-phenotype relationships, reintroduce wild-type arcB into mutants

  • The gene with predicted promoter can be PCR amplified and ligated into a broad-host-range vector like pFL122

  • Transfer the construct into mutants via triparental mating and compare growth ratios with the original mutant containing only the vector

Similar genes involved in metabolism showed differential expression at different pressures, suggesting that metabolic pathways including the ADI pathway might play important roles in pressure adaptation .

How does the structure of P. profundum ArcB relate to its function under high pressure conditions?

While the specific structural details of P. profundum ArcB have not been fully elucidated, research on other pressure-adapted proteins and related ornithine carbamoyltransferases provides valuable insights:

• Structural adaptations to pressure:

  • Proteins from piezophiles often show increased flexibility and reduced compactibility

  • Key strategies may include reduced ion pair interactions, decreased hydrophobic core packing, and increased surface hydration

  • Based on studies of other pressure-adapted enzymes, ArcB likely contains modifications in loop regions and at subunit interfaces to maintain function under pressure

• Comparative structural analysis:

  • Comparing ArcB with its homologs from non-piezophilic organisms can reveal pressure adaptations

  • For related enzymes, researchers have used techniques like US-align and mTM-align to highlight common core regions and determine if topology is shared among different types of related proteins

  • Protein structure models can be visualized with tools like PyMOL or Chimera

• Functional implications:

  • Structural changes likely affect substrate binding, catalytic efficiency, and oligomerization

  • For catabolic OTCs like ArcB, CP cooperativity is an important feature that may be affected by pressure

  • Studying the enzyme under various pressure conditions can reveal how structural adaptations translate to functional differences

What is known about the regulation of arcB expression in P. profundum under different environmental conditions?

The regulation of arcB expression in P. profundum appears to be complex and responsive to multiple environmental factors:

• Pressure-dependent regulation:

  • Proteomic studies have shown that many proteins in P. profundum are differentially expressed at different pressures

  • Transcriptome analysis indicates that P. profundum is under greater stress at atmospheric pressure than at elevated pressure, reflecting its deep-sea origin

  • The most actively transcribed genes are located on chromosome 1, where a larger percentage of loci required for growth at high pressure are also found

• Metabolic regulation:

  • The ADI pathway, including arcB, is typically regulated by anaerobic conditions and arginine availability

  • In some organisms, the catabolic ornithine carbamoyltransferase (ArcB) can be induced by specific substrates; for example, in S. aureus, exogenous ornithine induces arcB1 transcription

  • Transcription may also be influenced by related metabolites and cellular redox state

• Regulatory mechanisms:

  • Two-component regulatory systems likely play roles in pressure and temperature adaptation

  • Several pressure and temperature-sensitive mutants in P. profundum have been found to have mutations in transcriptional regulators

  • Proteins involved in RNA processing and translation, such as RNA helicases, also affect pressure and temperature adaptation

• Experimental approaches:

  • Northern blot analysis can be used to examine arcB expression under different conditions

  • RNA extraction protocols have been established for P. profundum grown at various temperatures and pressures

  • Example protocol: Extract total RNA using RNAzol B method, electrophorese through 1.2% formaldehyde agarose, blot onto nylon membrane, and probe with labeled arcB fragments

How can recombinant ArcB be utilized in adaptation studies comparing piezophilic and non-piezophilic organisms?

Recombinant ArcB from P. profundum offers a valuable tool for comparative studies of enzyme adaptation:

• Comparative enzymatic studies:

  • Express recombinant ArcB from P. profundum alongside homologs from non-piezophilic organisms

  • Compare kinetic parameters (Km, kcat, thermal stability, pressure stability) across different temperatures and pressures

  • Examine substrate specificity and the reversibility of the reaction under various conditions

• Chimeric protein approaches:

  • Create chimeric proteins by swapping domains between piezophilic and non-piezophilic ArcB

  • This approach has been successful with other P. profundum genes like fabF, where specific domains were found to be responsible for pressure adaptation

  • Test which regions confer pressure adaptation or temperature sensitivity

• Heterologous complementation:

  • Express P. profundum arcB in arcB mutants of non-piezophilic organisms and vice versa

  • Assess whether the piezophilic ArcB can restore function under different pressure conditions

  • This approach can help identify if the adaptation is at the protein level or requires cellular context

• Integrated multi-omics approaches:

  • Combine enzymatic studies with transcriptomics, proteomics, and metabolomics

  • A proteomics study of P. profundum identified differentially expressed proteins at different pressures, including transporters and metabolic enzymes

  • The differential expression of key metabolic pathways, including glycolysis/gluconeogenesis (up-regulated at high pressure) and oxidative phosphorylation (up-regulated at atmospheric pressure), suggests these pathways are important in pressure adaptation

What are the technical challenges in working with recombinant P. profundum ArcB and how can they be overcome?

Working with recombinant proteins from piezophilic and psychrophilic organisms presents several technical challenges:

• Expression and solubility issues:

  • Proteins from psychrophilic organisms often misfold at standard expression temperatures

  • Solution: Express at lower temperatures (15-18°C) and use solubility-enhancing tags (MBP, SUMO, GST)

  • Alternative approach: Use cell-free protein synthesis systems that can operate at lower temperatures

• Pressure effects on protein structure and function:

  • Standard laboratory equipment isn't designed to maintain samples under high pressure

  • Solution: Specialized pressure vessels for protein expression and activity assays

  • Example setup: Using sealed Pasteur pipettes in water-cooled pressure vessels operating at 0.1-40 MPa has been effective for P. profundum cultures

• Enzyme stability during purification:

  • Enzymes adapted to high pressure may be less stable at atmospheric pressure

  • Solution: Perform purification steps quickly and at low temperatures

  • Add stabilizing agents like glycerol (10-20%) or specific ions based on the enzyme's requirements

• Activity assay limitations:

  • Measuring enzyme kinetics under pressure requires specialized equipment

  • Solution: Design comparative assays that can be performed immediately after pressure treatment

  • Use stopped-flow techniques when immediate measurement after pressure release is needed

• Protein crystallization challenges:

  • Psychrophilic/piezophilic proteins often have increased flexibility, making crystallization difficult

  • Solution: Screen a wider range of crystallization conditions at lower temperatures

  • Consider alternative structural approaches like cryo-EM or small-angle X-ray scattering

How might recombinant ArcB be used in studying deep-sea adaptation mechanisms beyond pressure and temperature?

Recombinant ArcB provides opportunities to explore broader adaptations of deep-sea organisms:

• Nutrient limitation studies:

  • Deep-sea environments have distinct nutrient profiles compared to surface waters

  • Research has shown that different hydrostatic pressures represent distinct ecosystems with their own particular nutrient limitations and abundances

  • Investigating how ArcB activity and regulation responds to varying nutrient conditions could reveal adaptation strategies

• Energy metabolism adaptations:

  • The ADI pathway provides an alternative energy source through arginine catabolism

  • Examining how this pathway integrates with other metabolic systems under deep-sea conditions could reveal novel energy conservation strategies

  • The differential regulation of phosphate transport seen in P. profundum under different pressures suggests that basic metabolic processes are adjusted to deep-sea conditions

• Redox balance mechanisms:

  • Deep-sea environments often have different oxygen availability

  • Since the ArcB-containing ADI pathway functions under anaerobic conditions, it may play a role in maintaining redox balance

  • Studies with other sensor kinases like ArcB from E. coli have shown sensitivity to cellular redox state , suggesting potential similar mechanisms in P. profundum

• Ecological interactions:

  • Using recombinant ArcB to study how P. profundum interacts with other deep-sea organisms

  • The enzyme might play roles in competitive or cooperative interactions based on arginine metabolism

What potential biotechnological applications could utilize pressure-adapted enzymes like P. profundum ArcB?

Pressure-adapted enzymes offer unique properties for various biotechnological applications:

• Biocatalysis under extreme conditions:

  • Piezophilic enzymes may catalyze reactions more efficiently under high pressure

  • High-pressure biocatalysis can improve reaction rates, change reaction specificity, and reduce side reactions

  • Potential applications in chemical synthesis, pharmaceutical production, and food processing

• Novel enzyme engineering platforms:

  • Understanding the structural basis of pressure adaptation could inform the design of engineered enzymes with enhanced stability

  • The modularity seen in recombination systems like bridge RNA-directed recombinases suggests approaches for engineering novel functions

  • Integration of pressure-adaptation motifs into industrial enzymes could improve their stability and performance

• Biosensors for high-pressure environments:

  • Enzymes like ArcB that respond to environmental conditions could be developed into biosensors

  • Applications could include deep-sea monitoring, high-pressure industrial processes, or food processing

• Model systems for understanding environmental adaptation:

  • P. profundum and its enzymes can serve as models for understanding how organisms adapt to extreme environments

  • This knowledge could inform synthetic biology approaches to developing organisms for extreme environments on Earth or potentially beyond

How could structural comparisons between catabolic and anabolic ornithine carbamoyltransferases inform evolutionary studies of enzyme directionality?

The existence of distinct catabolic (ArcB) and anabolic (ArgF) ornithine carbamoyltransferases presents an interesting case for studying enzyme evolution:

• Structural determinants of reaction directionality:

  • Structural analysis of both enzymes can reveal key differences that determine reaction preference

  • Research on ornithine carbamoyltransferases has shown that catabolic OTCs display CP cooperativity while anabolic ones typically don't

  • Comparative structural studies could identify specific amino acid residues or domains responsible for these functional differences

• Evolutionary relationships:

  • Phylogenetic analysis combining sequence and structural data could reveal the evolutionary history of these enzymes

  • Did one form evolve from the other, or did they evolve independently?

  • The case of S. aureus, where catabolic ArcB can function in arginine biosynthesis , suggests possible evolutionary pathways

• Methodological approaches:

  • Structural analysis using X-ray crystallography, cryo-EM, or computational modeling

  • Ancestral sequence reconstruction to investigate evolutionary trajectories

  • Directed evolution experiments to explore potential evolutionary pathways between the two enzyme types

• Implications for enzyme engineering:

  • Understanding the structural basis for reaction directionality could enable engineering of enzymes with controlled directionality

  • Potential applications in metabolic engineering where pathway direction needs to be tightly controlled

Why might I observe inconsistent activity of recombinant P. profundum ArcB expressed in E. coli?

Several factors can contribute to inconsistent activity of recombinant ArcB:

• Expression conditions:

  • Temperature: P. profundum is psychrophilic; expression at higher temperatures may cause misfolding

  • Solution: Express at lower temperatures (15-18°C) and for longer periods

  • Induction level: Too high induction can lead to inclusion bodies

  • Solution: Use lower IPTG concentrations (0.1-0.2 mM) for gentler induction

• Post-translational modifications:

  • P. profundum may require specific modifications not present in E. coli

  • Solution: Try expression in alternative hosts closer to P. profundum, such as Vibrio species

• Pressure effects on folding:

  • Proteins adapted to high pressure may not fold correctly at atmospheric pressure

  • Solution: If available, try expression under pressure or use pressure treatment after cell disruption

  • Example system: water-cooled pressure vessels operating at 28 MPa have been used for P. profundum cultures

• Buffer composition:

  • Ions and co-factors may be critical for activity

  • Solution: Screen different buffer compositions, particularly testing marine-like salt concentrations

  • For P. profundum cultures, marine broth supplemented with glucose and HEPES buffer has been effective

• Stability concerns:

  • Storage conditions may affect stability

  • Solution: Test stabilizing agents (glycerol, specific ions) and store at appropriate temperatures

What controls should be included when designing experiments to study pressure adaptations of ArcB?

Proper experimental design with appropriate controls is crucial for studying pressure adaptation:

• Enzyme controls:

  • Include homologous enzymes from non-piezophilic organisms (e.g., E. coli or V. cholerae OTC)

  • Use enzymes known to be pressure-sensitive and pressure-resistant as benchmarks

  • Include heat-inactivated enzyme samples as negative controls

• Pressure treatment controls:

  • Perform assays at multiple pressure points (e.g., 0.1 MPa, 10 MPa, 28 MPa, 50 MPa)

  • Include pressure cycling experiments to distinguish between reversible and irreversible effects

  • Control for temperature changes during pressurization/depressurization

• Activity assay controls:

  • Run assays in both forward and reverse directions when possible

  • Include substrate-free and enzyme-free controls

  • Verify linearity of the assay under your specific conditions

• Expression system controls:

  • Compare proteins expressed at different temperatures

  • Use different tags and tag-free versions to rule out tag interference

  • Consider testing the same protein expressed in different host organisms

• Statistical considerations:

  • Perform experiments with at least three biological replicates

  • Include technical replicates within each biological replicate

  • Use appropriate statistical tests to determine significance of differences

How should I address potential contamination issues when working with recombinant P. profundum proteins?

Contamination can be a significant challenge when working with recombinant proteins:

• Expression host contamination:

  • E. coli host proteins may co-purify with your target

  • Solution: Use multiple purification steps with different principles (e.g., affinity chromatography followed by ion exchange and size exclusion)

  • Western blot analysis with anti-His tag or specific antibodies can confirm identity

  • Mass spectrometry can identify contaminating proteins

• Nucleic acid contamination:

  • DNA/RNA can affect enzyme assays and downstream applications

  • Solution: Treat samples with nucleases (DNase I, RNase A) during purification

  • Include additional wash steps with high salt to remove nucleic acids

• Endotoxin contamination:

  • Important consideration if proteins will be used in biological assays

  • Solution: Use endotoxin removal columns or specific detergents (Triton X-114)

  • Test final preparations with LAL (Limulus Amebocyte Lysate) assay

• Microbial contamination during growth:

  • Especially relevant for long, low-temperature expressions

  • Solution: Add antibiotics appropriate for your expression vector

  • Work aseptically and check cultures for contamination regularly

• Protease contamination:

  • Can lead to degradation and inconsistent results

  • Solution: Add protease inhibitors during purification

  • Keep samples cold and process quickly

  • Consider using protease-deficient expression strains

What genomic and sequence resources are available for P. profundum studies?

Several resources are available for researchers studying P. profundum genes:

• Genome sequence and annotation:

  • Complete genome sequence is available through GenBank

  • Genome browser specifically for P. profundum SS9 was available at http://SS9.cribi.unipd.it

  • Current genomic data can be accessed through standard databases like NCBI

• Sequence analysis tools:

  • BLASTN and BLASTP for identifying related sequences

  • COG database for classifying ORFs

  • SubLoc 1.0 for cellular localization prediction

  • SignalP for signal peptide analysis

• Genomic DNA sources:

  • Commercial sources: ATCC offers genomic DNA from P. profundum strain SS9 (catalog number BAA-1253D-5)

  • The bacterial strain itself is also available as ATCC catalog number BAA-1253

• Sequence repositories:

  • GenBank accession numbers for specific P. profundum genes (e.g., fabF sequence: AF188707)

  • Protein structure databases may contain models or structures of related enzymes

What specialized equipment is needed for research on pressure adaptation in P. profundum ArcB?

Research on pressure adaptation requires some specialized equipment:

• Pressure vessels:

  • Water-cooled pressure vessels capable of operating at 0.1-40 MPa or higher

  • Pressure vessels must be temperature-controlled for proper experiments

• Culture systems for pressure experiments:

  • Sealed containers that can withstand pressure (e.g., Pasteur pipettes sealed with a Bunsen burner and bag sealer)

  • Systems that exclude air to avoid uneven hydrostatic pressure distribution and ensure anaerobic conditions

• Specialized assay equipment:

  • High-pressure spectrophotometers for real-time enzyme assays under pressure

  • Rapid sampling systems for assays immediately after pressure release

  • Pressure cycling devices for testing effects of repeated pressurization/depressurization

• Analytical instruments:

  • HPLC or LC-MS/MS for metabolite analysis

  • Circular dichroism spectroscopy for protein structure analysis under pressure

  • Fluorescence spectroscopy for protein folding studies

• Computational resources:

  • Software for protein structure analysis (PyMOL, Chimera)

  • Tools for comparative genomics and sequence analysis

  • Molecular dynamics simulation packages capable of simulating high-pressure conditions

Are there established research collaborations or consortia studying P. profundum and deep-sea adaptations?

While the search results don't specifically mention formal consortia, the P. profundum research community appears to be active and collaborative:

• Research groups:

  • Multiple research groups have contributed to P. profundum studies, as evidenced by the publications

  • Collaborative efforts have led to genome sequencing, transcriptome analysis, and proteomics studies

• Resources sharing:

  • Strain repositories like ATCC maintain P. profundum strains and genomic DNA

  • Database resources are available for genome exploration

• Funding opportunities:

  • Deep-sea research often receives funding from organizations interested in marine biology, extremophiles, and biotechnology

  • National science foundations, space agencies (due to interest in extremophiles), and marine research institutes

• Collaborative approaches:

  • The nature of deep-sea research often requires collaborative efforts due to the specialized equipment needed

  • Interdisciplinary collaborations between microbiologists, biochemists, structural biologists, and marine scientists are common