Recombinant Photobacterium profundum Carbon storage regulator homolog (csrA)

What is Photobacterium profundum and why is its CsrA protein of interest?

Photobacterium profundum is a deep-sea gammaproteobacterium belonging to the family Vibrionaceae that has been isolated from various marine environments, including the Sulu Sea . It is a gram-negative rod with adaptations for growth across temperatures ranging from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa, depending on the strain . The bacterium has two circular chromosomes and demonstrates a remarkable ability to thrive under high-pressure conditions .

The Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that enables bacteria to respond to environmental changes by regulating various metabolic pathways, motility, biofilm formation, and virulence-associated genes . In P. profundum, which must adapt to extreme environments, the CsrA homolog likely plays a crucial role in regulating gene expression to optimize survival under varying conditions of pressure, temperature, and nutrient availability.

How does the CsrA protein function as a regulator at the molecular level?

CsrA functions as a post-transcriptional regulator by binding to specific RNA sequences, particularly those containing GGA motifs often located within the loops of hairpin structures . The protein forms a homodimer with two RNA-binding surfaces, allowing it to potentially interact with two binding sites simultaneously on a single transcript . This "bridging" can occur when binding sites are separated by ≥10 to ≤63 nucleotides, with an optimal intersite distance of >18 nucleotides .

The consensus sequence for a high-affinity CsrA binding site has been determined through systematic evolution of ligands by exponential enrichment (SELEX) as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved . The binding affinity is influenced by both the RNA primary sequence and its structural context, with very stable secondary structures potentially being disadvantageous for CsrA binding . When CsrA binds to mRNA, it can either repress translation by blocking ribosome access to the Shine-Dalgarno sequence or alter mRNA stability.

What experimental approaches are most effective for expressing recombinant P. profundum CsrA?

For expressing recombinant P. profundum CsrA, researchers should consider the following methodological approach:

• Vector selection: Use expression vectors with strong inducible promoters (such as T7) and include affinity tags (His6 or GST) to facilitate purification.

• Expression host optimization: While E. coli BL21(DE3) is commonly used, consider cold-adapted expression hosts for proteins from psychrophilic organisms like P. profundum. Expression at lower temperatures (15-18°C) may improve protein folding and solubility.

• Induction conditions: For proteins from pressure-adapted organisms, optimize induction parameters by testing various concentrations of inducer (0.1-1.0 mM IPTG) and induction temperatures (10-25°C) to maximize soluble protein yield.

• Purification strategy: Implement a multi-step purification protocol including:

  • Initial affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography to remove nucleic acid contaminants

  • Size exclusion chromatography to obtain highly pure dimeric protein

Given that CsrA forms dimers and binds RNA, special attention should be paid to removing nucleic acid contaminants during purification to ensure the recombinant protein is not pre-bound to E. coli RNA.

How do you verify the RNA-binding activity of purified recombinant CsrA?

Verification of RNA-binding activity for purified recombinant CsrA should involve multiple complementary approaches:

• Electrophoretic Mobility Shift Assay (EMSA): This is the gold standard for demonstrating direct binding of recombinant CsrA to target RNA sequences . Synthesize short RNA oligonucleotides containing the consensus binding sequence (with GGA motifs) and incubate with increasing concentrations of purified CsrA. RNA-protein complexes will migrate more slowly through native polyacrylamide gels compared to free RNA.

• Fluorescence anisotropy: Label RNA targets with fluorescent dyes and measure changes in anisotropy upon protein binding to determine binding kinetics and affinity constants.

• Surface Plasmon Resonance (SPR): Immobilize biotinylated RNA targets on streptavidin-coated sensor chips and flow purified CsrA protein at various concentrations to obtain real-time binding data.

• Filter binding assays: Use radiolabeled RNA and measure retention on nitrocellulose filters after incubation with CsrA to quantify binding.

• Functional validation: Test whether the recombinant CsrA can complement a csrA deletion mutant in a model organism, restoring phenotypes such as glycogen accumulation, motility, or biofilm formation.

These assays should be performed under conditions simulating P. profundum's natural environment, including testing the effects of high pressure and low temperature on binding efficiency where possible.

What approaches can be used to identify the complete regulon of CsrA in P. profundum?

Identifying the complete regulon of CsrA in P. profundum requires a multi-faceted approach combining various high-throughput methodologies:

• RNA-seq of mutant vs. wild-type strains: Generate a csrA deletion mutant and compare its transcriptome to wild-type P. profundum under various conditions (different pressures, temperatures, nutrient states). This approach has been successfully applied in other organisms such as Leptospira, where 575 transcripts were differentially expressed when csrA was overexpressed .

• CLIP-seq (Cross-linking Immunoprecipitation followed by sequencing): This technique involves:

  • In vivo UV cross-linking of RNA-protein complexes

  • Immunoprecipitation of CsrA-RNA complexes using CsrA-specific antibodies

  • RNase treatment to trim bound RNA fragments

  • Sequencing of protected RNA fragments to identify binding sites

• Ribosome profiling: Compare ribosome-protected mRNA fragments between wild-type and csrA mutant strains to identify transcripts with altered translation efficiency.

• In vitro screening using genomic SELEX: Incubate recombinant CsrA with a library of RNA fragments generated from the P. profundum genome, followed by selection and sequencing of bound RNAs.

• Bioinformatic prediction: Develop algorithms to scan the P. profundum genome for potential CsrA binding motifs based on the consensus sequence RUACARGGAUGU, followed by experimental validation .

Integration of these datasets would provide a comprehensive view of direct and indirect CsrA targets in P. profundum, especially when performed under conditions simulating deep-sea environments.

How does pressure affect CsrA-mediated regulation in P. profundum?

The effect of pressure on CsrA-mediated regulation in P. profundum likely involves complex adaptations specific to piezophilic bacteria:

• Pressure-dependent expression of CsrA: In P. profundum strain SS9 (a piezophile), several stress response genes are upregulated in response to atmospheric pressure, including htpG, dnaK, dnaJ, and groEL . Research should determine whether csrA itself is differentially expressed under varying pressure conditions using qRT-PCR and Western blotting.

• Pressure effects on CsrA binding affinity: High hydrostatic pressure can affect protein-nucleic acid interactions. Design experiments using fluorescence-based binding assays in pressure chambers to measure binding kinetics of CsrA to target RNAs under pressures ranging from 0.1 to 70 MPa.

• Pressure-specific regulon: Compare RNA-seq data from CsrA mutants grown under atmospheric versus high pressure (28 MPa for strain SS9) to identify pressure-specific targets of CsrA regulation.

• Structural adaptations: Investigate how the structure of P. profundum CsrA compares to homologs from non-piezophilic bacteria using techniques such as X-ray crystallography or cryo-EM under varying pressure conditions. Look specifically for amino acid substitutions that might confer pressure resistance.

• Membrane adaptation connection: P. profundum alters its fatty acid composition in response to pressure and temperature . Investigate whether CsrA directly or indirectly regulates genes involved in membrane lipid biosynthesis to facilitate adaptation to high pressure.

How do the mechanisms and targets of CsrA differ between piezophilic and non-piezophilic bacteria?

The mechanisms and targets of CsrA likely differ substantially between piezophilic bacteria like P. profundum and non-piezophilic bacteria due to their adaptation to different environmental pressures:

• Comparative genomics approach: Compare the genomes of P. profundum strains (especially SS9, which grows optimally at 28 MPa) with related non-piezophilic bacteria like Vibrio species to identify unique genes potentially regulated by CsrA in piezophiles .

• Heterologous expression studies: Express P. profundum CsrA in non-piezophilic model organisms (e.g., E. coli) and evaluate whether it can complement a csrA deletion and which phenotypes are restored versus those that remain altered.

• Domain swap experiments: Create chimeric CsrA proteins with domains from piezophilic and non-piezophilic bacteria to identify regions responsible for pressure-specific functions.

• RNA target comparison: Using RNA immunoprecipitation followed by sequencing (RIP-seq), compare the RNA targets of CsrA from P. profundum with those from non-piezophilic bacteria under various pressure conditions.

• Structural biology: Compare the three-dimensional structures of CsrA proteins from piezophilic and non-piezophilic bacteria to identify structural adaptations that might allow function under high pressure.

The differences observed may be analogous to those seen between pathogenic and non-pathogenic Leptospira strains, where the mechanisms of action and gene targets of CsrA differ significantly .

What role does CsrA play in regulating motility and flagellar gene expression in P. profundum?

Based on studies in other bacteria, including Leptospira, CsrA likely plays a significant role in regulating motility and flagellar gene expression in P. profundum:

• Flagellar gene regulation: In Leptospira, CsrA directly binds to flaB3 mRNA in non-pathogenic strains and to flaB4 mRNA in pathogenic strains . For P. profundum, investigate whether CsrA regulates flagellar filament proteins through:

  • Direct RNA binding assays with flagellar gene transcripts

  • Promoter-reporter fusion studies to quantify regulation

  • Motility assays comparing wild-type and csrA mutant strains

• Pressure-dependent motility regulation: Determine if CsrA differentially regulates motility genes under varying pressure conditions, as P. profundum has two flagella systems and motility is crucial for adaptation to changing environments .

• Competitive advantage investigation: Assess whether CsrA-mediated regulation of motility provides a competitive advantage in colonizing specific deep-sea niches through mixed culture competition experiments.

• Regulatory network mapping: Map the complete regulatory network connecting CsrA to flagellar gene expression, including potential intermediate regulators and environmental sensing mechanisms.

• Comparative analysis with pressure-adapted motility: Compare the CsrA-dependent motility regulation in P. profundum with that in non-piezophilic bacteria to identify pressure-specific adaptations.

A proposed model of this regulation based on available data is presented in Table 1.

Table 1: Comparative Model of CsrA-Mediated Flagellar Gene Regulation

How can CRISPR-Cas9 technology be optimized for studying CsrA function in P. profundum?

Optimizing CRISPR-Cas9 technology for P. profundum requires addressing several unique challenges related to its deep-sea origin:

• Pressure-stable delivery system: Develop electroporation protocols that account for the pressure-adapted membrane properties of P. profundum, potentially using high-pressure electroporation chambers.

• Temperature-adapted protocols: Modify standard CRISPR protocols to function efficiently at the lower optimal growth temperatures of P. profundum (15°C for strain SS9) .

• Guide RNA design considerations:

  • Account for the high GC content in targeting regions

  • Optimize for activity under low temperature conditions

  • Design multiple guides targeting different regions of the csrA gene

• Selective marker optimization: Select antibiotic resistance markers that function effectively under high-pressure conditions and are appropriate for marine bacteria.

• Inducible CRISPR systems: Develop pressure-inducible or temperature-inducible promoters to control Cas9 expression, allowing for conditional inactivation of csrA.

• Validation approaches:

  • Verify gene editing through sequencing

  • Confirm phenotypic changes through motility assays

  • Validate using Western blotting for CsrA protein levels

  • Perform RNA-seq to confirm downstream regulatory effects

• Base editing alternatives: Consider using CRISPR base editors rather than double-strand break repair systems if homologous recombination efficiency is low in P. profundum.

What structural features distinguish P. profundum CsrA from homologs in non-piezophilic bacteria?

The structural features of P. profundum CsrA likely reflect adaptations to function under high pressure and low temperature conditions:

• Amino acid composition analysis: Compare the amino acid composition of P. profundum CsrA with homologs from non-piezophilic bacteria, looking for:

  • Increased proportion of amino acids that confer pressure stability (such as glycine and alanine)

  • Decreased content of pressure-sensitive residues (such as proline)

  • Modified surface charge distribution to maintain protein-RNA interactions under pressure

• Structural flexibility determinants: Investigate regions of structural flexibility that might allow the protein to maintain function across a wide pressure range (0.1-70 MPa).

• Dimer interface analysis: Examine whether the dimer interface of P. profundum CsrA has specific adaptations to maintain stability under high pressure, potentially through increased hydrophobic interactions or additional salt bridges.

• RNA-binding pocket comparison: Analyze the RNA-binding pocket for adaptations that preserve RNA-binding specificity and affinity under high pressure conditions.

• Pressure effects on protein dynamics: Use molecular dynamics simulations to predict how high pressure affects the structural dynamics of P. profundum CsrA compared to non-piezophilic homologs.

These structural features should be experimentally validated through techniques such as X-ray crystallography or cryo-EM, ideally under various pressure conditions.

How does temperature interact with pressure to affect CsrA function in P. profundum?

The interplay between temperature and pressure is a key aspect of P. profundum adaptation and likely affects CsrA function:

• Combined temperature-pressure experiments: Design experiments that simultaneously vary temperature (0-25°C) and pressure (0.1-70 MPa) to identify optimal conditions for CsrA function in different P. profundum strains.

• Strain-specific adaptations: Compare CsrA function between P. profundum strains adapted to different conditions:

  • Strain SS9 (optimal growth at 15°C and 28 MPa)

  • Strain 3TCK (optimal growth at 9°C and 0.1 MPa)

  • Strain DSJ4 (optimal growth at 10°C and 10 MPa)

• RNA-binding thermodynamics under pressure: Measure binding constants of CsrA to target RNAs under different temperature-pressure combinations to generate a comprehensive activity landscape.

• Gene expression profiling: Use RNA-seq to identify genes regulated by CsrA under various temperature-pressure combinations, focusing on how the regulon changes with environmental conditions.

• Stability analysis: Assess the thermal and pressure stability of recombinant CsrA protein using techniques such as differential scanning calorimetry under pressure to determine how these environmental factors affect protein stability.

These studies would provide insights into how CsrA helps P. profundum adapt to its extreme environment through coordinated responses to both temperature and pressure changes.