Recombinant Photobacterium profundum Protein ProQ homolog (proQ)

What is ProQ and what is its primary function in bacterial cells?

ProQ is an RNA-binding protein belonging to the FinO/ProQ family that functions as a global post-transcriptional regulator and RNA chaperone . Unlike the well-characterized RNA chaperones Hfq and CsrA, ProQ binds to a distinct set of RNAs characterized by a high degree of structure . In bacteria such as P. profundum, ProQ plays crucial roles in adapting to environmental stresses, particularly high hydrostatic pressure conditions .

The primary function of ProQ is to bind and stabilize structured RNAs, including small regulatory RNAs (sRNAs) and certain mRNAs. It recognizes its targets in a sequence-independent manner through RNA structural motifs, particularly intrinsic terminators and stem-loop structures . ProQ can protect the 3'-ends of mRNAs from exonucleolytic degradation and facilitate sRNA-mRNA interactions, thereby regulating gene expression at the post-transcriptional level .

How does the structure of ProQ facilitate its RNA-binding function?

The ProQ protein contains a FinO domain with a distinctive fold that enables RNA binding. The solution NMR structure of Lpp1663, a minimal ProQ homolog from Legionella pneumophila, reveals that ProQ adopts a fold consisting of five alpha helices arranged to form a concave binding surface . This structure strongly resembles the prototypic ProQ/FinO domain fold with a Cα-rmsd of 1.8 Å to E. coli FinO and 1.9 Å to N. meningitidis NMB1681 .

The concave face of the FinO domain serves as the main RNA-binding site, with several conserved amino acid residues critical for RNA binding. These include R58, Y70, and R80, which are essential for binding all tested RNAs, while K54 and R62 show more moderate binding contributions . Interestingly, some evolutionary variable residues like K35 and R69 show varied effects on binding different RNAs, suggesting that these residues may tune interactions with specific RNA ligands .

ProQ recognizes single-stranded uridine-rich RNA sequences in the vicinity of stable stem-loop structures, with the single-stranded U-rich RNAs interacting mainly with the conserved RNA-binding surface on the concave site of the protein .

What experimental methods are commonly used to study ProQ-RNA interactions?

Several experimental approaches have been developed to study ProQ-RNA interactions:

• RNA Immunoprecipitation (RIP): This technique involves crosslinking RNA-protein complexes in vivo, immunoprecipitating ProQ with specific antibodies, and identifying bound RNAs through sequencing .

• Bacterial Three-Hybrid (B3H) Assay: This genetic screening approach can identify amino acid residues important for RNA binding. The assay has been used to confirm the importance of the concave face of ProQ in RNA recognition .

• Gel Shift Assays: Electrophoretic mobility shift assays (EMSAs) provide a direct method to probe the contributions of specific amino acids to RNA binding and can be quantified to determine binding affinities .

• RIL-seq (RNA Interaction by Ligation and sequencing): This method identifies RNA-RNA interactions mediated by RNA-binding proteins. RNAs are crosslinked to the protein (e.g., ProQ), the protein is immunoprecipitated, and proximal RNA ends are ligated, creating chimeric fragments that can be sequenced to identify interaction partners .

• Isothermal Titration Calorimetry (ITC): This biophysical technique can characterize the thermodynamics of ProQ-RNA binding interactions and determine binding affinities and stoichiometry .

• NMR Spectroscopy: Chemical shift perturbation experiments can map RNA binding surfaces on ProQ and identify key interacting residues .

How does P. profundum ProQ differ from ProQ homologs in other bacteria?

While ProQ homologs share structural similarities across bacterial species, there are important differences in their functions and regulatory networks:

Unlike ProQ in E. coli, which displays impaired biofilm formation when mutated, the ProQ mutant in D. dadantii shows increased adherence, illustrating species-specific regulatory networks . These differences likely result from the distinct sRNA landscapes produced by different bacterial species, with only small numbers of sRNA homologs overlapping between species .

How does ProQ contribute to pressure adaptation in P. profundum SS9?

P. profundum SS9 grows optimally at 28 MPa pressure, and its ability to adapt to high-pressure environments involves complex gene regulation networks in which ProQ plays a significant role. Proteomic analysis has revealed that P. profundum differentially expresses proteins involved in key metabolic pathways under different pressure conditions .

Under high pressure (28 MPa), proteins involved in the glycolysis/gluconeogenesis pathway are up-regulated, while under atmospheric pressure (0.1 MPa), several proteins involved in the oxidative phosphorylation pathway are up-regulated . ProQ likely contributes to these adaptations by regulating the expression of specific mRNAs and facilitating sRNA-mediated gene regulation in response to pressure changes.

The role of ProQ in pressure adaptation can be methodologically studied by:

• Creating ProQ deletion mutants and testing their growth under various pressure conditions

• Performing RNA-seq and proteomic analyses on wild-type and ΔproQ strains grown under different pressures

• Identifying ProQ-bound RNAs under various pressure conditions using RIP-seq or CLIP-seq

• Testing whether recombinant ProQ can complement pressure-sensitive phenotypes when expressed in ProQ-deficient strains

Research has shown that other genes like recD, which encodes a component of the DNA recombination and repair machinery, are also required for high-pressure growth in P. profundum SS9 . Future research should investigate potential connections between ProQ and the DNA repair machinery in pressure adaptation.

What is the relationship between ProQ and other RNA chaperones like Hfq in bacterial post-transcriptional regulation?

ProQ and Hfq are both RNA chaperones, but they generally bind different sets of RNAs and play distinct but sometimes overlapping roles in bacterial post-transcriptional regulation. Understanding their relationship is crucial for deciphering the complete RNA regulatory networks in bacteria.

Experimental approaches to investigate this relationship include:

• Comparative RIP-seq analysis: Immunoprecipitate both ProQ and Hfq from the same bacterial culture and sequence the bound RNAs to identify unique and overlapping targets.

• Double mutant phenotype analysis: Create single (ΔproQ and Δhfq) and double (ΔproQ Δhfq) mutants and compare their phenotypes to identify independent, competing, or additive roles.

• RIL-seq for both proteins: This can identify RNA-RNA interactions mediated by each chaperone, revealing their distinct regulatory networks.

Studies in D. dadantii have shown that deleting both hfq and proQ leads to more severe virulence defects than single deletions, suggesting they have both overlapping and independent functions . Interestingly, ProQ expression levels were increased in the hfq mutant, indicating that Hfq might regulate ProQ production directly or indirectly .

The distinct binding preferences of these chaperones also contribute to their different functions:

Although there is some overlap in the RNAs bound by these chaperones, they generally regulate different subsets of the bacterial transcriptome .

How can one design and optimize the expression and purification of recombinant P. profundum ProQ for structural and functional studies?

Methodological approach for expression and purification of recombinant P. profundum ProQ:

• Vector design: Clone the proQ gene from P. profundum SS9 into an expression vector with a suitable affinity tag (His6, GST, or MBP) to facilitate purification. Consider using a vector with an inducible promoter (e.g., T7) for controlled expression.

• Expression optimization:

  • Test multiple E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Optimize induction conditions (IPTG concentration, temperature, duration)

  • Perform small-scale expression tests at different temperatures (16°C, 25°C, 37°C) as lower temperatures may improve protein folding

  • Consider co-expression with chaperones if the protein shows poor solubility

• Purification strategy:

  • Perform affinity chromatography using the appropriate resin for the chosen tag

  • Include an ion-exchange chromatography step to remove nucleic acid contaminants

  • Employ size-exclusion chromatography for final polishing and buffer exchange

  • Verify protein purity by SDS-PAGE and western blotting

• Quality control:

  • Assess RNA contamination by measuring A260/A280 ratio (should be close to 0.57 for pure protein)

  • Verify proper folding using circular dichroism spectroscopy

  • Confirm RNA-binding activity using electrophoretic mobility shift assays with known RNA targets

For functional studies, it's critical to ensure the recombinant protein is free of bound RNA from the expression host. RNase treatment followed by heparin affinity chromatography can effectively remove contaminating nucleic acids.

What are the methodological challenges in identifying direct ProQ-regulated genes in P. profundum, and how can they be addressed?

Identifying direct ProQ-regulated genes in P. profundum presents several challenges and requires a multi-faceted approach:

Challenge 1: Growth under high pressure conditions

• Solution: Use specialized high-pressure cultivation systems that maintain P. profundum at its optimal growth pressure (28 MPa) while allowing for sample collection.

• Method: Culture P. profundum in sealed Pasteur pipettes or specialized pressure vessels at 28 MPa and 15°C as described in previous studies .

Challenge 2: Distinguishing direct from indirect regulation

• Solution: Combine multiple high-throughput techniques to identify both ProQ-bound RNAs and expression changes upon ProQ deletion.

• Method: Implement CLIP-seq (crosslinking immunoprecipitation followed by sequencing) to identify direct ProQ-RNA interactions, complemented by RNA-seq of wild-type and ΔproQ strains to detect expression changes.

Challenge 3: Confirming functional regulatory interactions

• Solution: Validate selected ProQ-RNA interactions using in vitro and in vivo approaches.

• Method: Perform gel shift assays with purified recombinant ProQ and candidate RNA targets, followed by reporter gene assays to confirm regulation in vivo.

Challenge 4: Accounting for pressure-specific regulation

• Solution: Compare ProQ-RNA interactions under different pressure conditions.

• Method: Perform CLIP-seq experiments with wild-type P. profundum grown at both atmospheric pressure (0.1 MPa) and high pressure (28 MPa).

A comprehensive workflow might include:

• Generation of a P. profundum strain expressing epitope-tagged ProQ for immunoprecipitation

• Parallel CLIP-seq analysis under different pressure conditions

• RNA-seq of wild-type and ΔproQ strains under matching conditions

• Bioinformatic integration of datasets to identify high-confidence direct targets

• Validation of selected targets using in vitro binding assays and genetic complementation studies

How does the RNA-binding specificity of ProQ influence its regulatory function in different bacterial species?

The RNA-binding specificity of ProQ varies somewhat between bacterial species, influencing its regulatory functions:

ProQ binds to RNA through its FinO domain, recognizing structural features rather than specific sequences. Biochemical and genetic dissection of the RNA-binding surface of ProQ has identified critical residues that contribute to RNA recognition . Some highly conserved residues (R58, Y70, and R80) are essential for binding all tested RNAs, while other more variable residues (K35, R69) show differential effects on binding different RNA targets .

These binding preferences lead to species-specific regulatory networks:

• In E. coli and Salmonella: ProQ binds numerous sRNAs and mRNAs, particularly focusing on structured RNAs like intrinsic terminators. This affects processes including osmotic regulation, virulence, and membrane homeostasis .

• In P. profundum: ProQ likely focuses on regulating genes involved in pressure adaptation, potentially including metabolic pathways that are differentially regulated under varying pressure conditions .

• In D. dadantii: ProQ regulates virulence-associated genes, but in patterns distinct from Hfq regulation. The proQ mutant exhibited decreased levels of fliC mRNA but repression of major virulence genes (pel, prt, cel) .

• In E. amylovora: ProQ influences exopolysaccharide production and has been linked to c-di-GMP metabolism, affecting biofilm formation and virulence .

To study these species-specific differences methodologically:

• Perform comparative CLIP-seq across multiple bacterial species expressing epitope-tagged ProQ

• Identify shared and species-specific binding motifs using computational analysis

• Test cross-complementation by expressing ProQ from one species in another species' proQ mutant

• Create chimeric ProQ proteins combining domains from different species to map functional specificity

What experimental approaches can be used to study pressure-dependent ProQ function in P. profundum?

Studying pressure-dependent ProQ function requires specialized equipment and methodologies:

• High-pressure cultivation systems:

  • Use stainless steel pressure vessels with temperature control

  • Employ sealed Pasteur pipettes placed within pressure chambers as described in previous studies

  • Maintain anaerobic conditions by excluding air to ensure even pressure distribution

• Genetic manipulation under pressure:

  • Generate ProQ variants with mutations in key RNA-binding residues

  • Create reporter gene fusions to monitor ProQ-dependent gene expression under different pressures

  • Perform complementation studies with wild-type and mutant ProQ variants

• Molecular analysis of pressure-dependent ProQ-RNA interactions:

  • Implement in vivo crosslinking immediately upon pressure release to capture interactions

  • Perform CLIP-seq or RIP-seq under different pressure conditions

  • Use structure probing techniques to analyze RNA conformational changes under pressure

• Proteomic approaches:

  • Apply label-free quantitative proteomic analysis to compare wild-type and ΔproQ strains under different pressures

  • Use SILAC (Stable Isotope Labeling by Amino acids in Cell culture) for more accurate quantification

  • Focus on proteins previously identified as pressure-regulated

A comparative analysis workflow might include:

This systematic approach would help identify pressure-specific functions of ProQ in P. profundum.

How can RNA sequencing-based approaches be applied to map the complete ProQ regulon in P. profundum?

Mapping the complete ProQ regulon in P. profundum requires a combination of RNA sequencing-based approaches:

• Differential RNA-seq (dRNA-seq) of wild-type vs. ΔproQ strains:

  • Grow both strains under identical conditions (atmospheric and high pressure)

  • Extract total RNA and prepare libraries for sequencing

  • Compare transcriptome profiles to identify differentially expressed genes

  • Focus on changes in both coding and non-coding RNAs

• ProQ-CLIP-seq (Cross-Linking Immunoprecipitation and sequencing):

  • Express epitope-tagged ProQ in P. profundum

  • Perform UV crosslinking to capture direct RNA-protein interactions

  • Immunoprecipitate ProQ-RNA complexes

  • Sequence bound RNAs to identify direct ProQ targets

• RIL-seq (RNA Interaction by Ligation and sequencing):

  • Crosslink RNA-ProQ-RNA complexes in vivo

  • Perform ProQ immunoprecipitation

  • Ligate proximal RNA ends to create chimeric fragments

  • Sequence to identify ProQ-mediated RNA-RNA interactions

• Term-seq for 3' end mapping:

  • Map the 3' termini of transcripts in wild-type and ΔproQ strains

  • Identify changes in termination patterns and RNA stability

  • Focus on intrinsic terminators that might serve as ProQ binding sites

Data integration and analysis:

The integration of these datasets would allow for the identification of:

• Directly bound ProQ targets

• Indirectly regulated genes

• ProQ-mediated sRNA-mRNA interactions

• Transcripts stabilized by ProQ binding at their 3' ends

This comprehensive approach would provide a detailed map of the ProQ regulon in P. profundum and insight into pressure-responsive regulation.

What is the role of ProQ in coordinating with other cellular processes such as DNA recombination and repair in P. profundum?

The connection between ProQ and DNA recombination/repair processes in P. profundum is an intriguing area for investigation, especially considering that recD, a DNA recombination and repair gene, is required for high-pressure growth .

Methodological approaches to investigate this connection:

• Comparative phenotypic analysis of single and double mutants:

  • Generate ΔproQ, ΔrecD, and ΔproQ ΔrecD double mutants

  • Compare growth curves under various pressure conditions

  • Assess DNA damage sensitivity using UV radiation or DNA-damaging chemicals

  • Measure mutation rates using rifampicin resistance assays

• Transcriptomic analysis of DNA repair pathways:

  • Perform RNA-seq on wild-type and ΔproQ strains grown at different pressures

  • Focus analysis on expression changes in DNA repair and recombination genes

  • Verify key findings with RT-qPCR

• ProQ-RNA interaction studies focused on repair machinery:

  • Use CLIP-seq to identify if ProQ directly binds mRNAs encoding DNA repair proteins

  • Test direct binding of recombinant ProQ to recD mRNA and other repair-related transcripts

  • Investigate if ProQ regulates sRNAs that target DNA repair machinery

• Proteomics of DNA repair complexes:

  • Perform co-immunoprecipitation with tagged ProQ to identify potential protein partners

  • Use mass spectrometry to detect DNA repair proteins that might associate with ProQ

  • Validate interactions with bacterial two-hybrid or co-immunoprecipitation experiments

The integration of these approaches would help elucidate whether ProQ plays a direct role in regulating DNA repair processes under high-pressure conditions, which could explain why both recD and ProQ are important for P. profundum growth at high pressures. This investigation might reveal novel regulatory pathways connecting RNA metabolism with DNA maintenance that are particularly important in extreme environments.

What are common pitfalls when expressing recombinant P. profundum ProQ in heterologous systems, and how can they be overcome?

Expressing recombinant P. profundum ProQ in heterologous systems can present several challenges:

Challenge 1: Poor solubility or inclusion body formation

• Problem: ProQ may fold incorrectly at standard expression temperatures.

• Solution: Lower the expression temperature to 16-18°C and induce with a reduced IPTG concentration (0.1-0.5 mM).

• Alternative approach: Fuse ProQ to solubility-enhancing tags such as MBP (maltose-binding protein) rather than smaller His-tags.

Challenge 2: RNA contamination during purification

• Problem: ProQ's natural RNA-binding ability leads to co-purification of host RNAs.

• Solution: Include high-salt washes (up to 1M NaCl) during purification and treat with RNase A followed by heparin affinity chromatography.

• Verification method: Check A260/A280 ratio; pure protein should have a ratio of approximately 0.57.

Challenge 3: Protein instability or degradation

• Problem: ProQ may be unstable in standard buffer conditions.

• Solution: Optimize buffer conditions by screening different pH values (6.5-8.5), salt concentrations (150-500 mM NaCl), and add stabilizing agents (5-10% glycerol, 1 mM DTT).

• Storage recommendation: Store purified protein in small aliquots at -80°C to avoid freeze-thaw cycles.

Challenge 4: Loss of RNA-binding activity

• Problem: Recombinant ProQ may not retain full activity after purification.

• Solution: Verify activity using electrophoretic mobility shift assays with known RNA targets.

• Activity rescue: Test different buffer conditions for binding assays, including the addition of magnesium ions (1-5 mM MgCl₂).

Challenge 5: Species-specific codon usage affecting expression

• Problem: P. profundum codon usage may not be optimal for E. coli expression.

• Solution: Use codon-optimized synthetic genes or express in Rosetta strains that supply rare tRNAs.

• Assessment method: Compare expression levels between standard BL21(DE3) and Rosetta strains.

A systematic optimization workflow comparing different expression conditions might include:

How can researchers address contradictory findings about ProQ function between different bacterial species?

Contradictory findings about ProQ function across bacterial species present a scientific challenge that requires systematic investigation. Here's a methodological framework to address such discrepancies:

• Standardize experimental conditions:

  • Use identical growth phases and media conditions when comparing ProQ function across species

  • Ensure genetic manipulations (knockouts, complementation) are performed using similar strategies

  • Apply consistent analytical methods across all species being compared

• Cross-species complementation studies:

  • Express ProQ from one species in another species' proQ mutant

  • Assess the degree of functional rescue for various phenotypes

  • Create a complementation matrix across multiple species to identify functional conservation and divergence

• Domain swap experiments:

  • Construct chimeric ProQ proteins with domains from different species

  • Express these chimeras in various proQ mutant backgrounds

  • Map functional specificity to particular protein domains

• Comparative omics approaches:

  • Perform parallel RNA-seq and ProQ-CLIP-seq in multiple species

  • Use consistent bioinformatic pipelines to analyze the data

  • Identify core conserved targets versus species-specific targets

• Evolutionary context analysis:

  • Conduct phylogenetic analysis of ProQ across bacterial lineages

  • Correlate functional differences with evolutionary distance

  • Consider horizontal gene transfer events that might explain functional divergence

A decision-making flowchart for addressing contradictory findings:

• Are the experimental conditions truly comparable?

  • If no: Standardize conditions and repeat key experiments

  • If yes: Proceed to step 2

• Is the contradiction at the phenotypic or molecular level?

  • Phenotypic: Test if the phenotype is directly or indirectly regulated by ProQ

  • Molecular: Compare direct binding targets using CLIP-seq or similar approaches

• Does cross-species complementation resolve the contradiction?

  • If yes: Differences may be due to expression levels or host factors

  • If no: Intrinsic functional differences exist between ProQ homologs

• Are differences explained by co-evolution with specific sRNA repertoires?

  • Analyze correlation between ProQ binding specificity and sRNA repertoire

  • Test if introducing species-specific sRNAs alters ProQ function

This systematic approach can help determine whether contradictory findings reflect true biological differences in ProQ function across species or are artifacts of experimental variation.

What are promising directions for engineering ProQ-based tools for synthetic biology applications?

ProQ's unique RNA-binding properties make it a promising candidate for engineering synthetic biology tools:

• RNA stability modulation system:

  • Engineer ProQ variants with tunable binding affinities

  • Design synthetic binding sites that can be inserted into target transcripts

  • Create an inducible system to control ProQ expression levels

  • Application: Stabilize mRNAs encoding difficult-to-express proteins or metabolic enzymes

• Synthetic RNA regulatory circuits:

  • Design artificial sRNAs with ProQ-binding motifs

  • Create synthetic mRNA targets with complementary sequences

  • Use ProQ as a mediator to facilitate specific sRNA-mRNA interactions

  • Application: Build genetic circuits with post-transcriptional control layers

• Pressure-responsive gene expression system:

  • Leverage P. profundum ProQ's involvement in pressure adaptation

  • Design pressure-sensitive ProQ variants through directed evolution

  • Create reporter systems that respond to pressure changes

  • Application: Develop biosensors for deep-sea environments or high-pressure bioprocessing

• RNA localization and scaffolding:

  • Fuse ProQ to localization domains or scaffold proteins

  • Design target RNAs with ProQ-binding motifs

  • Use the system to localize RNAs to specific cellular compartments

  • Application: Create synthetic RNA-protein granules or localize translation

• ProQ-based RNA purification system:

  • Develop high-affinity ProQ variants for specific RNA structures

  • Immobilize these variants on chromatography matrices

  • Use for selective purification of structured RNAs

  • Application: Purification of RNA therapeutics or diagnostic RNA biomarkers

Methodological considerations for these applications include:

• Protein engineering through directed evolution to enhance desired properties

• Rational design based on structural information from ProQ homologs

• Testing in multiple host organisms to ensure broad applicability

• Combining with other RNA-binding proteins for more complex functionality

How might environmental factors beyond pressure affect ProQ function in P. profundum, and what methodologies are appropriate to study these interactions?

P. profundum inhabits the deep sea, an environment characterized not only by high pressure but also by low temperature, limited nutrients, and other unique conditions. These factors may interact with ProQ function in complex ways:

Environmental Factor 1: Temperature

• Hypothesis: Low temperature may enhance ProQ's RNA-binding activity, compensating for pressure effects.

• Methodological approach:

  • Compare ProQ-RNA binding affinities at different temperatures using gel shift assays

  • Perform RNA-seq of wild-type and ΔproQ strains at various temperatures (4°C, 15°C, 28°C)

  • Use thermal shift assays to determine if pressure affects ProQ's thermal stability

Environmental Factor 2: Nutrient limitation

• Hypothesis: ProQ may regulate different sets of genes under nutrient-rich versus nutrient-limited conditions.

• Methodological approach:

  • Culture P. profundum in defined media with varying carbon or nitrogen sources

  • Perform comparative ProQ-CLIP-seq under these conditions

  • Identify condition-specific ProQ regulons using differential expression analysis

Environmental Factor 3: Osmolarity

• Hypothesis: Since ProQ affects osmotic regulation in E. coli, it may play a similar role in P. profundum.

• Methodological approach:

  • Test growth of wild-type and ΔproQ strains under different salt concentrations

  • Analyze the expression of osmoregulatory genes in both strains

  • Perform osmotic shock experiments and monitor transcriptome changes

Environmental Factor 4: Oxygen concentration

• Hypothesis: Oxygen availability may alter ProQ's regulatory network in P. profundum.

• Methodological approach:

  • Compare aerobic versus anaerobic growth of wild-type and ΔproQ strains

  • Perform RNA-seq under both conditions

  • Identify oxygen-dependent changes in the ProQ regulon

A comprehensive experimental design matrix for studying these interactions: