Recombinant Photobacterium profundum Ribosome maturation factor RimP (rimP)

What is the function of RimP in Photobacterium profundum?

RimP in P. profundum functions as a ribosomal maturation factor essential for the biogenesis of the 30S small ribosomal subunit. Similar to RimP proteins characterized in other bacteria, it plays a critical role in the assembly of functional ribosomes, particularly in facilitating the incorporation of the S12 ribosomal protein (RpsL) . This function is especially important in P. profundum, which must maintain proper protein synthesis under high hydrostatic pressure conditions found in the deep sea. Ribosomal assembly is known to be pressure-sensitive in mesophilic bacteria, suggesting that P. profundum's RimP may have evolved specific adaptations to function optimally under elevated pressure .

How is RimP characterized structurally in bacteria?

While no crystal structure of P. profundum RimP is currently available in the search results, insights can be drawn from structural studies of RimP homologs. The crystal structure of MSMEG_2624 (the RimP ortholog in Mycobacterium smegmatis) resolved at 2.2 Å reveals a protein with two distinct domains:

• An N-terminal domain

• A C-terminal domain

• An interdomain linker region containing highly conserved residues

The linker region is particularly significant as it coordinates both domains to bind with the small ribosomal protein S12 (RpsL) . Neither domain alone is sufficient for strong RpsL interaction, highlighting the importance of the complete protein architecture for proper function .

What phenotypes are observed when rimP is mutated in bacteria?

Mutation of rimP produces significant phenotypic changes across bacterial species:

In P. profundum, while specific rimP mutation studies are not detailed in the search results, the phenotypes would likely include pressure-dependent growth defects given its deep-sea habitat and the known pressure sensitivity of ribosomal assembly .

How might pressure affect RimP activity in Photobacterium profundum?

P. profundum SS9 exhibits optimal growth at 28 MPa (approximately equivalent to its isolation depth of 2500m) . While the search results don't explicitly describe pressure effects on RimP activity, several observations suggest likely mechanisms:

• Transcriptional regulation: In P. profundum, ToxRS is a major two-component signaling system that senses environmental pressure through membrane conformational changes and regulates gene expression in a pressure-responsive manner .

• Protein functionality: High pressure can cause irreversible depolymerization of protein complexes. For example, bacterial flagellar filaments depolymerize at approximately 340 MPa due to pressure-induced volume changes . RimP function likely requires structural adaptations to maintain activity under pressure.

• Energy metabolism adaptation: P. profundum shows different ATPase system utilization depending on pressure and growth medium, suggesting pressure affects energy availability for processes like protein synthesis and ribosome assembly .

Given these patterns, RimP in P. profundum likely exhibits pressure-optimized binding kinetics with its interaction partners, possibly through subtle structural modifications compared to shallow-water homologs.

What experimental approaches can be used to study RimP function under high pressure?

Several specialized approaches can be employed:

• High-pressure growth chambers: P. profundum strains can be cultured in stainless-steel pressure vessels at various pressures (e.g., 0.1 MPa to 30 MPa) to analyze growth phenotypes .

• High-pressure transcriptomics: RNA-seq analysis comparing wild-type and rimP mutant strains under various pressure conditions can reveal pressure-dependent gene expression patterns influenced by RimP .

• High-pressure microscopy: Direct observation of cell morphology and division can be performed using specialized high-pressure microscopic chambers (HPDS) coupled with image analysis software to measure cellular parameters under pressure .

• Polysome profiling: Sucrose gradient centrifugation can be used to analyze ribosome profiles (30S, 50S, 70S, and polysome fractions) in wild-type and rimP mutant strains at different pressures .

• Pressure-responsive reporter constructs: β-galactosidase reporter systems can be used to monitor pressure-responsive gene expression in vivo .

How can the interaction between RimP and RpsL be investigated in Photobacterium profundum?

Based on methodologies used for other bacterial species, the following approaches would be effective:

• Tandem affinity purification: Co-express His-tagged RimP and GST-tagged RpsL, followed by sequential purification through Ni²⁺ and GST affinity chromatography. This approach can identify direct protein-protein interactions as demonstrated with MSMEG_2624 and RpsL .

• Site-directed mutagenesis: Create mutations in conserved residues of the RimP linker region to assess their impact on RpsL binding. For example, deletion of specific residues (ΔP90–D93) in MSMEG_2624 significantly reduced RpsL binding .

• In vivo complementation assays: Complement a rimP knockout strain with various mutant versions of rimP to assess which domains and residues are essential for ribosome biogenesis under different pressure conditions .

• Mass spectrometry: Use quantitative MS to analyze the composition of 30S ribosomal subunits in wild-type versus rimP mutant strains to determine which ribosomal proteins are affected by RimP deficiency .

What expression systems are optimal for producing recombinant P. profundum RimP?

Based on successful approaches with other bacterial RimP proteins:

• E. coli expression system: Using BL21(DE3) strain with pRsfDuet-1 or similar expression vectors has proven effective for RimP homologs . Expression should be induced with 1 mM IPTG at low temperature (16°C) overnight to enhance proper folding.

• Co-expression strategy: For interaction studies, co-transform E. coli with plasmids encoding both RimP and its interaction partner (e.g., pGEX-6P-1-RpsL and pRsfDuet-1-RimP) .

• Growth conditions:

  • Medium: Luria-Bertani medium

  • Temperature: Grow at 37°C until A₆₀₀ = 0.6, then induce and shift to 16°C

  • Duration: Overnight induction (12-16 hours)

What purification protocol is recommended for recombinant P. profundum RimP?

A multi-step purification protocol is recommended:

• Affinity chromatography: Use His-tag purification with Ni-NTA resin for initial capture

  • Lysis buffer: 1× PBS with 0.5% Triton X-100

  • Wash buffer: PBS with 20 mM imidazole

  • Elution buffer: PBS with 250 mM imidazole

• Size-exclusion chromatography: Further purify using gel filtration to remove aggregates and obtain homogeneous protein preparations

  • Column: Superdex 75 or Superdex 200

  • Buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT

• Quality control steps:

  • SDS-PAGE to assess purity

  • Size-exclusion chromatography to confirm proper folding and absence of aggregation

  • Circular dichroism to verify secondary structure

How can rimP knockout mutants be generated in P. profundum?

Generation of rimP knockout mutants in P. profundum can follow established protocols for this organism:

• In-frame deletion approach:

  • Amplify ~1 kb upstream and downstream flanking regions of rimP

  • Ligate these regions using fusion PCR

  • Clone the construct into a suicide vector like pRE118 or pRL271 containing sacB for counter-selection

  • Introduce the plasmid into P. profundum via conjugation using helper strains like E. coli containing pRK2073

  • Select for first recombination event using antibiotic selection

  • Screen for second recombination (gene deletion) using sucrose counter-selection

• Verification methods:

  • Colony PCR with primers flanking the deletion site

  • Sequencing to confirm correct deletion junctions

  • Western blotting to verify absence of RimP protein

What complementation strategies are effective for functional analysis of rimP mutations?

For functional analysis of rimP mutations:

• Complementation vectors:

  • Broad-host-range plasmids like pGL10 are effective for complementation in P. profundum

  • Clone the wild-type or mutant rimP genes with their native promoters

• Site-directed mutagenesis strategies:

  • Target conserved residues in the linker region (based on alignment with other RimP proteins)

  • Create domain deletions to assess the importance of individual domains

  • Point mutations of charged residues that might be involved in protein-protein interactions

• Phenotypic assessment:

  • Compare growth rates at different pressures (0.1 MPa vs. 28 MPa)

  • Analyze ribosome profiles using sucrose gradient centrifugation

  • Measure swimming velocity using high-pressure microscopic chambers if motility is affected

How does RimP contribute to P. profundum's adaptation to deep-sea environments?

While the search results don't directly address this question for P. profundum RimP, we can infer likely mechanisms:

• Maintaining ribosome assembly: P. profundum must synthesize proteins under high pressure conditions where ribosome assembly is typically inhibited in mesophilic bacteria. RimP likely plays a critical role in ensuring proper 30S subunit maturation under these conditions .

• Pressure-optimized interactions: RimP in P. profundum may have evolved specific structural features that optimize its interactions with ribosomal components at elevated pressures, similar to other pressure-adapted proteins in this organism.

• Integration with pressure-sensing systems: P. profundum utilizes the ToxRS system to sense and respond to pressure changes . RimP function may be integrated with this regulatory network, potentially through transcriptional regulation or post-translational modifications.

• Energy efficiency: In the energy-limited deep sea, efficient ribosome assembly facilitated by RimP would provide a selective advantage, particularly given P. profundum's different ATP metabolism under pressure .

What techniques can assess the pressure stability of recombinant RimP?

Several biophysical techniques can evaluate pressure effects on RimP structure and function:

• High-pressure spectroscopy:

  • Circular dichroism under pressure to monitor secondary structure changes

  • Fluorescence spectroscopy to track tertiary structure alterations

  • FTIR spectroscopy to detect pressure-induced conformational changes

• High-pressure binding assays:

  • Surface plasmon resonance (SPR) under pressure to measure binding kinetics with RpsL

  • Isothermal titration calorimetry (ITC) to determine pressure effects on binding thermodynamics

• Molecular dynamics simulations:

  • Computational modeling of RimP under different pressure conditions to predict structural changes

  • In silico analysis of protein volume changes and compressibility

• Pressure stability analysis:

  • Compare pressure denaturation profiles of RimP from P. profundum with orthologs from shallow-water relatives like strain 3TCK

  • Measure enzymatic activity or binding capacity after pressure treatment