Recombinant Photobacterium profundum 50S ribosomal protein L10 (rplJ)

What is the function of 50S ribosomal protein L10 (rplJ) in Photobacterium profundum?

The 50S ribosomal protein L10 serves dual functions in bacterial cells. Primarily, it acts as a structural constituent of the ribosome, specifically binding to large ribosomal subunit rRNA and contributing to ribosomal assembly . Additionally, L10 functions as a translational repressor protein by controlling the translation of the rplJL-rpoBC operon through binding to its mRNA . In the deep-sea bacterium Photobacterium profundum, ribosomal proteins including L10 are particularly significant as they contribute to both low-temperature and high-pressure adaptation mechanisms .

The protein's role extends beyond basic translation, as ribosome assembly and function have been identified through transposon mutagenesis studies as critical elements for P. profundum growth under high-pressure conditions . This is consistent with the broader understanding that protein synthesis machinery must be adapted for functionality in extremophiles living in deep-sea environments.

How does the rplJ gene structure compare between P. profundum and other bacterial species?

While the complete sequence comparison specifically for P. profundum rplJ with other bacteria is not fully detailed in the available data, comparative genomics approaches reveal important patterns in ribosomal protein genes across bacterial species. Similar to what has been observed between Salmonella typhimurium, Klebsiella pneumoniae, and E. coli, we can expect several amino acid substitutions in the L10 protein sequence that reflect evolutionary adaptations to different environmental niches .

In closely related bacterial species, the rplJ and rplL genes typically maintain their organization in an operon structure, with the long-range coupling that provides for coordinated translation of L10 and L7/L12 cistrons . The genomic context of the rplJL genes is generally conserved, though pressure-adapted species like P. profundum may exhibit specific sequence modifications that contribute to protein stability and function under high hydrostatic pressure.

What methods are used to clone and express recombinant P. profundum rplJ?

The cloning and expression of recombinant P. profundum rplJ would typically follow protocols similar to those used for other bacterial ribosomal proteins, with modifications to account for the pressure-adapted nature of the source organism. Based on established methods:

• Gene Amplification: The rplJ gene can be PCR-amplified from P. profundum genomic DNA using specific primers designed based on the gene sequence . Standard PCR conditions might include: 92°C for 1 min, 48°C for 1 min, and 72°C for 1 min for 25 cycles .

• Cloning Strategy: The amplified gene can be cloned into vectors such as pCR2.1 for initial verification and then subcloned into expression vectors . For complementation studies in P. profundum, broad-host-range plasmids like pGL10 are appropriate .

• Expression Systems: Depending on the research goals, expression can be performed in:

  • E. coli for high-yield protein production

  • P. profundum itself for functional studies under pressure

  • Other bacterial hosts for comparative analysis

• Verification: Successful cloning can be verified through restriction digestion, PCR, and DNA sequencing to confirm the correct orientation and sequence of the insert .

How does hydrostatic pressure affect the expression and function of rplJ in P. profundum?

The expression of ribosomal proteins, including rplJ, in P. profundum is significantly influenced by hydrostatic pressure. Transcriptome studies using RNA-seq have revealed complex expression patterns of genes involved in pressure adaptation . The ToxR protein, a pressure-responsive transcriptional regulator in P. profundum, influences the expression of numerous genes in a pressure-dependent manner . While not explicitly stated in the available data, it is likely that rplJ expression is modulated as part of the cellular response to pressure changes.

Functionally, ribosomal proteins must maintain structural integrity and proper interactions under high pressure. The amino acid composition and structural features of L10 in P. profundum likely contain specific adaptations that allow it to function optimally under deep-sea conditions. These adaptations may include increased hydrophobic interactions, altered charge distributions, or modified flexibility in key regions of the protein.

For experimental assessment of pressure effects, researchers typically grow P. profundum cultures at varying pressures (e.g., atmospheric vs. 280 atm) and analyze changes in gene expression and protein function . Such experiments have revealed that genes involved in ribosome assembly and function are critical for both low-temperature and high-pressure growth .

What role does rplJ play in pressure adaptation mechanisms of P. profundum?

The 50S ribosomal protein L10, encoded by rplJ, contributes to pressure adaptation in P. profundum through several potential mechanisms:

• Ribosome Stability: As a structural component of the ribosome, L10 likely contains adaptations that help maintain ribosomal integrity under high pressure. Proper ribosome assembly has been identified as critical for growth under high-pressure conditions .

• Translational Regulation: Given L10's role as a translational repressor that controls the expression of the rplJL-rpoBC operon , it may serve as a regulatory switch in the pressure-responsive gene expression network.

• Integration with Other Adaptation Systems: Transposon mutagenesis studies have shown that numerous genes involved in chromosomal structure and function are important for pressure adaptation . L10 may interact with these systems as part of a coordinated response to pressure changes.

• Signal Transduction: Pressure adaptation in P. profundum involves various sensory and regulatory mechanisms . L10 could participate in signal transduction pathways that detect and respond to pressure changes.

Experimental approaches using gene knockouts or point mutations in rplJ could help elucidate its specific contributions to pressure adaptation, similar to studies performed with the recD gene .

How do mutations in rplJ affect the growth phenotype of P. profundum under varying pressure conditions?

While the available search results don't specifically address rplJ mutations in P. profundum, we can draw parallels from studies of other genes important for pressure adaptation. For example, mutations in the recD gene resulted in pressure-sensitive growth phenotypes, with the severity depending on the extent of gene disruption .

For rplJ, potential effects of mutations might include:

• Growth Rate Alterations: Mutations could lead to reduced growth rates specifically at high pressure while having minimal effect at atmospheric pressure.

• Temperature-Pressure Interaction: Since genes for ribosome assembly and function are important for both low-temperature and high-pressure growth , rplJ mutations might show synergistic negative effects when both stressors are combined.

• Morphological Changes: Changes in cell morphology, similar to the filamentation observed in E. coli at high pressure when carrying certain genes , might occur with rplJ mutations.

A methodological approach to study these effects would include:

• Creating targeted mutations in rplJ using gene disruption techniques

• Measuring growth curves at various pressures (atmospheric, intermediate, and high pressure)

• Microscopic examination of cell morphology

• Complementation studies to verify phenotype causality

What expression systems are most effective for producing recombinant P. profundum rplJ protein?

Based on established protocols for recombinant protein production and the specific characteristics of P. profundum proteins, several expression systems can be considered:

For heterologous expression in E. coli, vectors containing T7 promoters (pET series) are typically effective. When expressing in P. profundum itself, broad-host-range plasmids similar to those used in complementation studies would be appropriate .

Key methodological considerations include:

• Codon optimization if expressing in heterologous hosts

• Addition of solubility tags (e.g., MBP, SUMO) to enhance protein solubility

• Temperature reduction during induction to improve folding

• Purification under conditions that maintain protein stability

What methods are used to study rplJ gene expression regulation under varying pressure conditions?

The study of pressure-dependent gene expression in P. profundum involves several sophisticated methodological approaches:

• RNA-seq Analysis: Massively parallel cDNA sequencing provides a comprehensive view of the transcriptional landscape . This approach has been successfully used to identify pressure-responsive genes in P. profundum by comparing expression profiles at different pressures.

• Promoter Reporter Fusions: Fusing the rplJ promoter region to reporter genes such as lacZ or gfp allows quantitative assessment of promoter activity under varying pressure conditions.

• Quantitative RT-PCR: For targeted analysis of rplJ expression levels, qRT-PCR offers precise quantification of transcript abundance across different conditions.

• Transposon Mutagenesis: Screening transposon mutant libraries for altered pressure responses can identify regulators of rplJ expression . Mutants showing altered rplJ expression patterns may reveal regulatory pathways.

• Regulatory Protein Identification: Techniques such as DNA affinity chromatography or bacterial one-hybrid systems can identify proteins that bind to the rplJ promoter region.

When designing such experiments, it's critical to control for confounding variables such as temperature, growth phase, and media composition. High-pressure cultivation requires specialized equipment such as stainless steel pressure vessels connected to hydraulic pumps .

How can structural studies of recombinant rplJ provide insights into pressure adaptation?

Structural studies of recombinant rplJ from P. profundum can reveal the molecular basis of pressure adaptation through several approaches:

• Comparative Structural Analysis: Comparing the structure of P. profundum L10 with homologs from non-pressure-adapted organisms can highlight adaptive modifications. Key differences may include:

  • Altered distribution of charged residues

  • Modified hydrophobic core packing

  • Pressure-stable interaction surfaces

• Pressure-Resolved Structural Studies: Techniques such as high-pressure NMR, high-pressure X-ray crystallography, or high-pressure small-angle X-ray scattering (SAXS) can directly visualize structural changes under pressure.

• Molecular Dynamics Simulations: Computational approaches can model the behavior of L10 under varying pressure conditions, providing insights into conformational stability and flexibility.

• Functional Assays Under Pressure: Measuring L10's RNA binding capacity and regulatory functions at different pressures can correlate structural features with functional adaptations.

• Site-Directed Mutagenesis: Systematically altering key residues identified in structural studies and testing the effects on pressure tolerance can validate the role of specific structural features.

These approaches require purified recombinant protein, which can be obtained using the expression systems described earlier, followed by chromatographic purification techniques optimized to maintain the native structure.

How can RNA-seq data be used to analyze rplJ expression patterns in P. profundum?

RNA-seq provides a powerful approach for analyzing rplJ expression patterns in P. profundum under various conditions. Based on established methodologies:

• Comparative Expression Analysis: RNA-seq data can reveal differential expression of rplJ in response to pressure changes, temperature variations, or genetic modifications (e.g., toxR mutant vs. wild type) . This allows identification of conditions that specifically regulate rplJ expression.

• Operon Structure Determination: RNA-seq enables genome-wide prediction of operon structures, transcription start sites, and termination sites . For rplJ, this can reveal whether it is expressed as part of a polycistronic transcript and identify the complete set of genes co-regulated with it.

• UTR Analysis: RNA-seq can detect the presence and length of 5'-UTRs, which may contain cis-regulatory RNA structures that influence expression . The search results indicate that P. profundum has an unexpectedly high number of genes with large 5'-UTRs, suggesting complex regulatory mechanisms .

• sRNA Interaction: RNA-seq can identify small RNAs that might regulate rplJ expression post-transcriptionally . The identification of 460 putative small RNA genes in P. profundum suggests that sRNA regulation may be important in pressure adaptation .

• Comparative Genomics Integration: Combining RNA-seq data with comparative genomics can identify conserved regulatory elements across pressure-adapted bacteria.

When analyzing RNA-seq data specifically for rplJ, researchers should consider:

• Normalizing expression data appropriately for cross-condition comparisons

• Validating key findings with qRT-PCR

• Correlating expression changes with physiological parameters

• Integrating with proteomics data to account for post-transcriptional regulation

What techniques are used to assess the functional properties of recombinant rplJ protein?

The functional characterization of recombinant rplJ from P. profundum involves multiple complementary approaches:

• RNA Binding Assays: Since L10 binds to rRNA, electrophoretic mobility shift assays (EMSA) or filter binding assays can assess binding affinity and specificity under different pressure conditions.

• Translational Repression Assays: Given L10's role as a translational repressor , in vitro translation systems can test its regulatory function on target mRNAs.

• Complementation Studies: Introduction of recombinant rplJ into mutant strains can verify functional activity through restoration of growth phenotypes under pressure, similar to approaches used with other genes in P. profundum .

• Protein-Protein Interaction Analysis: Techniques such as pull-down assays, bacterial two-hybrid systems, or crosslinking followed by mass spectrometry can identify interaction partners that may be relevant to pressure adaptation.

• Ribosome Assembly Assays: In vitro ribosome reconstitution experiments can assess the ability of recombinant L10 to participate in proper ribosome assembly under varying pressure conditions.

When testing functionality under pressure, specialized high-pressure equipment is required. Researchers can adapt approaches used in other pressure studies with P. profundum, such as custom-designed stainless steel pressure vessels connected to hydraulic pumps .

What are the current limitations in studying recombinant P. profundum rplJ?

Several technical and conceptual challenges currently limit comprehensive study of recombinant P. profundum rplJ:

• High-Pressure Experimental Constraints: Conducting experiments under high pressure requires specialized equipment that isn't widely available, limiting research accessibility .

• Expression System Limitations: Expressing pressure-adapted proteins in standard laboratory systems may not replicate native folding and modifications.

• Complex Regulatory Networks: The regulation of ribosomal proteins involves multiple layers of control, including transcriptional regulation, post-transcriptional mechanisms, and protein-protein interactions . Dissecting these networks requires integrated approaches.

• Limited Comparative Data: While studies have characterized aspects of pressure adaptation in P. profundum , direct comparative data on L10 proteins from pressure-adapted versus non-adapted organisms remains sparse.

• Methodological Challenges in Structural Biology: Obtaining high-resolution structural data under pressure conditions presents significant technical hurdles.

How might future research on P. profundum rplJ contribute to our understanding of deep-sea adaptation?

Future research directions for P. profundum rplJ could significantly advance our understanding of deep-sea adaptation mechanisms:

• Systems Biology Integration: Combining transcriptomics, proteomics, and metabolomics approaches to place rplJ within the broader network of pressure-responsive elements .

• Synthetic Biology Applications: Engineering pressure-adapted features of rplJ into other organisms could create strains capable of functioning in high-pressure environments for biotechnological applications.

• Evolutionary Analyses: Comparative studies of rplJ across bacteria from different depth zones could reveal evolutionary patterns in pressure adaptation.

• Structural Insights: Solving high-resolution structures of P. profundum L10 under various pressure conditions would provide unprecedented insights into pressure-adaptive molecular mechanisms.

• Signal Transduction Exploration: Investigating how rplJ expression responds to pressure signals and how this connects to global stress responses could reveal novel sensing mechanisms .

These approaches will require continued development of high-pressure laboratory techniques, advanced computational methods, and interdisciplinary collaboration between microbiologists, biochemists, structural biologists, and evolutionary biologists.