Recombinant Photobacterium profundum 50S ribosomal protein L13 (rplM)

What is the biological function of 50S ribosomal protein L13 in Photobacterium profundum?

L13 in P. profundum, like its homolog in E. coli, likely serves as an essential component in the assembly of the 50S ribosomal subunit. Based on research in E. coli, L13 is characterized as an early assembly component that interacts with 23S rRNA . Given the importance of ribosomal assembly genes for both low-temperature and high-pressure growth in P. profundum, L13 likely plays a crucial role in the organism's adaptation to deep-sea environments . The protein's incorporation during ribosome assembly in vivo requires the assistance of the DEAD-box RNA helicase SrmB, which is needed to properly organize the L13 binding site on 23S rRNA by preventing the formation of incorrect alternative structures .

Beyond its structural role in ribosomes, L13 also functions as a regulatory protein. In E. coli, L13 acts as an autogenous repressor that regulates the expression of the rplM-rpsI operon at the translational level . This bifunctional nature of L13—involved in both ribosome assembly and regulation of gene expression—represents an important aspect of coordinated ribosomal component synthesis in bacteria.

How does P. profundum L13 differ from homologous proteins in mesophilic bacteria?

While the search results don't provide direct comparative data between P. profundum L13 and its mesophilic counterparts, we can infer likely differences based on P. profundum's adaptation to deep-sea environments. As a psychrotolerant and moderately piezophilic bacterium capable of growth at temperatures below 2°C and pressures up to nearly 90 MPa , P. profundum likely possesses ribosomal proteins with structural adaptations that maintain functionality under these extreme conditions.

Ribosomal proteins in extremophiles often exhibit modifications that enhance structural stability while maintaining necessary flexibility for proper function. For P. profundum L13, this might include:

These adaptations would be crucial since genes for ribosome assembly and function have been found to be important for both low-temperature and high-pressure growth in P. profundum .

What is the structure of the rplM-rpsI operon in P. profundum?

The operon is likely transcribed as a single mRNA that is subject to both transcriptional and translational regulation. The transcriptional regulation may involve ppGpp/DksA-dependent negative stringent control under amino acid starvation, similar to what has been observed in E. coli . At the translational level, the operon is likely regulated by L13 itself, which acts as an autogenous repressor by binding to a target within the rplM translation initiation region .

How does high hydrostatic pressure affect the expression and function of L13 in P. profundum?

The expression and function of L13 in P. profundum under high pressure conditions represents a fascinating area of research. While the search results don't provide direct experimental data on this specific question, we can draw insights from the broader findings about P. profundum's pressure adaptations.

P. profundum strain SS9 is capable of growth at pressures from 0.1 MPa to nearly 90 MPa, with optimal growth at 28 MPa . Large-scale transposon mutagenesis studies have revealed that genes for ribosome assembly and function are important for high-pressure growth in this organism . This suggests that the expression and function of ribosomal proteins, including L13, are likely pressure-regulated.

Methodologically, to investigate pressure effects on L13 expression, researchers could:

• Perform quantitative RT-PCR on the rplM gene at different pressures

• Use Western blotting to quantify L13 protein levels under varying pressure conditions

• Employ ribosome profiling to assess ribosome assembly and function at different pressures

• Create L13 mutants to identify pressure-sensitive regions of the protein

The data might reveal pressure-specific regulatory mechanisms for L13 expression that differ from those observed at atmospheric pressure, potentially involving unique transcription factors or RNA thermosensors adapted to function under high pressure.

What role does L13 play in P. profundum's adaptation to cold temperatures?

As a psychrotolerant bacterium capable of growth at temperatures below 2°C , P. profundum requires specialized cellular machinery that functions efficiently at low temperatures. While the search results don't provide specific information about L13's role in cold adaptation, we can infer its importance from the finding that genes for ribosome assembly and function are critical for low-temperature growth in P. profundum .

L13, as an early assembly component of the 50S ribosomal subunit , likely has structural adaptations that maintain proper folding and interactions with 23S rRNA at low temperatures. These adaptations might include:

• Cold-specific conformational flexibility

• Modified RNA-binding domains optimized for low-temperature interactions

• Potential involvement in cold-specific ribosome assembly pathways

To investigate these hypotheses, researchers could employ comparative structural analyses between L13 from P. profundum and mesophilic organisms, coupled with functional studies at different temperatures. Site-directed mutagenesis of key residues followed by growth assays at low temperatures would help identify regions of L13 critical for cold adaptation.

How does the autogenous regulation by L13 function under extreme conditions in P. profundum?

In E. coli, L13 serves as an autogenous repressor of the rplM-rpsI operon, regulating gene expression at the translational level . Given P. profundum's adaptation to extreme environments, the mechanisms of this autoregulation might show unique features adapted to function under high pressure and low temperature.

A methodological approach to investigating this question could include:

• Creating translational fusions of the P. profundum rplM-rpsI operon with reporter genes (similar to the lacZ fusions used in E. coli studies )

• Testing the activity of these reporter constructs under varying pressure and temperature conditions

• Performing in vitro binding assays with recombinant P. profundum L13 and its target RNA under different pressure and temperature conditions

• Comparing the regulatory RNA structures from P. profundum with those from mesophilic bacteria

These experiments would reveal whether the autogenous regulation by L13 in P. profundum employs pressure- or temperature-sensitive mechanisms that differ from those in mesophilic bacteria, potentially contributing to the organism's adaptation to the deep-sea environment.

What are the optimal conditions for expressing recombinant P. profundum L13 in heterologous systems?

Expression of recombinant proteins from extremophiles often presents unique challenges. For recombinant P. profundum L13, researchers should consider the following methodology:

Expression System Selection:

• E. coli BL21(DE3) remains a standard choice, but cold-adapted strains like Arctic Express may be more suitable given P. profundum's psychrotolerant nature

• Consider using P. profundum's own codon preferences in the construct design

Optimization Parameters:

Purification Considerations:

• Include stabilizing agents like glycerol (10-20%) in all buffers

• Maintain low temperature throughout purification

• Consider testing both native and tagged versions (His-tag, GST) as tags may affect folding

When expressing L13 from P. profundum, it's important to note that excessive expression might be toxic to the host cells, as observed with E. coli L13 . Therefore, tight control of expression levels is recommended.

What experimental approaches can be used to study the interaction between P. profundum L13 and 23S rRNA?

Studying the interaction between L13 and 23S rRNA in P. profundum requires specialized approaches that account for the protein's adaptations to extreme conditions. Several methodological approaches are recommended:

In Vitro Binding Assays:

• RNA Electrophoretic Mobility Shift Assays (EMSAs)

  • Use recombinant P. profundum L13 with labeled 23S rRNA fragments

  • Perform at various temperatures (2-20°C) and pressures if equipment allows

  • Include controls with E. coli L13 for comparison

• Surface Plasmon Resonance (SPR)

  • Immobilize either L13 or 23S rRNA fragments

  • Measure binding kinetics at different temperatures

  • Determine Kd values under various conditions

Structural Studies:

• Cryo-electron microscopy of P. profundum ribosomes

• Nuclear Magnetic Resonance (NMR) studies of L13-rRNA complexes

• X-ray crystallography of the L13-binding domain of 23S rRNA

In Vivo Approaches:

• UV crosslinking followed by immunoprecipitation

• CRISPR-based mutagenesis of the L13 binding site on 23S rRNA

• Complementation studies with chimeric L13 proteins

These methods should be performed under conditions that mimic P. profundum's native environment when possible, including lower temperatures and, ideally, high pressure.

How can researchers investigate the role of the DEAD-box RNA helicase SrmB in L13 incorporation in P. profundum ribosomes?

In E. coli, the incorporation of L13 into the 50S ribosomal subunit requires the DEAD-box RNA helicase SrmB . Investigating this relationship in P. profundum would provide valuable insights into ribosome assembly under extreme conditions. A comprehensive methodological approach would include:

Genetic Approaches:

• Create a P. profundum SrmB deletion mutant using techniques similar to those described for P. profundum mutagenesis

• Assess growth phenotypes under various temperature and pressure conditions

• Examine ribosome profiles to identify assembly intermediates

Biochemical Methods:

• Purify recombinant P. profundum SrmB and L13

• Perform in vitro reconstitution assays with 23S rRNA

• Measure ATP hydrolysis by SrmB in the presence and absence of L13 and rRNA

Structural Biology:

• Determine the structure of P. profundum SrmB alone and in complex with RNA

• Compare with mesophilic homologs to identify cold- or pressure-adapted features

In Vivo Analysis:

• Use fluorescently tagged L13 and SrmB to track their localization

• Employ ribosome profiling to examine translation defects in SrmB mutants

• Identify suppressor mutations that restore L13 incorporation in SrmB mutants

This multi-faceted approach would provide comprehensive insights into the role of SrmB in L13 incorporation under the extreme conditions that P. profundum experiences in its deep-sea habitat.

How does the regulatory function of L13 in P. profundum compare to that in other bacteria adapted to different extreme environments?

The regulatory function of L13 appears to be conserved across bacterial species, but may exhibit adaptations specific to different extreme environments. In E. coli, L13 functions as an autogenous repressor of the rplM-rpsI operon at the translational level . Comparing this function across extremophiles would provide insights into the evolution of regulatory mechanisms.

A methodological approach to this question would include:

• Sequence analysis of the rplM-rpsI operons from bacteria adapted to various extreme environments (psychrophiles, thermophiles, piezophiles, halophiles)

• Structural prediction of the 5' UTRs to identify potential regulatory elements

• In vitro binding assays comparing L13 proteins from different extremophiles with their respective mRNA targets

• Reporter gene assays to compare the regulatory efficiency across species

Expected findings might reveal how the L13 regulatory mechanism has evolved to function under different extreme conditions, with potential adaptations in RNA structure stability, protein-RNA binding kinetics, or regulatory thresholds.

These comparisons would provide valuable insights into the evolutionary plasticity of ribosomal protein regulatory mechanisms.

What are the methodological challenges in studying recombinant P. profundum L13 under high pressure conditions?

Studying recombinant proteins under high pressure presents several methodological challenges that researchers must address:

Equipment Limitations:

• Specialized high-pressure vessels are required for both expression and functional studies

• Most standard laboratory equipment cannot operate under high pressure

• Real-time monitoring of biological processes under pressure is technically difficult

Experimental Design Considerations:

• Pressure must be applied and released gradually to prevent cellular damage

• Control experiments at atmospheric pressure are essential for comparison

• Temperature must be carefully controlled, as pressure changes can affect sample temperature

Analytical Challenges:

• Sampling from high-pressure vessels without disturbing the pressure environment

• Developing assays that can be performed under pressure or immediately after decompression

• Distinguishing direct pressure effects from secondary responses

Solutions and Workarounds:

• Use of pressure-resistant optical windows for spectroscopic measurements

• Development of microfluidic devices compatible with high-pressure systems

• Flash-freezing samples immediately upon decompression to preserve molecular states

• Computational modeling to predict pressure effects before experimental validation

When studying P. profundum L13 specifically, researchers should consider designing experiments that can compare protein function across a pressure range from 0.1 MPa to the organism's optimal pressure of 28 MPa , as well as comparing with homologous proteins from non-piezophilic bacteria.

How might understanding P. profundum L13 contribute to the development of cold-adapted or pressure-resistant biotechnology applications?

Understanding the structural and functional properties of P. profundum L13 could inform the development of biotechnological applications requiring activity at low temperatures or high pressures. Several potential applications include:

Cold-Adapted Protein Expression Systems:

• Engineered expression systems incorporating elements from P. profundum's translational machinery could enhance protein production at low temperatures

• This would be particularly valuable for expressing proteins that misfold or aggregate at higher temperatures

Pressure-Resistant Enzymatic Processes:

• Insights from P. profundum L13's pressure adaptations could guide the engineering of industrial enzymes for high-pressure bioprocessing

• Potential applications include high-pressure fermentation or biocatalysis under non-standard conditions

Methodological Approach to Translation:

• Identify specific amino acid residues or structural features of L13 that confer cold or pressure adaptation

• Incorporate these features into designer proteins through site-directed mutagenesis

• Test engineered proteins under varying temperature and pressure conditions

• Develop expression systems that mimic P. profundum's ribosomal environment for improved function under extreme conditions

The study of P. profundum L13 may also provide insights into general principles of protein adaptation to extreme conditions, potentially informing broader protein engineering strategies.

What can comparative analysis of P. profundum L13 with homologs from other extremophiles reveal about ribosomal protein evolution?

Comparative analysis of L13 across extremophiles can provide valuable insights into the evolution of ribosomal proteins under diverse selective pressures. A methodological approach would include:

Sequence Analysis:

• Construct phylogenetic trees of L13 sequences from diverse extremophiles

• Identify convergently evolved features associated with specific environmental adaptations

• Calculate evolutionary rates in different lineages to identify regions under selection

Structural Comparison:

• Model L13 structures from various extremophiles using homology modeling or experimental determination

• Identify structural adaptations specific to different extreme environments

• Map conserved vs. variable regions to functional domains

Functional Analysis:

• Express recombinant L13 proteins from different extremophiles

• Test their functionality in heterologous ribosome assembly systems

• Evaluate their regulatory capacity with their native mRNA targets

This comparative approach could reveal whether different extreme environments drive similar or distinct adaptations in ribosomal proteins, and how conserved functions (structural and regulatory) are maintained despite these adaptations.

Such comparative analyses would contribute significantly to our understanding of protein evolution under extreme conditions.