Recombinant Photorhabdus luminescens subsp. laumondii 50S ribosomal protein L35 (rpmI)

What is the amino acid sequence of the Photorhabdus luminescens rpmI protein?

The full amino acid sequence of the 50S ribosomal protein L35 (rpmI) from Photorhabdus luminescens subsp. laumondii is: MPKIKTVRGA AKRFKKTASG GFKRKRANLR HILTKKSTKR KRHLRPKGMV SKGDLGLVVA CLPYA. This 65-amino acid sequence represents the complete protein, spanning the expression region 1-65. For experimental work, researchers should note the high proportion of basic residues (lysine and arginine), which suggests a role in nucleic acid binding common to ribosomal proteins.

What is the biological function of the 50S ribosomal protein L35 in Photorhabdus luminescens?

The 50S ribosomal protein L35 in P. luminescens, like in other bacteria, is a critical component of the large ribosomal subunit. While the search results don't specify its exact role in P. luminescens, ribosomal protein L35 typically participates in the assembly of the 50S subunit and contributes to protein synthesis by stabilizing rRNA structure. In the context of P. luminescens's complex lifecycle - transitioning between symbiosis with nematodes and pathogenicity in insects - ribosomal proteins play essential roles in supporting the translation of various virulence factors and metabolic enzymes required for these different life stages.

How is the rpmI protein related to P. luminescens pathogenicity?

While rpmI itself is not directly identified as a virulence factor in the search results, it plays a supportive role in P. luminescens pathogenicity by enabling the translation of various toxins and virulence factors. P. luminescens produces four major groups of toxins: toxin complexes (Tcs), Photorhabdus insect related (Pir) proteins, "makes caterpillars floppy" (Mcf) toxins, and Photorhabdus virulence cassettes (PVC). As a ribosomal protein, rpmI would be involved in the translation machinery producing these toxins, which are essential for the bacterium's ability to kill insect hosts rapidly.

What are the optimal storage conditions for recombinant P. luminescens rpmI protein?

The shelf life and stability of recombinant rpmI depend on several factors including storage state, buffer ingredients, and temperature. For optimal results:

• Liquid form: Store at -20°C/-80°C for up to 6 months

• Lyophilized form: Store at -20°C/-80°C for up to 12 months

• Working aliquots: Store at 4°C for up to one week

Important note: Repeated freezing and thawing is not recommended as it may compromise protein integrity. Researchers should aliquot the protein upon receipt to minimize freeze-thaw cycles.

What is the recommended reconstitution protocol for lyophilized rpmI protein?

For optimal reconstitution of lyophilized rpmI protein:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended as default)

• Aliquot for long-term storage at -20°C/-80°C

This protocol ensures proper solubilization and stability of the protein for subsequent experimental applications.

What expression systems are used to produce recombinant P. luminescens rpmI protein?

The recombinant rpmI protein described in the search results is produced in a mammalian cell expression system. This contrasts with many bacterial proteins that are often expressed in E. coli systems. Mammalian expression likely provides certain advantages for this particular protein, potentially including proper folding, solubility, or post-translational modifications that might be important for structural studies or functional assays. Researchers considering alternative expression systems should evaluate whether mammalian-specific features are critical for their experimental needs.

How can rpmI protein be used to study P. luminescens ribosomal assembly?

To investigate ribosomal assembly in P. luminescens using rpmI:

• Perform in vitro binding assays between purified rpmI and rRNA fragments to identify specific interaction sites

• Use fluorescently labeled rpmI to track incorporation into ribosomal subunits during assembly

• Employ cryo-electron microscopy to visualize the structural position of rpmI within the assembled ribosome

• Create rpmI mutants through site-directed mutagenesis to identify critical residues for incorporation into the ribosome

These approaches would provide insights into P. luminescens-specific aspects of ribosome assembly, potentially revealing adaptations related to its unique lifecycle as both a symbiont and pathogen.

What interactions might exist between rpmI and the PhoP-PhoQ regulatory system in P. luminescens?

While direct interactions between rpmI and the PhoP-PhoQ system are not explicitly mentioned in the search results, this represents an intriguing research question. The PhoP-PhoQ two-component system in P. luminescens responds to environmental magnesium availability and has been linked to virulence, motility, and antimicrobial peptide resistance.

A methodological approach to investigate potential relationships would include:

• Comparative proteomics analysis of wild-type vs. phoP mutant strains to determine if rpmI expression is altered

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify if PhoP binds to the rpmI promoter region

• Construction of reporter fusions to measure rpmI expression under different conditions that activate PhoP-PhoQ

• Co-immunoprecipitation studies to detect physical interactions between rpmI and components of the PhoP-PhoQ system

These experiments would help determine whether ribosomal proteins like rpmI are regulated as part of the PhoP-PhoQ virulence response in P. luminescens.

How does the structure of P. luminescens rpmI compare to homologous proteins in related pathogenic bacteria?

To conduct a comprehensive structural comparison:

• Perform multiple sequence alignment of rpmI proteins from P. luminescens, other Enterobacteriaceae members, and more distant bacterial species

• Generate homology models based on existing ribosomal protein structures

• Identify conserved vs. variable regions that might indicate P. luminescens-specific adaptations

• Use X-ray crystallography or NMR spectroscopy to determine the actual tertiary structure of P. luminescens rpmI

• Compare binding interfaces with rRNA and adjacent proteins to identify potential functional differences

What quality control measures should be used when working with recombinant rpmI protein?

Rigorous quality control is essential when working with recombinant rpmI:

Researchers should perform these quality control steps before using the protein in downstream applications to ensure reproducible results. The recombinant protein should have a purity of at least 85% as determined by SDS-PAGE.

What are the main technical challenges in studying interactions between rpmI and other ribosomal components?

Studying ribosomal protein interactions presents several methodological challenges:

• Maintaining native conformations outside the ribosomal context

• Distinguishing direct interactions from those mediated by rRNA

• Capturing transient interactions during ribosome assembly

• Ensuring protein solubility during interaction studies

• Developing appropriate binding assays that mimic physiological conditions

To overcome these challenges, researchers might employ techniques such as surface plasmon resonance with carefully designed buffer conditions, microscale thermophoresis for detecting interactions in solution, or hydrogen-deuterium exchange mass spectrometry to map interaction interfaces with high resolution.

How can rpmI be used as a target for studying novel antimicrobials against P. luminescens?

Given P. luminescens's role as both an insect pathogen and an emerging human pathogen, developing antimicrobials targeting ribosomal proteins presents an interesting research direction. A methodological approach would include:

• High-throughput screening of compound libraries against purified rpmI

• In silico docking studies to identify potential binding pockets

• Testing rpmI-binding compounds for growth inhibition of P. luminescens cultures

• Assessing selectivity by comparing binding to homologous proteins from other species

• Ribosome profiling to confirm the mechanism of action targets translation

• Structure-activity relationship studies to optimize lead compounds

This research could be particularly valuable given increasing resistance to conventional antibiotics and the need for species-selective antimicrobial approaches.

How does rpmI expression change during different stages of the P. luminescens lifecycle?

P. luminescens undergoes phase variation with distinct phenotypic variants during its lifecycle. A comprehensive approach to study rpmI expression across these phases would include:

• Quantitative RT-PCR to measure rpmI transcript levels in:

  • Phase I (primary) variants found in the infective juvenile stage

  • Phase II (secondary) variants that appear after extended incubation

  • Bacteria isolated directly from insect hemolymph at different infection stages

  • Bacteria within the nematode gut

• Western blotting with rpmI-specific antibodies to confirm protein-level changes

• Ribosome profiling to determine if rpmI incorporation into ribosomes varies across phases

• Fluorescent reporter constructs to visualize expression patterns in real-time during lifecycle transitions

What role might rpmI play in the adaptation of P. luminescens to different host environments?

P. luminescens transitions between nematode and insect hosts, requiring adaptation to different environmental conditions. To investigate rpmI's potential role in this adaptation:

• Compare rpmI sequence conservation across P. luminescens strains with different host specificities

• Conduct structural studies of rpmI under different conditions mimicking host environments (varying pH, temperature, salt concentrations)

• Perform comparative ribosome profiling between bacteria grown in vitro versus isolated from different host tissues

• Create rpmI mutants with altered stability characteristics and assess their impact on growth in different host-mimicking conditions

These studies would help determine whether ribosomal proteins like rpmI contribute to the remarkable environmental adaptability of P. luminescens.

What are the most pressing unanswered questions about P. luminescens rpmI protein?

Several key research questions remain to be addressed:

• How does the structure of P. luminescens rpmI differ from well-characterized ribosomal proteins in model organisms?

• Is rpmI expression or incorporation into ribosomes regulated during host infection?

• Could structural features of rpmI explain any aspects of P. luminescens's unique biology as both symbiont and pathogen?

• Are there post-translational modifications of rpmI that affect its function in different environmental conditions?

• Could rpmI serve as a potential therapeutic target for controlling P. luminescens infections in humans?

Answering these questions would significantly advance our understanding of both P. luminescens biology and bacterial ribosome function more broadly.

What emerging technologies could advance our understanding of rpmI function?

Several cutting-edge methodologies could drive new discoveries about rpmI:

• Cryo-electron microscopy to visualize P. luminescens ribosomes at near-atomic resolution

• Ribosome profiling coupled with RNA-seq to assess translation dynamics during infection

• CRISPR interference systems adapted for P. luminescens to create conditional knockdowns of rpmI

• Proximity labeling approaches to identify the interactome of rpmI in living P. luminescens cells

• Single-molecule fluorescence techniques to visualize ribosome assembly in real-time