Recombinant Bacillus licheniformis 50S ribosomal protein L11 (rplK)

What is the primary function of 50S ribosomal protein L11 in bacterial systems?

The 50S ribosomal protein L11 forms a critical part of the ribosomal stalk that helps the ribosome interact with GTP-bound translation factors during protein synthesis . This interaction is essential for proper translational efficiency and accuracy. Additionally, ribosomal protein L11 has been shown to bind to and suppress the E3 ligase function of HDM2, thus activating p53 in certain systems . In bacterial ribosomes, L11 belongs to the universal ribosomal protein uL11 family, indicating its highly conserved and essential nature across bacterial species .

What expression systems are most effective for recombinant production of B. licheniformis rplK?

For effective recombinant expression of B. licheniformis rplK, E. coli-based expression systems typically provide good yields for bacterial proteins. Key considerations include:

• Vector selection: pET series vectors with T7 promoter systems offer high expression levels for bacterial proteins

• Host strain optimization: BL21(DE3) derivatives designed for expression of proteins from Gram-positive bacteria often improve yields

• Expression conditions: Induction at lower temperatures (16-25°C) may improve solubility

• Purification approach: Histidine-tagged constructs allow for efficient purification via nickel affinity chromatography

When designing expression constructs, researchers should account for the protein's natural characteristics - similar to the reference L11 protein which has a length of 142 amino acids and mass of 15.2 kDa .

What are optimal conditions for purifying recombinant B. licheniformis rplK while maintaining functional integrity?

Purification of recombinant rplK requires careful optimization to preserve its native structure and function:

• Lysis buffer composition: Use buffers containing 20-50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 5-10% glycerol, and 1-5 mM β-mercaptoethanol

• Purification strategy:

  • Initial capture: Immobilized metal affinity chromatography (IMAC)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

• Quality control metrics:

  • SDS-PAGE for purity assessment (expected band at ~15-16 kDa)

  • Western blotting with anti-L11 antibodies

  • Mass spectrometry for identity confirmation

During purification, monitor the protein's functional integrity through binding assays with GTP-bound translation factors to verify that the ribosomal stalk interaction capability remains intact .

How can researchers effectively assess the interaction between rplK and translation factors?

To characterize interactions between rplK and translation factors:

• Binding assays:

  • Surface plasmon resonance (SPR) to determine binding kinetics (kon, koff) and affinity (KD)

  • Microscale thermophoresis (MST) for solution-based interaction analysis

  • Isothermal titration calorimetry (ITC) for thermodynamic profiling

• Structural analysis methods:

  • X-ray crystallography of the rplK-factor complex

  • Cryo-electron microscopy to visualize interaction in near-native state

  • NMR spectroscopy for dynamic interaction mapping

• Functional validation:

  • In vitro translation assays measuring the impact of wild-type versus mutant rplK

  • GTPase activity assays to assess factor activation by rplK

Document all experimental parameters thoroughly, including buffer compositions, protein concentrations, and instrument settings to ensure reproducibility.

How does B. licheniformis rplK contribute to bacterial stress response and antibiotic resistance mechanisms?

The role of rplK in stress response can be investigated using these methodological approaches:

• Stress induction experiments:

  • Heat shock (42-45°C)

  • Nutrient limitation

  • Antibiotic exposure (sublethal concentrations)

• Quantitative analyses:

  • qRT-PCR to measure changes in rplK expression under stress conditions

  • Ribosome profiling to assess ribosome composition changes

  • Proteomics to identify stress-dependent interaction partners

• Functional investigations:

  • Generation of conditional rplK mutants to assess growth under stress

  • Complementation studies with wild-type versus mutant rplK

This approach parallels methods used to study B. licheniformis responses in other contexts, such as those described for volatile compound production and antifungal activity .

What methodologies are most effective for studying post-translational modifications of B. licheniformis rplK?

To investigate post-translational modifications (PTMs) of rplK:

• PTM identification strategy:

  Method	Application	Resolution	Sample requirement
  LC-MS/MS	Global PTM mapping	High	1-10 μg purified protein
  2D-PAGE	Visual PTM screening	Medium	50-100 μg protein extract
  Western blot	Targeted PTM detection	Medium	10-50 μg protein extract
  Top-down proteomics	Intact protein analysis	Very high	1-5 μg highly purified protein

• Site-directed mutagenesis: Generate point mutations at putative modification sites to assess functional impact

• Enrichment techniques: Develop specific enrichment methods for phosphorylation, methylation, or acetylation to increase detection sensitivity

These approaches should be complemented with functional assays to determine how specific modifications impact rplK's role in translation.

How can researchers connect rplK function to B. licheniformis antifungal properties?

B. licheniformis has demonstrated significant antifungal activity through volatile compounds . To explore potential connections between rplK and these properties:

• Comparative expression analysis:

  • Measure rplK expression levels during antifungal volatile production

  • Compare ribosome composition during active volatile production versus non-producing conditions

• Functional genomics approach:

  • Create rplK conditional mutants and assess impact on volatile production

  • Perform ribosome profiling to identify translational changes affecting antifungal pathways

• Protein-focused investigations:

  • Immunoprecipitation of rplK to identify interaction partners involved in secondary metabolite production

  • Structural analysis of ribosomes during volatile compound synthesis

This research direction connects to established methods for B. licheniformis volatile collection and analysis as described in the research literature .

What experimental design best addresses contradictions in published data on bacterial ribosomal protein functions?

When addressing contradictory findings about rplK function:

• Systematic literature review:

  • Apply FINERMAPS criteria (feasible, interesting, novel, ethical, relevant, manageable, appropriate, potential value, publishability, systematic) to formulate precise research questions

  • Categorize contradictions by experimental system, conditions, and methodologies

• Comparative experimental approach:

  • Design experiments that simultaneously test multiple hypotheses

  • Implement standardized protocols across different B. licheniformis strains

  • Include positive and negative controls from other bacterial species

• Statistical validation:

  • Power analysis to determine appropriate sample sizes

  • Multiple statistical tests to evaluate significance

  • Meta-analysis techniques when combining results from different experiments

• Verification strategy:

  • Employ multiple orthogonal techniques for each key finding

  • Validate in different growth conditions and genetic backgrounds

  • Collaborate with independent laboratories for confirmation

This methodological framework follows established research question formulation principles that emphasize clarity, focus, complexity, and feasibility .

How might structural analysis of B. licheniformis rplK inform ribosome-targeting antibiotic development?

Future research connecting rplK structural studies to antibiotic development should:

• Structural determination approaches:

  • X-ray crystallography of purified B. licheniformis rplK (alone and in ribosomal context)

  • Cryo-EM structures of intact ribosomes with focus on L11-stalk region

  • NMR studies of dynamic regions that may differ from other bacterial species

• Comparative analysis workflow:

  • Superimpose B. licheniformis rplK structures with pathogenic bacterial counterparts

  • Identify structural differences that could be exploited for selective targeting

  • Molecular dynamics simulations to reveal unique binding pocket characteristics

• Structure-based drug design approach:

  • Virtual screening against identified unique structural features

  • Fragment-based design targeting L11-specific conformations

  • Development of peptide mimetics that disrupt specific L11 interactions

These approaches build upon demonstrated roles of L11 in ribosomal function while applying methodologies to identify species-specific features that could be therapeutically relevant .

What are the methodological considerations for integrating rplK research into systems biology studies of B. licheniformis?

For systems biology integration:

• Multi-omics experimental design:

  • Transcriptomics: RNA-seq during various growth conditions

  • Proteomics: Quantitative proteomics with focus on translation machinery

  • Metabolomics: Targeted analysis of metabolites affected by translational regulation

  • Ribosome profiling: To connect rplK function to translational efficiency

• Data integration framework:

  • Correlation networks linking rplK expression to global cellular processes

  • Pathway enrichment analysis to identify biological processes most affected by rplK

  • Predictive modeling of translation efficiency based on rplK status

• Validation experiments:

  • CRISPR interference to modulate rplK expression

  • Targeted metabolic flux analysis to verify predicted changes

  • Growth phenotyping under diverse conditions to validate model predictions

This systems approach connects the molecular function of rplK to broader cellular phenotypes, including potentially the antifungal properties observed in B. licheniformis .