Recombinant Bacillus licheniformis 50S ribosomal protein L6 (rplF)

What is Bacillus licheniformis 50S ribosomal protein L6 (rplF) and where is it located in the genome?

Bacillus licheniformis 50S ribosomal protein L6 (rplF) is a component of the large ribosomal subunit that contributes to the structural integrity and functionality of the ribosome during protein translation. In the Bacillus licheniformis ATCC 14580 genome, the rplF gene is located at position 140516..141052, encoding a protein of 178 amino acids with a GC content of 41.90% . The gene is annotated with the synonym BL01036 and has been assigned the protein ID 161760690 . It is one of several ribosomal proteins found in a cluster in the B. licheniformis genome, which is characteristic of the organization of ribosomal protein genes in prokaryotes.

Why is rplF considered significant for phylogenetic studies of Bacillus species?

Ribosomal protein L6 has emerged as one of the most reliable phylogenetic markers among ribosomal proteins. Research has demonstrated that rplF belongs to a select group of ribosomal proteins (including L6, L7/12, L9, L13, L24, L32, S3, S9, S12, S15, S16, S17, and S18) that can accurately reproduce the phylogenetic positions of different Bacillus species in alignment with the gold standard 16S rRNA phylogeny . Unlike some other ribosomal proteins that struggle to differentiate between closely related species such as B. licheniformis and B. pumilus, rplF maintains sufficient sequence diversity to chronicle the evolutionary relationships between Bacillus species with high fidelity . This makes it particularly valuable for researchers seeking alternative phylogenetic markers or complementary approaches to 16S rRNA analysis.

What expression systems are recommended for recombinant production of B. licheniformis rplF?

For recombinant production of B. licheniformis rplF, E. coli expression systems remain the most widely utilized approach due to their efficiency and versatility. Recommended methodological approaches include:

• Vector selection: pET series vectors under the control of T7 promoter systems offer high expression levels for ribosomal proteins.

• Host strain optimization: BL21(DE3) derivatives with reduced proteolytic activity such as BL21(DE3)pLysS are preferred to minimize degradation of the recombinant protein.

• Expression conditions: Induction with 0.5-1.0 mM IPTG at lower temperatures (16-25°C) often improves proper folding and solubility of ribosomal proteins.

• Codon optimization: Adapting the B. licheniformis rplF coding sequence to E. coli codon usage can significantly enhance expression levels, especially considering the 41.90% GC content of the native gene .

• Fusion tags: His-tag or GST fusion constructs facilitate downstream purification while potentially improving solubility.

For researchers encountering solubility issues, alternative approaches include expression in B. subtilis systems, which may provide a more native-like environment for proper folding of Bacillus ribosomal proteins.

How can recombinant B. licheniformis rplF be used in comparative phylogenetic analysis?

Recombinant B. licheniformis rplF offers significant advantages for comparative phylogenetic analysis through the following methodological approaches:

• Multi-protein phylogenetic reconstruction: Researchers can use purified recombinant rplF alongside other category 1 ribosomal proteins (those that accurately reproduce phylogenetic positions) to create robust phylogenetic trees that validate or complement 16S rRNA phylogeny .

• Sequence-structure-function relationship analysis: The following experimental workflow is recommended:

  • Express recombinant rplF from multiple Bacillus species

  • Perform comparative structural analysis through circular dichroism or crystallography

  • Correlate structural differences with sequence divergence

  • Map evolutionary changes to functional domains

• Evolutionary rate calibration: Researchers can utilize the evolutionary rates of rplF to establish molecular clocks for dating divergence events within the Bacillus genus.

Experimental evidence demonstrates that ribosomal protein L6 consistently reproduces phylogenetic relationships between B. amyloliquefaciens, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis with high accuracy, making it an excellent candidate for species differentiation and evolutionary studies .

What purification strategies yield the highest purity recombinant B. licheniformis rplF?

Obtaining high-purity recombinant B. licheniformis rplF requires a systematic purification approach:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns for His-tagged rplF constructs, with optimized imidazole gradients (typically 20-250 mM) to minimize non-specific binding.

• Secondary purification: Ion exchange chromatography utilizing the theoretical pI of rplF to select appropriate column chemistry and buffer conditions.

• Polishing step: Size exclusion chromatography to remove aggregates and achieve >95% purity, which is critical for structural studies or antibody production.

• Endotoxin removal: For immunological applications, endotoxin removal using polymyxin B columns or phase separation techniques.

Purification efficiency can be monitored through SDS-PAGE and Western blotting using antibodies against the fusion tag or the rplF protein itself. Mass spectrometry analysis should be used to confirm protein identity and integrity.

What experimental approaches can detect interactions between recombinant rplF and other ribosomal components?

Investigating interactions between recombinant B. licheniformis rplF and other ribosomal components requires sophisticated biophysical and biochemical techniques:

• Pull-down assays: Utilizing tagged recombinant rplF as bait to identify interacting partners from B. licheniformis lysates, followed by mass spectrometry identification.

• Surface Plasmon Resonance (SPR): Quantitative assessment of binding kinetics between purified rplF and other purified ribosomal proteins or rRNA fragments.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Mapping interaction interfaces through differential solvent accessibility.

• Crosslinking Mass Spectrometry (XL-MS): Identification of proximity relationships between rplF and other ribosomal components through chemical crosslinking.

• Fluorescence Resonance Energy Transfer (FRET): Real-time monitoring of interactions in reconstituted systems using fluorescently labeled components.

What are the best practices for designing experiments to study B. licheniformis rplF conservation and variation?

Comprehensive experimental design for studying B. licheniformis rplF conservation requires multiple approaches:

• Sampling strategy: Include multiple strains of B. licheniformis alongside representatives of other Bacillus species, particularly B. subtilis as a close relative.

• Genomic analysis workflow:

  • Extract genomic DNA using standardized protocols

  • Amplify rplF using genus-specific and species-specific primers

  • Sequence using both Sanger and next-generation methods

  • Employ RFLP analysis for rapid screening of variations

• Replication requirements: Following principles of sound experimental design, a minimum of three biological replicates and technical replicates should be included to ensure statistical validity .

• Control selection: Appropriate controls should include:

  • Highly conserved ribosomal proteins (L5, L36, S2, S10, and S21) as negative controls

  • Variable ribosomal proteins (L7Ae, L17, L20) as positive controls

• Constraint analysis: Determine selective pressures by calculating dN/dS ratios across different functional domains of the protein.

How can recombinant B. licheniformis rplF be used to study the impact of small RNAs on ribosome function?

Recent discoveries regarding small RNA regulation in B. licheniformis provide a novel context for studying ribosomal protein function. Researchers can explore potential connections between small RNAs and rplF through:

• Co-immunoprecipitation: Using antibodies against recombinant rplF to pull down potentially associated small RNAs.

• RNA-protein binding assays: Employing electrophoretic mobility shift assays (EMSAs) or RNA immunoprecipitation followed by sequencing (RIP-seq) to identify small RNAs directly interacting with rplF.

• Functional studies: Investigating whether small RNAs like AprAs impact translation efficiency or ribosome assembly through interactions with ribosomal proteins.

• Expression correlation analysis: Examining whether conditions that affect small RNA expression also influence rplF expression levels or post-translational modifications.

This research direction is particularly intriguing given the discovery of the AprAs antisense small RNA in B. licheniformis and its role in regulating subtilisin production . While direct connections between small RNAs and rplF have not been established, this represents an emerging frontier for investigation.

What are the primary challenges in obtaining functionally active recombinant B. licheniformis rplF?

Researchers face several technical challenges when working with recombinant B. licheniformis rplF:

• Proper folding: Ribosomal proteins often misfold when expressed outside their native ribosomal assembly context.

  • Solution: Co-expression with chaperones (GroEL/ES, DnaK/J) and slower expression rates at reduced temperatures.

• Solubility issues: Recombinant rplF may form inclusion bodies.

  • Solution: Fusion to solubility enhancers like MBP (maltose-binding protein) or SUMO, with subsequent tag removal.

• Functionality assessment: Confirming that recombinant rplF retains native function.

  • Solution: In vitro ribosome reconstitution assays with rplF-depleted 50S subunits.

• RNA binding properties: Ensuring recombinant rplF maintains proper RNA interactions.

  • Solution: RNA binding assays with rRNA fragments corresponding to the rplF binding site.

• Stability during purification: Preventing degradation during extraction and purification.

  • Solution: Addition of protease inhibitors and maintaining cold temperatures throughout purification.

How can researchers address data interpretation challenges when studying recombinant B. licheniformis rplF phylogeny?

When using recombinant B. licheniformis rplF for phylogenetic studies, researchers should address several analytical challenges:

• Methodological inconsistencies: Different phylogenetic algorithms may produce varying results.

  • Solution: Apply multiple phylogenetic methods (Maximum Likelihood, Bayesian, Neighbor-Joining) and compare for consistency .

• Bootstrap validation: Ensuring statistical support for phylogenetic placements.

  • Solution: Implement bootstrap analysis with at least 1000 replicates and consider branches reliable only when bootstrap values exceed 70%.

• Horizontal gene transfer detection: Identifying potential evolutionary artifacts.

  • Solution: Compare rplF phylogeny with multiple reference genes and search for incongruence patterns.

• Sequence quality assurance: Preventing sequencing errors from affecting phylogenetic placement.

  • Solution: Sequence verification through bidirectional Sanger sequencing and coverage analysis for NGS data.

• Species boundary determination: Defining thresholds for species discrimination.

  • Solution: Calculate intra- and interspecific sequence variation and establish statistically supported cutoff values.

What quality control measures should be implemented for recombinant B. licheniformis rplF production?

Rigorous quality control is essential for reproducible research with recombinant B. licheniformis rplF:

• Sequence verification: Confirm the recombinant construct through DNA sequencing before expression.

• Protein identity confirmation: Validate protein identity using:

  • Western blotting with specific antibodies

  • Peptide mass fingerprinting via tryptic digestion and mass spectrometry

  • N-terminal sequencing for unambiguous identification

• Purity assessment: Employ multiple methods to determine protein purity:

  • SDS-PAGE with densitometry analysis (target >95% purity)

  • Size exclusion chromatography to detect aggregates

  • Capillary electrophoresis for high-resolution purity assessment

• Functional validation: Confirm biological activity through:

  • RNA binding assays

  • Contribution to in vitro ribosome assembly

  • Circular dichroism to verify proper secondary structure

• Batch consistency: Implement batch-to-batch comparison using:

  • Standardized activity assays

  • Precise protein concentration determination using amino acid analysis

How might structural studies of recombinant B. licheniformis rplF inform antibiotic development?

Structural studies of recombinant B. licheniformis rplF have significant implications for antibiotic research:

• Structure determination methodology:

  • X-ray crystallography of purified recombinant rplF

  • Cryo-EM of intact B. licheniformis ribosomes with focus on the L6 region

  • NMR studies of specific domains with potential antibiotic binding sites

• Comparative structural analysis:

  • Identification of structural differences between B. licheniformis rplF and human ribosomal proteins

  • Mapping of species-specific pockets that could serve as selective antibiotic targets

• In silico screening:

  • Molecular docking of compound libraries against identified binding pockets

  • Molecular dynamics simulations to assess stability of potential antibiotic-rplF complexes

• Validation studies:

  • Site-directed mutagenesis of residues in potential binding sites

  • Binding assays with candidate compounds

  • Growth inhibition assays using B. licheniformis strains with wild-type and mutant rplF

This research direction is particularly relevant since ribosomal proteins are established targets for various antibiotics, and species-specific structural features could enable selective targeting of pathogenic Bacillus species without affecting beneficial ones.

What novel applications might emerge from understanding the coevolution of rplF with other ribosomal components?

The demonstrated phylogenetic accuracy of rplF opens several innovative research directions:

• Coevolution network mapping:

  • Correlation analysis between rplF sequence variation and other ribosomal components

  • Identification of coevolving residue networks through statistical coupling analysis

  • Experimental validation of predicted coevolutionary relationships through compensatory mutation studies

• Synthetic biology applications:

  • Design of chimeric ribosomes with components from different Bacillus species

  • Engineering of ribosomes with enhanced properties based on understanding of natural variation

  • Development of species-specific translation systems for biotechnology applications

• Evolutionary adaptation mechanisms:

  • Investigation of how rplF variation contributes to adaptation to different ecological niches

  • Experimental evolution studies to track rplF changes under selective pressure

  • Correlation of rplF sequence variants with growth under different environmental conditions

Understanding the evolutionary constraints and adaptations of rplF will provide insights into ribosome evolution and potentially enable the rational design of ribosomes with novel properties for biotechnological applications.