Recombinant Acinetobacter sp. 50S ribosomal protein L28 (rpmB)

What expression systems are most effective for recombinant production of Acinetobacter sp. rpmB?

For effective recombinant production of Acinetobacter sp. rpmB, several expression systems can be employed, each with specific advantages:

E. coli-based expression systems:

• BL21(DE3) or Rosetta strains are preferred for small ribosomal proteins

• Codon optimization may be necessary when expressing Acinetobacter genes in E. coli

• Expression vector selection should incorporate affinity tags (His6, GST) for simplified purification

• Temperature modulation (typically 18-25°C) after induction improves solubility

Purification strategy:

• Cell lysis using sonication or pressure-based disruption in buffer containing protease inhibitors

• Initial clarification via centrifugation (14,000-20,000 × g)

• Affinity chromatography (Ni-NTA for His-tagged constructs)

• Size exclusion chromatography for final polishing and buffer exchange

Quality control includes SDS-PAGE analysis, mass spectrometry verification, and functional assays to ensure proper folding. Typical yields range from 5-15 mg/L of bacterial culture when optimization is performed .

How can researchers validate the functionality of recombinant rpmB protein?

Validating functionality of recombinant rpmB requires multiple approaches to ensure the protein retains its native properties:

In vitro translation assays:

• Reconstitution of ribosome with and without L28 to assess impact on translation efficiency

• Monitoring peptide synthesis rates using radiolabeled amino acids

• Assessing binding to 23S rRNA through filter binding or electrophoretic mobility shift assays

Structural integrity assessment:

• Circular dichroism to confirm proper secondary structure formation

• Thermal shift assays to determine protein stability

• Limited proteolysis to evaluate folding quality

Functional complementation:

• Expression of recombinant rpmB in L28-deficient strains (if viable)

• Assessment of growth restoration under various conditions

• Ribosome profiling to evaluate translation fidelity

Researchers should note that ribosomal proteins often function in complex with other components, necessitating contextual validation beyond isolated protein characterization.

What methodologies determine the essentiality of rpmB in Acinetobacter species?

Determining gene essentiality requires sophisticated genetic approaches that can be applied to investigate rpmB in Acinetobacter:

High-efficiency recombination methods:
Gene replacement techniques employing PCR-generated cassettes with antibiotic resistance markers flanked by 40-50 nucleotide homology regions to the target gene allow precise replacement of the chromosomal rpmB open reading frame . This approach requires:

• Design of primers containing:

  • 40-50 nucleotide sequence homologous to regions surrounding the rpmB ORF

  • 22-25 nucleotide priming sequence complementary to antibiotic resistance gene

• PCR verification of recombinants using:

  • Colony PCR with primers flanking the insertion site

  • Sequencing confirmation of successful replacements

• Essentiality assessment strategies:

  • Inability to obtain gene knockouts suggests essentiality

  • Creation of partial diploids (merodiploids) to test conditional essentiality

  • Evaluation of segregation rates under non-selective conditions

Conditional knockout systems:

• Inducible promoter replacement to control expression

• Temperature-sensitive plasmids carrying functional copies

• CRISPR interference (CRISPRi) for targeted knockdown

Phenotypic microarray analysis can subsequently characterize conditional mutants for metabolic changes, as demonstrated in studies of other ribosomal genes .

How does rpmB expression correlate with antibiotic resistance mechanisms in Acinetobacter?

While direct evidence for rpmB's role in antibiotic resistance is limited, investigations can follow methodologies established for studying ribosome-targeting antibiotics:

MALDI-TOF MS analytical approach:
Mass spectrometry has proven valuable for detecting carbapenemase activity in Acinetobacter species and similar approaches can be adapted for studying ribosomal modifications:

• Sample preparation protocol:

  • Bacterial inoculum optimization (1×10^9 to 2.5×10^10 CFU/ml)

  • Incubation with antibiotic of interest (e.g., macrolides, lincosamides)

  • Matrix preparation with α-cyano-4-hydroxycinnamic acid

• Spectrum analysis parameters:

  • Monitoring specific peaks associated with antibiotic modification

  • Comparative analysis between resistant and susceptible strains

  • Evaluation of peak intensity changes after enzyme inhibitor treatment

Correlation analysis framework:

• RNA sequencing to quantify rpmB expression levels across resistant isolates

• Proteomic analysis to determine L28 abundance and modifications

• Site-directed mutagenesis of rpmB to assess impact on minimum inhibitory concentrations

Researchers should design experiments that distinguish between direct effects (L28 modification) and indirect effects (altered expression of resistance determinants) .

What are the optimal protocols for studying rpmB interactions with other ribosomal components?

Investigating L28 interactions within the ribosomal complex requires specialized approaches:

Cryo-electron microscopy:

• Sample preparation with purified 50S subunits or complete 70S ribosomes

• Data collection at 300kV with direct electron detectors

• Processing with motion correction and CTF estimation

• 3D reconstruction and focused refinement around L28 binding site

Cross-linking mass spectrometry (XL-MS):

• Chemical cross-linking protocol:

  • Treatment with bissulfosuccinimidyl suberate (BS3) or disuccinimidyl suberate (DSS)

  • Quenching with primary amines (e.g., Tris buffer)

  • Digestion with trypsin and/or other proteases

  • Enrichment of cross-linked peptides

• MS data analysis workflow:

  • Identification of cross-linked peptide pairs

  • Mapping interactions to 3D structural models

  • Validation through mutagenesis of key residues

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Maps protein dynamics and solvent accessibility

• Identifies binding interfaces between L28 and rRNA or other proteins

• Compares conformational changes upon complex formation

These methods provide complementary structural information to build comprehensive interaction models of L28 within the ribosomal architecture.

How can researchers investigate the potential role of rpmB in regulating drug resistance similar to RPL28 in cancer cells?

The human RPL28 gene has been implicated in sorafenib resistance in hepatocellular carcinoma (HCC) , suggesting potential parallels for investigating bacterial rpmB in antibiotic resistance:

Experimental approach for bacterial systems:

• Development of resistant strains:

  • Serial passage in increasing antibiotic concentrations

  • Measurement of IC50 values for parent and resistant strains

  • RNA sequencing to identify differentially expressed genes

• Functional validation:

  • Gene knockdown using antisense RNA or CRISPR interference

  • Complementation with wild-type and mutant alleles

  • Assessment of resistance phenotype reversion

• Mechanistic investigation:

  • Ribosome profiling to assess translation efficiency changes

  • Proteomics to identify altered stress response pathways

  • Metabolomics to detect adaptive metabolic shifts

Comparative analysis framework:
When examining rpmB's potential role in resistance, researchers should consider parallels from eukaryotic systems while acknowledging fundamental differences in ribosome structure and function.

Researchers should employ isogenic strains differing only in rpmB expression to avoid confounding factors when evaluating resistance phenotypes .

What bioinformatic approaches are most effective for analyzing rpmB sequence conservation across Acinetobacter species?

Comprehensive bioinformatic analysis of rpmB requires multiple computational approaches:

Phylogenetic analysis protocol:

• Sequence retrieval:

  • Extract rpmB sequences from complete Acinetobacter genomes

  • Include diverse clinical and environmental isolates

  • Incorporate reference strains (ATCC 17978, etc.)

• Multiple sequence alignment:

  • MUSCLE or MAFFT alignment with refinement

  • Visualization with Jalview or similar tools

  • Conservation scoring using Scorecons or AMAS

• Phylogenetic tree construction:

  • Maximum likelihood methods (RAxML, IQ-TREE)

  • Bayesian inference (MrBayes)

  • Tree visualization with iTOL or FigTree

Structural bioinformatics approaches:

• Homology modeling using existing bacterial L28 structures

• Identification of conserved surface patches for interaction mapping

• Electrostatic potential calculation to identify functional surfaces

Selection pressure analysis:

• dN/dS ratio calculation using PAML or HyPhy

• Identification of sites under positive or purifying selection

• Correlation with antibiotic resistance phenotypes across species

The high conservation of rpmB across Acinetobacter species suggests critical functional roles, while variable regions may indicate species-specific adaptations or resistance mechanisms.

What emerging technologies show promise for advancing rpmB research in Acinetobacter?

Several cutting-edge technologies offer significant potential for advancing our understanding of rpmB in Acinetobacter species:

Single-molecule approaches:

• Fluorescence resonance energy transfer (FRET) to monitor L28 dynamics during translation

• Optical tweezers to measure force generation during ribosome function

• Zero-mode waveguides for real-time observation of single-ribosome translation

Integrative structural biology:

• Combination of cryo-EM, X-ray crystallography, and NMR data

• Molecular dynamics simulations to predict functional movements

• Artificial intelligence-based structure prediction methods

Systems biology approaches:

• Ribosome profiling coupled with transcriptomics and proteomics

• Network analysis of L28 interactions within cellular pathways

• Genome-wide CRISPR screens to identify genetic interactions

These technologies will enable researchers to address outstanding questions about L28's role in antibiotic resistance, ribosome assembly, and translation regulation in Acinetobacter species.

How should researchers design experiments to resolve contradictory findings about rpmB function?

When confronted with contradictory findings regarding rpmB function, researchers should implement:

Experimental design strategies:

• Standardization of experimental conditions:

  • Defined growth media and growth phase for sampling

  • Consistent antibiotic concentrations and exposure times

  • Uniform genetic backgrounds for mutant construction

• Multiple methodological approaches:

  • Complementary in vivo and in vitro systems

  • Orthogonal measurement techniques

  • Independent validation in different laboratories

• Systematic controls:

  • Positive and negative controls for each experiment

  • Complementation studies to confirm phenotype causality

  • Dose-response relationships rather than single-point measurements

Data integration framework:

• Meta-analysis of published findings with statistical rigor

• Bayesian approaches to weigh evidence quality

• Development of computational models to reconcile divergent data

By implementing these strategies, researchers can develop a more coherent understanding of rpmB's functions and its potential as a therapeutic target in Acinetobacter species.