Recombinant Acinetobacter sp. 50S ribosomal protein L36 (rpmJ)

What is the basic structure and size of RpmJ protein in Acinetobacter species?

RpmJ (L36) is one of the smallest 50S ribosomal proteins, typically consisting of approximately 38 amino acids. As observed in related bacterial species, RpmJ plays a crucial role in 23S rRNA folding and ribosomal assembly . The small size of this protein makes it particularly interesting for structural studies, as it represents a minimalistic functional unit within the complex ribosomal machinery. Comparative analyses between Acinetobacter and E. coli RpmJ proteins reveal high conservation of key functional domains despite evolutionary distance between these bacterial genera.

How does RpmJ contribute to ribosomal assembly in Acinetobacter?

RpmJ functions as an integral component in the assembly of the 50S ribosomal subunit, specifically contributing to proper 23S rRNA folding . In experimental models using related bacteria, RpmJ knockout mutants display altered translation fidelity, indicating that this small protein plays a disproportionately important role in maintaining ribosomal structural integrity. Research methodologies to study this process typically involve cryo-electron microscopy of ribosomes at various assembly stages, comparing wild-type and RpmJ-deficient strains. Evidence suggests that RpmJ acts as a nucleation point for other ribosomal proteins during the hierarchical assembly process of the 50S subunit.

What are the recommended methods for generating recombinant RpmJ from Acinetobacter species?

For recombinant expression of Acinetobacter RpmJ, researchers typically employ the following protocol:

• Gene amplification: PCR amplification of the rpmJ gene from Acinetobacter genomic DNA using specific primers with appropriate restriction sites.

• Vector construction: Cloning the amplified gene into an expression vector (commonly pET-based systems) with a histidine or other affinity tag.

• Expression optimization: Testing expression in E. coli BL21(DE3) or similar strains using varying IPTG concentrations (0.1-1.0 mM) and temperatures (16-37°C).

• Purification strategy: Employing metal affinity chromatography followed by size exclusion chromatography to obtain pure protein.

When working with such a small protein (38 amino acids), special attention must be paid to fusion tag design and potential interference with protein folding. Some researchers prefer TEV protease-cleavable tags to obtain native protein post-purification. Western blotting with anti-His antibodies or custom antibodies against RpmJ provides validation of successful expression.

What are effective approaches for studying RpmJ knockout phenotypes in Acinetobacter?

Based on methodologies employed in related bacterial studies, researchers should consider:

• Generation of clean deletion mutants using homologous recombination or CRISPR-Cas9 approaches to avoid polar effects.

• Complementation studies: Reintroducing rpmJ on a plasmid to confirm phenotype restoration.

• Phenotypic characterization should include:

  • Growth curve analysis under standard and stress conditions

  • Ribosome profiling to assess translation activity

  • Antibiotic sensitivity testing, particularly with translation inhibitors

  • Metal sensitivity/resistance assays, especially for zinc

Researchers have observed that rpmJ mutants in E. coli display increased sensitivity to protein synthesis inhibitors including chloramphenicol, erythromycin, clarithromycin, and tetracycline, suggesting altered ribosomal function . Similar approaches could be applied to Acinetobacter to determine conservation of these phenotypes across species.

How can researchers effectively measure translation fidelity in RpmJ mutants?

Translation fidelity in RpmJ mutants can be assessed using dual luciferase reporter systems, similar to methodologies employed in E. coli studies . The protocol involves:

• Transforming wild-type and rpmJ mutant strains with plasmids containing Renilla (Rluc) and Firefly (Fluc) luciferase genes separated by:

  • Stop codons (to measure readthrough)

  • Frameshift mutations (to measure frameshifting)

• Measuring luciferase activities in cell lysates using a luminometer

• Calculating the Fluc/Rluc (F/R) ratio, where higher values indicate decreased translation fidelity

In E. coli rpmJ mutants, increased F/R values were observed particularly with UGA stop codon readthrough, demonstrating that this small ribosomal protein significantly impacts translation accuracy . Implementing appropriate controls, including multiple biological replicates and statistical analysis, is crucial for reliable interpretation of results.

What is the relationship between RpmJ and zinc resistance in bacteria?

One of the most intriguing findings regarding RpmJ is its unexpected role in zinc homeostasis. Research shows that knockout of rpmJ leads to zinc resistance in E. coli . This resistance mechanism operates through:

• Decreased intracellular zinc concentration under excess zinc conditions

• Dependence on ZntA, a zinc efflux pump

• Altered expression of genes involved in stress response and metabolism

The table below summarizes key findings on zinc resistance in ribosomal protein mutants:

To study this phenomenon in Acinetobacter, researchers should employ metal sensitivity assays using gradient plates or broth dilution methods with varying zinc concentrations, followed by intracellular zinc concentration measurements using fluorescent zinc probes or atomic absorption spectroscopy.

How does RpmJ influence intracellular zinc concentration in Acinetobacter?

While specific mechanisms in Acinetobacter require further investigation, insights from E. coli suggest that RpmJ's influence on zinc homeostasis likely operates through indirect mechanisms rather than direct zinc binding . Methodological approaches to investigate this include:

• Measurement of intracellular zinc concentration using fluorescent probes (FluoZin-3)

• Expression analysis of zinc transport systems (importers and exporters)

• Epistasis studies with zinc transport system knockouts (e.g., ZntA) to determine pathway interactions

• Proteomics and transcriptomics to identify differentially expressed genes related to metal homeostasis

In E. coli, the rpmJ mutant showed decreased intracellular zinc concentration under excess zinc conditions, and this phenotype was dependent on the zinc efflux pump ZntA . This suggests complex regulatory relationships between ribosomal function and metal homeostasis systems that may be conserved in Acinetobacter species.

How does RpmJ knockout affect antibiotic susceptibility profiles in Acinetobacter?

Given that rpmJ knockout in E. coli increased sensitivity to protein synthesis inhibitors , similar effects might be expected in Acinetobacter. To investigate this:

• Determine minimum inhibitory concentrations (MICs) of various antibiotic classes:

  • Protein synthesis inhibitors (tetracyclines, macrolides, aminoglycosides)

  • Cell wall synthesis inhibitors (β-lactams, carbapenems)

  • DNA replication inhibitors (fluoroquinolones)

• Perform time-kill assays to assess the rate of bacterial killing

• Analyze expression of antibiotic resistance genes and efflux pumps

This research is particularly relevant given the high incidence of extreme drug resistance (XDR) in Acinetobacter baumannii, which is associated with poor clinical outcomes . Understanding how ribosomal proteins like RpmJ influence antibiotic susceptibility could potentially lead to novel therapeutic strategies.

How do transcriptomic profiles differ between wild-type and RpmJ-deficient Acinetobacter?

RNA sequencing (RNA-Seq) analysis represents a powerful approach to understand the global impact of RpmJ deficiency. Based on findings in E. coli, researchers might expect:

• Altered expression of genes involved in:

  • Translation and ribosomal function

  • Energy metabolism

  • Stress response pathways

  • Metal homeostasis (particularly zinc-related genes)

• Methodological considerations:

  • Sample multiple growth phases (early log, mid-log, stationary)

  • Include stress conditions (metal excess, antibiotic exposure)

  • Perform adequate biological replicates (minimum 3)

  • Consider ribosome profiling in parallel to distinguish transcriptional from translational effects

In E. coli, RNA sequencing revealed 195 upregulated and 275 downregulated genes in the rpmJ mutant compared to wild-type, affecting diverse functional categories . Notably, genes encoding iron-sulfur cluster synthases were downregulated, potentially contributing to zinc resistance by reducing the availability of toxic targets for zinc.

Can structural biology approaches reveal how RpmJ impacts ribosomal function in Acinetobacter?

Advanced structural biology techniques can provide mechanistic insights into RpmJ function:

• Cryo-electron microscopy (cryo-EM) of ribosomes from wild-type and rpmJ mutant strains to visualize structural alterations

• Structural analysis of translation in progress using ribosome stalling techniques

• Molecular dynamics simulations to predict conformational changes in the absence of RpmJ

• Cross-linking mass spectrometry to identify protein-protein and protein-RNA interactions involving RpmJ

These approaches can help determine whether RpmJ's effects are due to direct structural contributions to the ribosome or indirect regulatory mechanisms. Given RpmJ's role in 23S rRNA folding, structural changes in the mutant ribosomes might explain the observed altered translation fidelity and antibiotic sensitivity.