Recombinant Acinetobacter sp. 50S ribosomal protein L21 (rplU)

What methods are used for recombinant expression of Acinetobacter rplU protein?

Recombinant expression of Acinetobacter rplU typically employs bacterial expression systems, particularly E. coli, using the following methodological approach:

• Gene isolation and vector preparation: The rplU gene (309 bp) is PCR-amplified from Acinetobacter genomic DNA using specifically designed primers that incorporate restriction sites for directional cloning.

• Expression system selection: Common expression vectors include pET series plasmids with T7 promoters for high-level expression in E. coli BL21(DE3) or similar strains.

• Expression optimization: Expression conditions must be optimized for temperature (typically 18-30°C), IPTG concentration (0.1-1.0 mM), and duration (4-24 hours) to maximize soluble protein yield.

• Purification strategy: His-tagged rplU can be purified using nickel affinity chromatography followed by size exclusion chromatography to obtain high purity protein.

The process typically yields 10-15 mg of purified protein per liter of bacterial culture, with costs starting at approximately $99 plus $0.30 per amino acid for custom expression services .

How does rplU compare across different Acinetobacter species?

When analyzing rplU conservation across Acinetobacter species, researchers should employ comparative genomic approaches similar to those used for rpoB gene analysis . While specific rplU comparison data across all Acinetobacter species is not extensively documented, the approach can be modeled after successful phylogenetic analyses conducted using rpoB and 16S rRNA genes.

The methodological approach should include:

• Sequence alignment of rplU genes from various Acinetobacter species using MAFFT or similar alignment tools

• Phylogenetic analysis using programs such as MEGA with Neighbor-Joining (NJ) tree construction

• Calculation of sequence similarities and genetic distances

Based on related ribosomal protein studies, we would expect high conservation of rplU within the Acinetobacter baumannii-calcoaceticus complex, which includes A. baumannii, A. pittii, A. calcoaceticus, and A. nosocomialis, as these species are phylogenetically closely related .

How can rplU be used in Acinetobacter species identification and taxonomic studies?

While rpoB gene sequencing is currently considered the most accurate method for Acinetobacter species identification , rplU has potential as an alternative or complementary molecular marker for taxonomic and identification studies.

Methodological approach for implementing rplU in species identification:

• Primer design and optimization: Design degenerate primers targeting conserved regions flanking variable segments of the rplU gene, similar to the approach used for rpoB .

• Comparative sequence analysis: Evaluate the discriminatory power of rplU by:

  • Calculating interspecies diversity using similarity percentages between reference strains

  • Comparing with established markers like rpoB and 16S rRNA genes

  • Constructing phylogenetic trees to visualize taxonomic relationships

• Validation against type strains: Test the rplU sequencing method against the collection of reference Acinetobacter species, particularly focusing on the challenging Acinetobacter baumannii-calcoaceticus complex members that are difficult to distinguish by traditional methods .

The limitations of 16S rRNA gene sequencing for closely related Acinetobacter species (e.g., A. pittii, A. nosocomialis, A. calcoaceticus, and A. baumannii having nearly identical 16S rRNA sequences) suggest that alternative markers like rplU could potentially offer improved resolution, though this would require extensive validation studies.

What role might rplU play in antibiotic resistance mechanisms of Acinetobacter species?

Investigating the potential role of rplU in antibiotic resistance requires sophisticated molecular and biochemical approaches:

• Comparative expression analysis: Quantify rplU expression levels in antibiotic-resistant vs. sensitive strains using RT-qPCR or RNA-seq under various antibiotic exposures.

• Mutation studies: Generate site-directed mutants of rplU to identify residues critical for ribosome function and antibiotic interaction.

• Structural biology approaches: Use cryo-EM to visualize the 50S ribosomal subunit with bound antibiotics in wild-type vs. resistant strains.

Research suggests that ribosomal proteins can contribute to resistance mechanisms, particularly against antibiotics targeting the ribosome. The significant difference in carbapenem resistance rates between A. baumannii (high resistance) and non-A. baumannii species (only 2.6% resistance) presents an opportunity to investigate whether differential ribosomal protein structure or expression contributes to this disparity.

What experimental approaches are most effective for studying rplU-23S rRNA interactions?

Since rplU binds to 23S rRNA specifically in the presence of protein L20 , the following methodological approaches are recommended for characterizing these interactions:

• In vitro reconstitution assays:

  • Express and purify recombinant rplU and L20 proteins

  • Synthesize or extract 23S rRNA

  • Perform binding assays under varying conditions using techniques such as:

    • Electrophoretic mobility shift assay (EMSA)

    • Filter binding assays

    • Surface plasmon resonance (SPR)

• Crosslinking studies:

  • Use UV or chemical crosslinking to capture transient interactions

  • Identify interaction sites by mass spectrometry after nuclease/protease digestion

• Structural biology approaches:

  • Cryo-electron microscopy of assembled complexes

  • X-ray crystallography of sub-complexes

  • NMR studies of specific interaction domains

• Computational modeling:

  • Molecular dynamics simulations of rplU-L20-23S rRNA interactions

  • Prediction of interaction sites based on conservation analysis

The interaction dependency on protein L20 suggests potential cooperative binding or conformational changes that should be carefully considered in experimental design.

How can rplU be leveraged for novel antimicrobial development against multidrug-resistant Acinetobacter?

The development of novel antimicrobials targeting rplU would follow these research stages:

• Target validation:

  • Generate conditional knockdown mutants of rplU in Acinetobacter to confirm essentiality

  • Identify unique structural features of Acinetobacter rplU not present in human ribosomal proteins

• High-throughput screening approaches:

  • Develop an in vitro translation system using Acinetobacter ribosomes

  • Screen compound libraries for specific inhibition of translation

  • Utilize thermal shift assays to identify compounds that bind directly to rplU

• Structure-based drug design:

  • Determine high-resolution structures of Acinetobacter rplU alone and in complex with 23S rRNA

  • Identify potential binding pockets for small molecules

  • Design compounds that specifically disrupt rplU function or its assembly into the ribosome

• Validation in clinical isolates:

  • Test candidate compounds against diverse clinical isolates, particularly those from multidrug-resistant Acinetobacter baumannii infections in hospital settings

  • Evaluate efficacy against different infection types (respiratory, urinary tract, bloodstream, and wound infections)

This approach could be particularly valuable given the significant clinical challenge posed by multidrug-resistant Acinetobacter baumannii in healthcare settings .

What are the implications of rplU sequence variations on ribosomal function across Acinetobacter species?

Investigating the functional consequences of rplU sequence variations requires sophisticated comparative analyses:

• Comprehensive sequence comparison:

  • Collect rplU sequences from diverse Acinetobacter isolates

  • Identify conserved vs. variable regions using multiple sequence alignment

  • Map variations to functional domains and interaction surfaces

• Structure-function analysis:

  • Model the impact of sequence variations on protein structure

  • Predict effects on interactions with 23S rRNA and L20

  • Generate recombinant proteins with specific variations for functional testing

• Translational efficiency studies:

  • Reconstitute ribosomes with variant rplU proteins

  • Measure translation rates and accuracy using reporter systems

  • Compare stress responses in strains with different rplU variants

• Evolutionary analysis:

  • Calculate selection pressures on different regions of the rplU gene

  • Identify potential adaptive mutations in clinical vs. environmental isolates

This research could provide insights into how ribosomal protein variations might contribute to Acinetobacter adaptation to different environments, including the development of antibiotic resistance and virulence in clinical settings.

What are common challenges in purifying recombinant Acinetobacter rplU and how can they be addressed?

Researchers often encounter several challenges when purifying recombinant Acinetobacter rplU:

• Protein solubility issues:

  • Problem: rplU may form inclusion bodies when overexpressed

  • Solution: Optimize expression conditions by lowering temperature (16-18°C), reducing inducer concentration, or using solubility-enhancing fusion tags (SUMO, MBP, or GST)

• Co-purification of bacterial RNA:

  • Problem: rplU's natural RNA-binding activity leads to contamination with host RNA

  • Solution: Implement high-salt washes (500 mM - 1 M NaCl) during purification and/or treatment with RNase A followed by an additional purification step

• Protein instability:

  • Problem: Purified rplU may aggregate or degrade during storage

  • Solution: Optimize buffer conditions (typically 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol), add stabilizing agents (1-5 mM DTT or 0.5-1 mM TCEP), and store at -80°C in small aliquots

• Low expression yield:

  • Problem: Poor expression levels of functional protein

  • Solution: Optimize codon usage for E. coli, co-express with molecular chaperones, or try alternative expression hosts like Acinetobacter itself (though transformation protocols may be more challenging)

A systematic approach to optimization, testing multiple conditions in parallel, is recommended to efficiently resolve these challenges.

How can researchers distinguish between Acinetobacter species when studying rplU in clinical isolates?

Accurate species identification is critical when studying rplU in clinical Acinetobacter isolates. Researchers should implement a multi-method approach:

• Molecular identification methods:

  • Primary: rpoB gene sequencing, which has been demonstrated as the most accurate method for Acinetobacter species identification

  • Secondary: 16S rRNA gene sequencing (though limited in resolving closely related species)

  • Confirmatory for A. baumannii: Detection of blaOXA-51-like gene, which serves as a reliable genetic marker for A. baumannii identification

• Phenotypic verification:

  • VITEK 2 or VITEK MS systems can provide supporting evidence but should not be relied upon exclusively due to demonstrated limitations in discrimination ability

  • Implement specific biochemical tests to distinguish between closely related species

• Whole genome sequencing:

  • For definitive identification in research requiring absolute certainty

  • Particularly useful for distinguishing members of the Acinetobacter baumannii-calcoaceticus complex (A. baumannii, A. pittii, A. calcoaceticus, and A. nosocomialis)

• Strategic approach for working with clinical isolates:

  • Always maintain original isolates and documentation of identification methods

  • Include reference strains in experiments as controls

  • Report species identification methods clearly in publications

This comprehensive approach helps avoid the significant confusion that can result from misidentification, particularly within the closely related Acinetobacter baumannii-calcoaceticus complex .