Recombinant Acinetobacter sp. 30S ribosomal protein S14 (rpsN)

What is the role of rpsN (30S ribosomal protein S14) in Acinetobacter species?

The 30S ribosomal protein S14, encoded by the rpsN gene, serves several critical functions in Acinetobacter species. Primarily, this protein binds to 16S rRNA and is essential for the proper assembly of 30S ribosomal subunits . Research indicates that rpsN likely plays a crucial role in determining the conformation of the 16S rRNA at the A site, which is fundamental for accurate tRNA positioning during translation . This protein belongs to the universal ribosomal protein uS14 family, underscoring its evolutionary conservation and functional importance across bacterial species .

How conserved is the rpsN gene across different Acinetobacter species?

The rpsN gene exhibits considerable conservation across Acinetobacter species, reflecting its essential role in ribosomal function. Sequence analysis demonstrates that while there are minor variations in the gene sequence, the functional domains of rpsN remain highly preserved across the genus . This conservation is particularly evident within the Acinetobacter calcoaceticus–Acinetobacter baumannii (Acb) complex, which includes clinically significant species such as A. baumannii, A. pittii, A. nosocomialis, and A. calcoaceticus .

The conservation pattern of rpsN reflects evolutionary pressure to maintain ribosomal functionality while allowing for species-specific adaptations. While the exact sequence identity percentages across all Acinetobacter species are not provided in the search results, the consistent annotation and description of rpsN across multiple species in database entries suggest substantial conservation of this gene .

What methods are most reliable for identifying Acinetobacter species that express the rpsN gene?

Accurate identification of Acinetobacter species expressing the rpsN gene requires sophisticated molecular approaches, as phenotypic methods often lack sufficient discriminatory power. Based on comparative studies, the following methodological hierarchy has been established:

• rpoB gene sequencing has demonstrated superior discriminatory power for Acinetobacter species identification, particularly within the closely related Acb complex . The interspecies diversity of rpoB sequences provides reliable differentiation between species, with interspecies similarities ranging from 84.8% to 95.6% .

• 16S rRNA gene sequencing offers reliable identification at the genus level but shows limited resolution at the species level, especially within the Acb complex where sequence similarities are exceptionally high .

• Phenotypic identification methods (VITEK 2, VITEK MS) provide inconsistent results compared to genotypic methods and are generally less reliable for accurate species determination .

• blaOXA-51-like gene detection serves as a reliable genetic marker specifically for A. baumannii identification, as this gene is absent in non-A. baumannii isolates .

For researchers specifically interested in studying rpsN, a multifaceted approach combining rpoB gene sequencing for species identification followed by rpsN-specific PCR amplification and sequencing would provide the most comprehensive characterization.

What experimental challenges exist when working with recombinant rpsN from Acinetobacter species?

Working with recombinant rpsN protein from Acinetobacter species presents several experimental challenges that researchers should anticipate and address:

• Protein solubility issues: Ribosomal proteins like rpsN often have highly charged surfaces designed for RNA interactions, which can lead to aggregation when expressed recombinantly without their natural RNA binding partners.

• Maintaining native conformation: Ensuring that recombinant rpsN adopts its native structure is crucial, particularly when the protein is expressed outside its normal ribosomal context.

• Species differentiation: Accurately distinguishing between closely related Acinetobacter species is essential when studying species-specific variations in rpsN function or expression .

• Functional validation: Developing appropriate assays to confirm that recombinant rpsN retains its natural RNA-binding and ribosome assembly capabilities.

• Contamination concerns: Acinetobacter species, particularly those within the Acb complex, are clinically significant pathogens that may carry antibiotic resistance genes, necessitating appropriate biosafety measures .

Addressing these challenges requires careful optimization of expression systems, purification protocols, and functional validation approaches specific to ribosomal proteins.

How can researchers design experiments to study the interaction between rpsN and 16S rRNA in Acinetobacter species?

Investigating the interaction between rpsN and 16S rRNA requires sophisticated experimental approaches that can capture both structural and functional aspects of this relationship. The following methodological framework is recommended:

Experimental Design Table for rpsN-16S rRNA Interaction Studies:

For optimal results, researchers should employ multiple complementary approaches. Initial in silico analysis of predicted interaction sites based on homology models can guide the design of targeted mutagenesis experiments. This should be followed by in vitro binding studies and structural analysis, with final validation in cellular systems.

What approaches are recommended for studying species-specific variations in rpsN expression across the Acinetobacter genus?

Investigating species-specific variations in rpsN expression requires careful consideration of both species identification and gene expression analysis. The following comprehensive approach is recommended:

• Accurate Species Identification:

  • Implement rpoB gene sequencing for definitive species identification within the Acinetobacter genus .

  • Verify species identity using multiple genetic markers, including potentially blaOXA-51-like gene presence for A. baumannii .

  • Construct phylogenetic trees to visualize relationships between studied isolates.

• Expression Analysis Strategy:

  • Design species-specific primers for rpsN based on sequence alignments from verified strains.

  • Validate primer specificity against a panel of well-characterized Acinetobacter species.

  • Utilize RT-qPCR with appropriate reference genes validated for each Acinetobacter species.

  • Consider RNA-Seq for comprehensive transcriptomic comparison across species.

• Experimental Controls:

  • Include reference strains for each species under investigation.

  • Normalize expression data to multiple reference genes to ensure accurate quantification.

  • Control for growth phase and environmental conditions that may influence ribosomal protein expression.

• Statistical Analysis:

  • Apply appropriate statistical methods to assess significance of expression differences.

  • Consider hierarchical clustering to identify patterns of expression across species.

  • Correlate expression data with physiological or clinical parameters of interest.

This integrated approach accounts for the taxonomic complexity of Acinetobacter while providing robust expression data that can be meaningfully interpreted across species.

How do mutations in the rpsN gene potentially affect antibiotic resistance profiles in Acinetobacter species?

The relationship between rpsN mutations and antibiotic resistance in Acinetobacter species represents an important research area, particularly given the clinical significance of these pathogens. The potential mechanisms and experimental approaches include:

• Mechanistic Relationships:

  • Mutations in rpsN may alter the binding sites for antibiotics that target the 30S ribosomal subunit, particularly aminoglycosides.

  • Conformational changes in the A site of 16S rRNA, which is influenced by rpsN, could affect translation accuracy and antibiotic efficacy.

  • Species-specific effects may exist, as non-A. baumannii isolates demonstrate significantly lower resistance rates to carbapenems (2.6%) compared to A. baumannii isolates .

• Experimental Approaches:

  • Site-directed mutagenesis to introduce specific mutations identified in resistant clinical isolates.

  • Minimum inhibitory concentration (MIC) determination for various antibiotics across wild-type and mutant strains.

  • Ribosome profiling to assess translation accuracy and efficiency in mutant strains.

  • Structural studies to visualize how mutations affect antibiotic binding.

• Analysis Framework:

  • Correlate specific rpsN mutations with resistance profiles across clinical isolates.

  • Assess the impact of mutations on bacterial fitness and growth rates.

  • Determine whether mutations affect resistance to multiple antibiotic classes or show specificity.

Understanding the relationship between rpsN mutations and antibiotic resistance could potentially inform new therapeutic strategies targeting ribosomal function in multidrug-resistant Acinetobacter species.

What methods provide the highest resolution for studying the structure-function relationship of rpsN in Acinetobacter species?

For high-resolution studies of the structure-function relationship of rpsN, researchers should consider a complementary suite of advanced structural biology techniques:

Table: Structural Analysis Methods for rpsN Studies

A comprehensive structural investigation would ideally combine multiple methods. For example, high-resolution static structures from Cryo-EM or X-ray crystallography could be complemented by dynamic information from NMR or smFRET, with computational modeling bridging any gaps and generating testable hypotheses about structure-function relationships.

What experimental design considerations are crucial when studying the role of rpsN in ribosomal A site conformation?

Investigating the role of rpsN in determining ribosomal A site conformation requires careful experimental design:

• Structure-Based Mutagenesis Approach:

  • Identify rpsN residues that interact with the A site region of 16S rRNA based on structural data.

  • Design a systematic mutagenesis strategy targeting:
    a) Residues directly contacting 16S rRNA
    b) Residues involved in protein-protein interactions with neighboring ribosomal proteins
    c) Conserved vs. variable residues across Acinetobacter species

  • Express and purify mutant proteins for in vitro reconstitution studies.

• RNA Structure Analysis:

  • Implement chemical probing techniques (SHAPE, DMS) to assess conformational changes in 16S rRNA.

  • Compare accessibility patterns of the A site region in:
    a) Wild-type ribosomes
    b) Ribosomes with mutant rpsN
    c) Ribosomes lacking rpsN (if viable)

  • Map structural changes to functional domains within the A site.

• Functional Translation Assays:

  • Develop reporter systems to quantify translation fidelity with wild-type vs. mutant rpsN.

  • Measure:
    a) Misincorporation rates at specific codons
    b) Frameshifting frequency
    c) Stop codon readthrough efficiency

  • Correlate functional defects with structural alterations.

• Randomized Block Design for Controlling Variables:

  • Implement a randomized block design as described in experimental design literature to control for variables .

  • Block factors should include:
    a) Protein preparation batch
    b) RNA preparation batch
    c) Experimental day

  • Analysis of variance (ANOVA) can then be used to assess significance while accounting for these blocking factors.

This comprehensive experimental approach combines structural, biochemical, and functional analyses to establish causal relationships between rpsN sequence, A site conformation, and ribosomal function in Acinetobacter species.

What are the optimal conditions for expressing and purifying recombinant Acinetobacter sp. rpsN protein?

Optimizing expression and purification of recombinant Acinetobacter sp. rpsN requires careful consideration of expression systems, growth conditions, and purification strategies:

Expression System Selection:

• E. coli BL21(DE3) typically provides high yields for ribosomal proteins.

• Consider codon optimization based on the specific Acinetobacter species source.

• Fusion tags (His6, MBP, GST) can enhance solubility and facilitate purification.

Expression Optimization Table:

Purification Strategy:

• Affinity chromatography using appropriate tag (His6-tag recommended for minimal interference)

• Ion exchange chromatography (typically cation exchange due to basic nature of ribosomal proteins)

• Size exclusion chromatography for final polishing and buffer exchange

Critical Quality Controls:

• Circular dichroism (CD) spectroscopy to verify secondary structure

• RNA binding assays to confirm functional activity

• Mass spectrometry to verify protein integrity and modifications

This systematic approach addresses the challenges specific to ribosomal proteins while providing research-grade material for subsequent functional and structural studies.

How can researchers effectively analyze data from comparative studies of rpsN across different Acinetobacter species?

Data analysis for comparative studies of rpsN across Acinetobacter species requires rigorous statistical approaches and careful consideration of biological context:

• Sequence Analysis Framework:

  • Multiple sequence alignment using MAFFT or similar algorithms, as implemented in previous Acinetobacter studies .

  • Phylogenetic analysis employing Neighbor-Joining (NJ) methods to establish evolutionary relationships .

  • Calculation of sequence identity/similarity matrices with defined thresholds (97% identity has been suggested as appropriate for species delineation in some Acinetobacter genes) .

• Expression Data Analysis:

  • Normalization strategies should account for:
    a) Reference gene stability across species
    b) RNA quality variations
    c) PCR efficiency differences

  • Statistical analysis using ANOVA with post-hoc tests for multiple comparisons.

  • Implementation of randomized block designs to control for experimental batch effects .

• Structural Comparison Approaches:

  • Homology modeling based on high-resolution ribosome structures.

  • Root Mean Square Deviation (RMSD) calculations to quantify structural differences.

  • Conservation mapping onto structural models to identify functionally important regions.

• Functional Data Integration:

  • Correlation analysis between sequence variation and functional parameters.

  • Principal Component Analysis (PCA) to identify patterns across multiple variables.

  • Path analysis to establish potential causal relationships between sequence, structure, and function.

This comprehensive analytical framework ensures robust interpretation of comparative data while accounting for the taxonomic complexity of Acinetobacter species.

What considerations are important when designing primers for rpsN amplification across diverse Acinetobacter species?

Designing primers for rpsN amplification across diverse Acinetobacter species requires careful consideration of sequence conservation, specificity, and technical parameters:

• Sequence Analysis for Primer Design:

  • Align rpsN sequences from multiple Acinetobacter species.

  • Identify conserved regions flanking variable segments for universal primer design.

  • For species-specific primers, target unique regions with at least 3-4 nucleotide differences at the 3' end.

• Primer Design Parameters:

  • Optimal length: 18-25 nucleotides

  • GC content: 40-60%

  • Melting temperature (Tm): 55-65°C with <5°C difference between primer pairs

  • Avoid secondary structures and primer-dimers

  • Include degenerate bases at variable positions for universal primers

• Validation Strategy:

  • Test primers against a panel of well-characterized Acinetobacter species.

  • Sequence amplicons to confirm specificity.

  • Optimize PCR conditions for each species or for universal application.

• Application-Specific Considerations:

  • For expression studies: design primers within the coding region

  • For evolutionary studies: include flanking regions

  • For full-length cloning: add appropriate restriction sites

This systematic approach to primer design ensures reliable amplification while accounting for the genetic diversity within the Acinetobacter genus, similar to the approach used for rpoB amplification in previous studies .

How can rpsN be utilized as a marker for evolutionary studies within the Acinetobacter genus?

The rpsN gene offers potential as a phylogenetic marker for evolutionary studies within Acinetobacter, though with important considerations:

When properly implemented within a multi-locus approach, rpsN can contribute valuable evolutionary insights, particularly regarding the conservation of essential ribosomal functions across diverse Acinetobacter lineages.

What are the implications of rpsN variation for antibiotic development strategies targeting Acinetobacter species?

The implications of rpsN variation for antibiotic development targeting Acinetobacter species are multifaceted and potentially significant:

• Target Site Conservation Analysis:

  • Regions of high conservation in rpsN across Acinetobacter species represent potential broad-spectrum antibiotic targets.

  • Variable regions may offer opportunities for species-specific targeting, particularly relevant given the differing resistance profiles between A. baumannii and non-A. baumannii species (resistance rates of 2.6% for non-A. baumannii vs. significantly higher for A. baumannii) .

• Structure-Based Drug Design Considerations:

  • Identify pockets or interfaces involving rpsN that could be targeted by small molecules.

  • Focus on rpsN interactions with 16S rRNA at the A site that are essential for ribosomal function.

  • Consider species-specific structural features that might influence drug binding.

• Resistance Mechanism Anticipation:

  • Catalog naturally occurring variations in rpsN across clinical isolates.

  • Identify positions prone to mutation that might confer resistance.

  • Design inhibitors with multiple binding modes to reduce resistance development potential.

• Validation Strategy:

  • Test candidate compounds against panels of diverse Acinetobacter species.

  • Evaluate efficacy against clinical isolates with different rpsN variants.

  • Assess resistance development rates under selective pressure.

This approach leverages detailed understanding of rpsN structural and functional properties to inform rational antibiotic design strategies that account for natural variation within the Acinetobacter genus.

What are the key considerations for designing comprehensive research projects focused on Acinetobacter sp. rpsN?

Designing comprehensive research projects focused on Acinetobacter sp. rpsN requires integration of multiple experimental approaches and careful consideration of the taxonomic complexity within this genus. Researchers should prioritize accurate species identification using rpoB gene sequencing, as this has demonstrated superior discriminatory power compared to 16S rRNA sequencing or phenotypic methods . The experimental design should incorporate randomized block designs to control for variables that might influence results .

For functional studies, it's essential to consider the dual role of rpsN in ribosome assembly and 16S rRNA conformation at the A site . This necessitates both structural and functional readouts in experimental designs. Additionally, researchers should be aware of the clinical significance of different Acinetobacter species, particularly the distinction between A. baumannii and non-A. baumannii isolates in terms of antibiotic resistance profiles .