Recombinant Acinetobacter baumannii Elongation factor G (fusA), partial

What is Elongation factor G (fusA) in Acinetobacter baumannii and what is its biological significance?

Elongation factor G (EF-G), encoded by the fusA gene in Acinetobacter baumannii, is a critical protein involved in the translocation step of bacterial protein synthesis. This GTPase works by catalyzing the movement of mRNA and tRNAs during translation, facilitating the elongation of the nascent peptide chain. In A. baumannii, which is a significant pathogen of clinical concern due to its multidrug resistance capabilities, EF-G serves as an essential component of the cellular machinery . The protein is also known as fusA based on its gene designation, referring to its historical connection to fusidic acid resistance in some bacterial species. Understanding EF-G's structure and function provides insights into bacterial translation mechanisms and potential antimicrobial targets, particularly relevant given the emergence of resistant A. baumannii strains documented in recent clinical studies .

What are the standard methods for characterizing recombinant Acinetobacter baumannii Elongation factor G?

Characterization of recombinant A. baumannii Elongation factor G typically employs multiple complementary techniques. SDS-PAGE is the primary method used to verify protein purity, with commercial preparations typically achieving >85% purity . Mass spectrometry provides precise molecular weight determination and can confirm post-translational modifications. Functional characterization often involves GTPase activity assays to confirm the protein retains enzymatic activity.

For structural studies, researchers commonly employ:

• Circular dichroism (CD) to assess secondary structure integrity

• Differential scanning fluorimetry to evaluate thermal stability

• Size exclusion chromatography to confirm proper folding and oligomeric state

Researchers should systematically document protein concentration, buffer conditions, and experimental temperatures, as these parameters significantly influence assay reproducibility. When reporting characterization data, include both raw values and normalized results to facilitate cross-laboratory validation.

How should recombinant Acinetobacter baumannii Elongation factor G be reconstituted and stored for maximum stability?

For optimal results when working with recombinant A. baumannii Elongation factor G, follow this methodological approach to reconstitution and storage:

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to bring all content to the bottom

• Reconstitute the lyophilized protein using deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimal recommendation is 50%) to enhance stability

• Aliquot into single-use volumes to minimize freeze-thaw cycles

Storage Guidelines:

• Short-term working aliquots can be maintained at 4°C for up to one week

• For medium-term storage, maintain at -20°C

• For extended storage and maximum stability, store at -80°C

Critically, repeated freeze-thaw cycles must be avoided as they significantly compromise protein integrity through mechanical stress and increased exposure to reactive oxygen species. Experimental design should account for the differential shelf life between liquid preparations (approximately 6 months at -20°C/-80°C) and lyophilized forms (approximately 12 months at -20°C/-80°C) . Researchers should maintain detailed records of storage conditions and reconstitution dates to ensure experimental reproducibility.

What experimental designs are most effective for studying the role of Elongation factor G in antibiotic resistance mechanisms?

When investigating Elongation factor G's role in antibiotic resistance mechanisms in A. baumannii, a multi-tiered experimental approach is essential. Fractional factorial design offers particular advantages, allowing researchers to systematically evaluate multiple factors with fewer experimental runs while preserving statistical power . This approach is ideal for complex biological systems where multiple variables may influence antibiotic resistance.

A comprehensive experimental strategy should include:

• Genetic approaches:

  • Site-directed mutagenesis of key fusA residues

  • Complementation studies with wild-type versus mutant fusA

  • CRISPR-Cas9 gene editing to introduce or correct specific mutations

• Functional assays:

  • In vitro translation assays comparing wild-type and mutant EF-G

  • GTPase activity measurements under various antibiotic pressures

  • Ribosome binding assays with fluorescently labeled components

• Structural studies:

  • 3D protein modeling to predict how mutations disrupt protein structure

  • X-ray crystallography or cryo-EM to confirm structural changes

  • Molecular dynamics simulations to understand conformational changes

For antibiotic susceptibility testing, researchers should employ standardized methods such as microdilution or E-test, using iron-depleted media to better simulate in vivo conditions. Recent studies have demonstrated that antibiotic uptake experiments combined with LC-MS/MS analysis provide robust quantitative data on how mutations in transport proteins affect antibiotic accumulation in bacterial cells . This integrated approach allows for mechanistic insights connecting genotypic changes to phenotypic resistance.

How can whole-genome sequencing be effectively utilized to identify mutations in the fusA gene associated with phenotypic changes?

Whole-genome sequencing (WGS) offers a powerful methodology for identifying fusA mutations associated with phenotypic changes in A. baumannii, particularly in the context of antibiotic resistance. A systematic approach should follow these methodological steps:

• Strain selection and isolation:

  • Isolate phenotypically distinct strains (e.g., resistant vs. susceptible)

  • Ensure pure cultures through multiple passages

  • Document phenotypic characteristics using standardized methods

• Sequencing strategy:

  • Employ paired-end sequencing with at least 100x coverage

  • Include both short-read (Illumina) and long-read (PacBio/Nanopore) technologies

  • Sequence multiple biological replicates to confirm findings

• Bioinformatic analysis pipeline:

  • Perform quality control (FastQC, Trimmomatic)

  • Map reads to reference genome (BWA, Bowtie2)

  • Conduct variant calling with multiple algorithms (GATK, FreeBayes, VarScan)

  • Focus on non-synonymous mutations in fusA and related genes

• Validation and functional correlation:

  • Confirm mutations through Sanger sequencing

  • Correlate specific mutations with phenotypic data

  • Perform comparative analysis across multiple strains

This approach has proven successful in identifying novel chromosomal mutations responsible for antibiotic resistance. For example, recent research identified a mutation in a TonB-dependent receptor homolog that resulted in a premature stop codon, impairing receptor function and contributing to cefiderocol resistance in A. baumannii . Such variant calling analysis is essential for detecting emerging mutations associated with antibiotic resistance and can complement traditional susceptibility testing in clinical settings.

What methodologies can accurately measure the impact of fusA mutations on protein function and antibiotic resistance?

To accurately measure how fusA mutations affect EF-G function and antibiotic resistance in A. baumannii, researchers should implement a multi-faceted analytical approach:

Protein Function Assessment:

• Translation efficiency assays:

  • Cell-free translation systems with purified components

  • Measurement of peptide synthesis rates using fluorescent or radioactive markers

  • Comparative analysis between wild-type and mutant EF-G proteins

• Ribosome interaction studies:

  • Pull-down assays to quantify EF-G-ribosome binding affinity

  • FRET-based approaches to measure conformational changes during translocation

  • Cryo-EM visualization of EF-G-ribosome complexes

Antibiotic Resistance Evaluation:

• Minimum Inhibitory Concentration (MIC) determination:

  • Broth microdilution in iron-depleted conditions

  • E-test methods on standardized media

  • Time-kill assays to assess bactericidal effects

• Antibiotic uptake and accumulation:

  • LC-MS/MS quantification of intracellular antibiotic concentrations

  • Normalization to bacterial cell density (OD600)

  • Time-course measurements (e.g., 5 and 20 minutes post-exposure)

The experimental design should include appropriate controls, including:

• Isogenic strains differing only in fusA sequence

• Complementation with wild-type fusA to confirm causality

• Laboratory reference strains with known antibiotic susceptibility profiles

This integrated approach provides comprehensive data linking genotypic changes to functional outcomes and resistance phenotypes. For example, a recent study employed LC-MS/MS to demonstrate reduced cefiderocol uptake in a resistant A. baumannii strain, confirming the functional impact of a mutation in a TonB-dependent receptor . Similar methodologies can be applied to study fusA mutations, providing mechanistic insights into how structural changes affect protein function and antimicrobial resistance.

How can mass spectrometry techniques be optimally applied to study Elongation factor G interactions and modifications?

Mass spectrometry offers powerful capabilities for studying EF-G interactions and modifications in A. baumannii research. For optimal application, researchers should implement the following methodological framework:

Sample Preparation Protocols:

• In-solution digestion with multiple proteases (trypsin, chymotrypsin) to maximize sequence coverage

• FASP (Filter-Aided Sample Preparation) for complex samples

• Enrichment strategies for post-translational modifications (phosphoenrichment, GlycoCapture)

Instrumentation Selection and Parameters:

• High-resolution instruments (Orbitrap, Q-TOF) for precise mass determination

• Targeted approaches (PRM, SRM) for quantifying specific peptides

• Data-independent acquisition for comprehensive peptidome analysis

Data Analysis Workflow:

• Database searching against A. baumannii proteomes and common contaminants

• Manual validation of important peptide spectrum matches

• Statistical analysis of quantitative data (p < 0.05 threshold)

This approach has been validated in antibiotic uptake studies where LC-MS/MS successfully quantified intracellular concentrations of antibiotics like cefiderocol in A. baumannii strains . For studying EF-G specifically, researchers should standardize bacterial growth conditions (using iron-depleted media when relevant) and cell lysis procedures (lysozyme treatment with 10 mM EDTA followed by sonication cycles) . The resulting data should be normalized to cell density measurements (OD600) to ensure accurate comparison across samples.

A comprehensive experimental design should include time-course measurements to capture dynamic changes in protein interactions or modifications, as demonstrated in studies showing differential antibiotic accumulation at 5 and 20 minutes post-exposure .

What are the most effective 3D protein modeling approaches for predicting how mutations affect Elongation factor G structure and function?

For predicting how mutations affect EF-G structure and function in A. baumannii, researchers should employ a systematic 3D protein modeling workflow that integrates multiple computational approaches:

Modeling Methodology Selection:

• Template-based modeling:

  • Select templates with >30% sequence identity to A. baumannii EF-G

  • Prioritize bacterial EF-G structures, especially from Gram-negative organisms

  • Create multiple models using different alignment algorithms

• Ab initio and hybrid approaches:

  • Apply for regions lacking template coverage

  • Implement AlphaFold2 or RoseTTAFold for full-protein predictions

  • Compare results from multiple methods to assess consistency

• Model refinement and validation:

  • Energy minimization to resolve steric clashes

  • Molecular dynamics simulations to assess stability

  • Validation using PROCHECK, VERIFY3D, and MolProbity metrics

Mutation Impact Analysis:

• Structure comparison techniques:

  • RMSD calculations between wild-type and mutant models

  • Analysis of secondary structure disruptions

  • Identification of altered hydrogen bonding networks

• Functional site prediction:

  • GTP binding pocket geometry assessment

  • Ribosome interaction interface analysis

  • Domain movement and conformational flexibility evaluation

This integrated approach has proven effective in recent A. baumannii research, where 3D protein modeling successfully demonstrated that a 10-base deletion disrupted secondary protein structure in a TonB-dependent receptor, compromising its function as a transporter and contributing to antibiotic resistance . For EF-G specifically, emphasis should be placed on modeling the five structural domains and identifying how mutations might affect interdomain movements critical for translocation function.

What experimental controls and validation methods are essential when studying Elongation factor G's role in antibiotic resistance mechanisms?

When investigating EF-G's role in antibiotic resistance mechanisms in A. baumannii, implementing rigorous experimental controls and validation methods is essential for generating reliable and reproducible results:

Essential Controls for Experimental Validity:

Validation Methodologies:

• Genetic validation:

  • Sanger sequencing confirmation of mutations

  • RT-qPCR for expression level verification

  • RNA-seq for broader transcriptional impact

• Functional validation:

  • In vitro translation assays with purified components

  • Site-directed mutagenesis to create/revert specific mutations

  • Heterologous expression in model organisms

• Biochemical validation:

  • Purification and activity assays of wild-type and mutant proteins

  • Mass spectrometry to confirm protein modifications

  • Binding affinity measurements (ITC, SPR)

This comprehensive approach ensures that observed phenotypes can be confidently attributed to specific fusA mutations rather than confounding variables. Recent research on A. baumannii antibiotic resistance has demonstrated the value of integrating multiple validation methods, including functional experiments in iron-depleted media to simulate in vivo conditions and LC-MS/MS analysis to confirm mechanisms like reduced antibiotic uptake .

What are the current methodological challenges in studying Elongation factor G in multidrug-resistant Acinetobacter baumannii strains?

Researchers investigating EF-G in multidrug-resistant A. baumannii face several significant methodological challenges that require innovative approaches:

Technical Challenges and Potential Solutions:

• Genetic manipulation difficulties:

  • Challenge: A. baumannii is notoriously difficult to transform, particularly in clinical isolates

  • Solution: Optimize electroporation protocols with glycine pre-treatment to weaken cell walls; employ bacteriophage-based delivery systems; utilize CRISPR-Cas delivery via conjugation

• Protein expression and purification:

  • Challenge: EF-G often forms inclusion bodies or exhibits low solubility

  • Solution: Optimize expression using baculovirus systems ; employ fusion tags with enhanced solubility properties; develop refolding protocols from inclusion bodies

• Distinguishing direct from indirect effects:

  • Challenge: Mutations in fusA may cause pleiotropic effects beyond EF-G function

  • Solution: Implement carefully designed controls; use ribosome profiling to assess global translation impacts; employ systems biology approaches to map interaction networks

• Standardization across laboratories:

  • Challenge: Variable growth conditions and resistance testing methods limit comparability

  • Solution: Adopt standardized iron-depleted media preparation protocols ; implement consistent antibiotic susceptibility testing methods; establish reference strain repositories

• Complex data integration:

  • Challenge: Connecting genomic, structural, and functional data requires sophisticated analysis

  • Solution: Develop integrated bioinformatic pipelines combining variant calling with functional prediction ; implement machine learning approaches to identify resistance patterns; create centralized databases for A. baumannii resistance mutations

These challenges highlight the need for multidisciplinary approaches and standardized methodologies in A. baumannii research. As demonstrated in recent studies, the integration of genomic techniques with functional validation provides the most robust framework for understanding resistance mechanisms .

How do variant calling methodologies impact the identification of significant fusA mutations in clinical isolates?

Variant calling methodologies significantly impact the identification of clinically relevant fusA mutations in A. baumannii, with important implications for resistance mechanism research:

Critical Methodological Considerations:

• Sequencing technology selection:

  • Short-read technologies may miss structural variants and repeat regions

  • Long-read sequencing improves detection of genomic rearrangements

  • Hybrid approaches combining both technologies provide most comprehensive coverage

• Alignment algorithm impact:

  • Different aligners (BWA-MEM, Bowtie2, HISAT2) show variable performance with A. baumannii genomes

  • Local vs. global alignment strategies affect detection of indels in GC-rich regions

  • Multiple alignment approaches should be compared to avoid algorithm-specific biases

• Variant caller optimization:

  • Caller sensitivity varies by variant type (SNPs vs. indels)

  • Parameter tuning significantly affects detection thresholds

  • Ensemble approaches combining multiple callers increase confidence in detected variants

• Reference genome selection:

  • Using inappropriate reference strains leads to false positives/negatives

  • Multi-reference approaches better capture A. baumannii genomic diversity

  • De novo assembly followed by gene-specific alignment may identify novel variants

What emerging technologies show promise for advancing our understanding of Elongation factor G function in the context of antimicrobial resistance?

Several emerging technologies demonstrate significant potential for advancing our understanding of EF-G function in antimicrobial resistance in A. baumannii:

Cutting-Edge Methodological Approaches:

• Single-molecule techniques:

  • Single-molecule FRET to visualize EF-G conformational changes during translocation

  • Optical tweezers to measure forces generated during EF-G-mediated translocation

  • These approaches provide unprecedented insights into the dynamics of translation at nanometer resolution

• Cryo-electron microscopy advancements:

  • Time-resolved cryo-EM to capture transient states during EF-G function

  • Cryo-electron tomography of whole cells to visualize ribosomes in native contexts

  • These methods are revealing previously unobservable structural conformations relevant to antibiotic interactions

• Integrated OMICS approaches:

  • Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics)

  • Spatial transcriptomics to map expression patterns within bacterial populations

  • These comprehensive approaches connect genotype to phenotype across multiple biological levels

• Advanced computational methods:

  • Deep learning for predicting functional impacts of fusA mutations

  • Molecular dynamics simulations with enhanced sampling to model conformational changes

  • Network analysis to map resistance mechanism interactions

• Novel functional screening approaches:

  • CRISPR interference (CRISPRi) libraries for systematic functional analysis

  • Microfluidic devices for single-cell analysis of heterogeneous populations

  • High-throughput automated screening platforms for antimicrobial discovery

The integration of these technologies promises to overcome current limitations in understanding EF-G's role in antimicrobial resistance. For example, combining advanced LC-MS/MS technologies with genomic analyses has already demonstrated utility in tracking antibiotic uptake in resistant A. baumannii strains . As these technologies become more accessible, they will enable more precise characterization of resistance mechanisms and potentially identify novel therapeutic strategies targeting EF-G function.

What are the most significant research gaps in our understanding of Elongation factor G in Acinetobacter baumannii?

Despite advances in A. baumannii research, significant knowledge gaps remain regarding EF-G's role in bacterial physiology and antimicrobial resistance. Researchers should prioritize addressing these fundamental questions to advance the field:

• Structure-function relationships:

  • Complete characterization of domain-specific functions in A. baumannii EF-G

  • Mapping of species-specific structural features that might serve as targeted therapeutic sites

  • Understanding how post-translational modifications regulate EF-G activity

• Resistance mechanism integration:

  • Elucidating how fusA mutations interact with other resistance mechanisms

  • Determining whether EF-G mutations serve as primary resistance determinants or compensatory adaptations

  • Understanding the fitness costs of fusA mutations in different environmental contexts

• Evolutionary dynamics:

  • Tracking the emergence and spread of fusA mutations in clinical settings

  • Determining the role of horizontal gene transfer in fusA evolution

  • Understanding selective pressures driving EF-G diversification

• Translation regulation networks:

  • Mapping interactions between EF-G and other translation factors in A. baumannii

  • Characterizing strain-specific variation in translation regulation

  • Understanding how translation modulation contributes to stress responses

Addressing these gaps requires integrated approaches combining genomic, structural, and functional methodologies. Recent research has demonstrated the value of such integration, with studies successfully connecting genomic mutations to functional changes in antibiotic uptake mechanisms in A. baumannii . Similar approaches applied to EF-G research would significantly advance our understanding of this essential bacterial protein.

How can researchers optimize experimental protocols for studying recombinant Elongation factor G to ensure reproducibility and reliability?

To ensure reproducibility and reliability in recombinant EF-G research, investigators should implement standardized protocols addressing key variables throughout the experimental workflow:

Protocol Optimization Framework:

• Protein production standardization:

  • Consistent expression systems (baculovirus recommended for complex proteins)

  • Standardized purification protocols with quality control checkpoints

  • Comprehensive documentation of batch-to-batch variation

• Storage and handling procedures:

  • Implementation of single-use aliquoting to prevent freeze-thaw degradation

  • Standardized reconstitution protocols with optimal buffer compositions

  • Consistent glycerol concentrations (recommend 50%) for long-term storage

• Functional assay standardization:

  • Establish minimum reporting standards for assay conditions

  • Implement internal controls for normalization across experiments

  • Define acceptance criteria for assay validity

• Data management practices:

  • Comprehensive metadata collection for all experiments

  • Implementation of electronic laboratory notebooks for improved documentation

  • Use of standardized data reporting formats

This standardized approach addresses the significant challenge of reproducibility in protein-based research. The shelf life considerations for recombinant proteins (6 months for liquid preparations, 12 months for lyophilized forms at -20°C/-80°C) should be incorporated into experimental planning . Additionally, reconstitution procedures should follow established guidelines, including centrifugation prior to opening, sterile water reconstitution to 0.1-1.0 mg/mL, and appropriate glycerol addition .