Recombinant Acinetobacter sp. Regulatory protein recX (recX)

What is RecX and what is its primary function in Acinetobacter species?

RecX is a regulatory protein that functions as a negative regulator of RecA, a protein central to homologous recombination (HR). In Acinetobacter species, RecX helps control RecA-mediated recombination activities, which is crucial for maintaining genomic stability. RecX was identified as a protein required to overcome the effects of RecA overexpression, confirming its role as a negative regulator .

The protein achieves this regulation through direct interaction with the RecA nucleoprotein filament, binding within its helical groove. This interaction leads to local dissociation of RecA protomers, destabilizing the filament and inhibiting homologous recombination . This regulation is particularly important in pathogenic species like Acinetobacter baumannii, where proper DNA repair mechanisms are essential for bacterial survival during infection and antibiotic exposure.

What is the structural basis of RecX function?

RecX exhibits a modular architecture consisting of tandem repeats of three-helix domains that resemble the helix-turn-helix (HTH) DNA-binding motif. Crystallographic analysis reveals that RecX is assembled from three tandem repeats of this three-helix motif . The relative arrangement of these repeats generates an elongated and curved shape that is well-suited for binding within the helical groove of the RecA filament.

The protein has a distinct concave surface lined with conserved positively charged residues that are critical for its function. These basic residues participate in a network of cation-π interactions, which constrain the basic side chains in a conformation that facilitates interaction with the RecA filament and nucleic acids. This structural arrangement allows RecX to make extensive contacts with both protein and nucleic acid components of the filament through its concave surface .

How does Acinetobacter sp. differ from other bacterial species in terms of recX function?

While the fundamental regulatory role of RecX is conserved across many bacterial species, Acinetobacter species show some unique characteristics. In particular, the RecX protein in Acinetobacter has evolved to function effectively in a bacterium that frequently acquires antibiotic resistance and persists in hospital environments.

The RecX from Acinetobacter shares the basic structural framework with RecX proteins from other bacteria like E. coli, but sequence analysis suggests variations that may be adapted to the specific RecA homolog in Acinetobacter. Some Acinetobacter RecX sequences contain additional repeats in their N- and C-terminal tails, resulting in proteins that are approximately 100 amino acids longer than the consensus size, potentially indicating expanded functionality .

These differences may contribute to Acinetobacter's remarkable ability to rapidly develop antibiotic resistance through horizontal gene transfer and recombination events, which are processes influenced by RecA-RecX interactions.

What are the key amino acid residues in RecX that mediate its interaction with the RecA filament?

Structure-based mutational analysis has identified several conserved basic residues on the concave surface of RecX that are crucial for its inhibitory function. Specifically, residues R25, K35, R127, K128, and R145 have been identified as functionally important. When these residues were mutated to reverse their charge (to glutamic acid) or to maintain hydrophobicity while removing charge (to methionine), the ability of RecX to inhibit RecA's ATPase and strand-exchange activities was significantly reduced .

The mutations can be ranked in order of decreasing efficacy as: R25E > R145M > K128M ≈ K35E > R127E. The R25E mutation was the most effective, almost completely eliminating RecX-dependent inhibition even at high concentrations (0.8 μM; 1:2.5 ratio of RecX to RecA). The double mutation R25E and R127E showed the strongest effect, rendering the protein virtually inactive in both ATPase and strand-exchange assays .

These findings suggest that these conserved basic residues form an extensive interaction interface with the RecA filament, mediating RecX's regulatory function through electrostatic and cation-π interactions.

How can the recombineering system for Acinetobacter be optimized for studying recX function?

The recombineering system for Acinetobacter (Rec Ab) provides an efficient method for genome editing, allowing researchers to establish gene-phenotype relationships including those involving recX. Optimization of this system for studying recX requires careful consideration of several parameters:

• Homology length: PCR products with at least 125 bp of homology flanking the target gene show optimal recombination efficiency. Products with less than 75 bp of homology do not produce recombinants under standard conditions, while those with 100 bp show limited efficiency. PCR products with 125 bp homology regions demonstrate a 1-2 log increase in recombination efficiency compared to 100 bp homology .

• DNA concentration: A minimum of 5 μg of PCR product is required for efficient recombination. Using 10 μg does not further increase efficiency compared to 5 μg .

• Cell density: Optimal results are achieved with electrocompetent cells at a density of 10^10 CFU/reaction .

• Plasmid curing: The plasmid carrying Rec Ab is unstable in the absence of ampicillin selection, with 50-70% of colonies losing the plasmid after overnight growth without selection. This allows for easy curing of the recombineering plasmid after successful gene replacement .

• Verification: Sequencing of the recombination site is essential to validate the fidelity of the synthesized DNA, as errors in the region of chemically synthesized homology have been observed .

Following these optimization parameters typically yields approximately 100 colonies per transformation (range: 20-200 colonies), depending on the specific gene being targeted.

What mechanisms does RecX employ to regulate RecA filament dynamics?

RecX regulates RecA filament dynamics through multiple mechanisms:

• Active promotion of filament disassembly: RecX binds within the helical groove of the RecA nucleoprotein filament and actively promotes the dissociation of RecA protomers, leading to filament destabilization and inhibition of homologous recombination .

• Capping mechanism: One model suggests that RecX can cap the growing 3'-end of the RecA filament. This capping results in net disassembly of RecA protomers from the opposite end and eventual dissolution of the filament .

• Steric interference: RecX may cause steric clash with the strand-exchange reaction when bound within the helical groove of the RecA filament .

• Allosteric inhibition: RecX binding may prevent the ATP-coupled allosteric changes in RecA that are required for recombination .

• DNA interaction: Molecular docking studies suggest that filament-bound RecX can make extensive contacts with the phosphate backbone of the postsynaptic strand of DNA, potentially interfering with DNA binding and strand exchange .

These mechanisms collectively contribute to RecX's ability to regulate RecA-mediated homologous recombination, ensuring proper control of this process in Acinetobacter species.

What are the recommended protocols for expressing and purifying recombinant RecX from Acinetobacter species?

Based on established methods for RecX proteins, the following protocol is recommended for expressing and purifying recombinant RecX from Acinetobacter species:

• Cloning strategy:

  • Amplify the recX gene from Acinetobacter genomic DNA using high-fidelity PCR

  • Clone into an expression vector (pET system recommended) with an N-terminal His-tag for purification

  • Transform into an E. coli expression strain (BL21(DE3) or similar)

• Expression conditions:

  • Grow cultures in LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induce protein expression with 0.5-1.0 mM IPTG

  • Continue growth at a reduced temperature (18-25°C) for 12-16 hours to enhance soluble protein production

• Purification procedure:

  • Harvest cells by centrifugation and lyse using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

  • Clarify lysate by centrifugation at 20,000 × g for 30 minutes

  • Purify using Ni-NTA affinity chromatography with imidazole gradient elution

  • Further purify by ion-exchange chromatography on a Mono Q column

  • Final polishing step using size exclusion chromatography on a Superdex 75 column

• Quality control:

  • Verify purity by SDS-PAGE (>95% purity)

  • Confirm protein identity by Western blot and mass spectrometry

  • Test functionality using RecA filament assembly assays

This protocol typically yields 5-10 mg of purified RecX protein per liter of bacterial culture, suitable for structural and biochemical studies.

What assays can be used to measure RecX activity and its effects on RecA functions?

Several complementary assays can be employed to measure RecX activity and its effects on RecA functions:

• ATP hydrolysis assay:

  • Measures RecA's ATPase activity in the presence of ssDNA

  • RecX inhibition is quantified by reduced ATP hydrolysis rates

  • Typically conducted using a coupled enzymatic assay with pyruvate kinase and lactate dehydrogenase, monitoring NADH oxidation spectrophotometrically at 340 nm

  • Standard conditions: 25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 1 μM RecA, 3 μM (nucleotides) ssDNA, varying concentrations of RecX (0-1 μM)

• DNA strand-exchange assay:

  • Evaluates RecA's ability to catalyze strand exchange between homologous DNA molecules

  • Typically uses circular ssDNA and linear dsDNA substrates

  • Products are analyzed by agarose gel electrophoresis

  • RecX inhibition is measured as decreased formation of strand-exchange products

  • Standard conditions: 25 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM ATP, 1 μM RecA, 3 μM (nucleotides) circular ssDNA, 3 μM (nucleotides) linear dsDNA, varying concentrations of RecX (0-1 μM)

• RecA filament dynamics assay:

  • Monitors assembly and disassembly of fluorescently labeled RecA filaments on DNA

  • Can be performed using total internal reflection fluorescence (TIRF) microscopy

  • Allows real-time visualization of RecX effects on filament stability

• Electrophoretic mobility shift assay (EMSA):

  • Detects direct binding of RecX to RecA-DNA filaments

  • Uses fluorescently labeled DNA and native gel electrophoresis

  • RecX binding alters the migration pattern of RecA-DNA complexes

• Surface plasmon resonance (SPR):

  • Measures direct binding kinetics between RecX and RecA

  • Allows determination of association and dissociation rate constants

These assays collectively provide comprehensive insights into the inhibitory mechanisms of RecX on RecA functions and can be used to characterize wild-type and mutant RecX proteins.

How can one study the effect of RecX on Acinetobacter antibiotic resistance mechanisms?

Studying the effect of RecX on Acinetobacter antibiotic resistance mechanisms requires a multifaceted approach:

• Generation of recX knockout and overexpression strains:

  • Create recX deletion mutants using the recombineering system with 125 bp homology regions

  • Develop complementation strains with wild-type recX and various mutants (R25E, K35E, etc.)

  • Construct RecX overexpression strains under inducible promoters

• Antibiotic susceptibility testing:

  • Determine minimum inhibitory concentrations (MICs) for various antibiotics using broth microdilution method

  • Compare wild-type, recX mutant, and complemented strains

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

• Evolution experiments:

  • Subject wild-type and recX mutant strains to sub-inhibitory antibiotic concentrations

  • Monitor the rate of resistance development over multiple generations

  • Sequence evolved strains to identify mutations contributing to resistance

• DNA damage response analysis:

  • Expose strains to DNA-damaging agents (UV, mitomycin C)

  • Quantify survival rates and mutation frequencies

  • Measure SOS response activation using reporter constructs

• Gene expression profiling:

  • Perform RNA-seq to identify genes differentially expressed in recX mutants

  • Focus on genes involved in DNA repair, recombination, and antibiotic resistance

  • Validate key findings with RT-qPCR

• Horizontal gene transfer assessment:

  • Measure rates of natural transformation, conjugation, and transduction

  • Determine if RecX affects the acquisition of resistance genes

• Biofilm formation analysis:

  • Quantify biofilm formation using crystal violet staining

  • Assess antibiotic tolerance in biofilms of wild-type and recX mutant strains

This comprehensive approach will elucidate how RecX-mediated regulation of RecA affects Acinetobacter's ability to develop and maintain antibiotic resistance, potentially identifying new targets for combating this problematic pathogen.

How should researchers interpret contradictory data regarding RecX function in different experimental systems?

When faced with contradictory data regarding RecX function across different experimental systems, researchers should follow this systematic approach:

• Examine experimental conditions:

  • Compare buffer compositions, including pH, salt concentration, and presence of divalent cations

  • Assess protein concentrations and ratios of RecX to RecA

  • Review DNA substrates (length, sequence, single- vs. double-stranded)

  • Consider incubation times and temperatures

• Evaluate protein quality:

  • Verify protein purity and proper folding

  • Check for the presence of tags that might affect function

  • Assess storage conditions and potential degradation

• Consider strain-specific differences:

  • RecX proteins from different Acinetobacter species or strains may exhibit varying activities

  • Compare sequence homology between RecX proteins used in different studies

  • Identify key amino acid differences that might explain functional variations

• Integrate multiple assays:

  • Recognize that different assays measure different aspects of RecX function

  • ATP hydrolysis inhibition may not perfectly correlate with strand exchange inhibition

  • In vitro results may not fully predict in vivo behavior

• Statistical analysis:

  • Apply appropriate statistical tests to determine if differences are significant

  • Consider sample sizes and experimental replicates

  • Calculate effect sizes to quantify the magnitude of differences

• Molecular modeling:

  • Use structural information to model how differences in RecX proteins might affect interaction with RecA

  • Predict how mutations might alter binding affinity or functional inhibition

A data integration table (Table 1) can help visualize contradictions and identify patterns:

Table 1: Integration framework for analyzing contradictory RecX functional data

By systematically analyzing contradictions using this framework, researchers can identify the variables most likely responsible for discrepancies and design targeted experiments to resolve them.

What bioinformatic approaches are most effective for analyzing RecX homologs across Acinetobacter species?

Effective bioinformatic analysis of RecX homologs across Acinetobacter species requires a multi-layered approach:

• Sequence retrieval and database development:

  • Extract RecX sequences from completely sequenced Acinetobacter genomes in NCBI

  • Include clinical and environmental isolates to capture diversity

  • Create a curated database of RecX sequences with associated metadata

• Multiple sequence alignment (MSA):

  • Use MUSCLE or MAFFT algorithms optimized for distantly related sequences

  • Manually refine alignments focusing on conserved domains

  • Consider structure-guided alignment using available crystal structures

• Phylogenetic analysis:

  • Construct maximum likelihood trees using RAxML or IQ-TREE

  • Apply appropriate substitution models (LG+G+F recommended)

  • Perform bootstrap analysis (1000 replicates) to assess branch support

  • Root trees using distant bacterial RecX homologs as outgroups

• Domain and motif identification:

  • Use HMMER to identify conserved domains

  • Develop position-specific scoring matrices for RecX-specific motifs

  • Map key functional residues (R25, K35, R127, K128, R145) across species

• Structural prediction and comparison:

  • Generate homology models using AlphaFold2 or SWISS-MODEL

  • Compare predicted structures using root-mean-square deviation (RMSD)

  • Visualize conservation patterns on structural models

• Selective pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Use PAML, HYPHY, or MEME for codon-based analyses

  • Correlate selection patterns with functional domains

• Genomic context analysis:

  • Examine genes flanking recX across Acinetobacter genomes

  • Identify operonic structures and potential co-regulated genes

  • Compare with genomic organization in other bacterial genera

Table 2: Key bioinformatic tools for RecX analysis

This comprehensive bioinformatic workflow will reveal evolutionary patterns in RecX across Acinetobacter species and help identify key residues that may contribute to species-specific functions or adaptations.

How do variations in RecX expression levels affect Acinetobacter pathogenicity and survival in hospital environments?

The relationship between RecX expression levels and Acinetobacter pathogenicity is complex and can be analyzed using the following research framework:

• Expression profiling under different conditions:

  • Measure recX expression using RT-qPCR in response to various stressors:

    • Antibiotics (sub-inhibitory concentrations)

    • Disinfectants (quaternary ammonium compounds, alcohols)

    • Desiccation

    • Nutrient limitation

    • Host immune factors (antimicrobial peptides, reactive oxygen species)

  • Compare expression patterns between clinical and environmental isolates

• Construction of expression variants:

  • Develop strains with titratable recX expression:

    • Deletion mutants (ΔrecX)

    • Complemented strains with native promoter

    • Overexpression strains with inducible promoters

    • Point mutants affecting key functional residues

• Stress tolerance assessment:

  • Challenge strains with varying RecX levels to hospital-relevant stressors:

    • Survival on dry surfaces (0-28 days)

    • Resistance to disinfectants

    • Tolerance to UV radiation

    • Persistence in nutrient-limited conditions

• Virulence phenotype characterization:

  • Measure key virulence traits:

    • Biofilm formation capacity

    • Adherence to epithelial cells

    • Resistance to serum killing

    • Survival within macrophages

    • Production of virulence factors (OmpA, phospholipases)

• In vivo infection models:

  • Test pathogenicity in relevant animal models:

    • Galleria mellonella (wax moth) larvae

    • Mouse pneumonia model

    • Wound infection model

    • Measure bacterial burden, dissemination, and host survival

• Genomic stability analysis:

  • Assess mutation rates under stress conditions

  • Measure frequency of mobile genetic element transfer

  • Quantify genomic rearrangements

Table 3: Effect of RecX expression levels on Acinetobacter survival traits

Understanding how RecX expression levels affect these traits will provide insights into Acinetobacter's success as a hospital pathogen and may identify new strategies for control and prevention of infections.

What are the most promising approaches for targeting RecX-RecA interactions to combat antibiotic resistance in Acinetobacter?

Based on current understanding of RecX structure and function, several approaches show promise for therapeutic development:

• Small molecule inhibitors of RecX-RecA interaction:

  • Design compounds that bind to the concave surface of RecX

  • Focus on mimicking the interaction interface of RecA filaments

  • Target the highly conserved basic residues (R25, K35, R127, K128, R145)

  • Develop high-throughput screening assays using fluorescence polarization

• Peptide-based inhibitors:

  • Design peptides that mimic the RecA regions that interact with RecX

  • Develop stapled peptides to improve stability and cell penetration

  • Create peptidomimetics that maintain key interaction points while improving pharmacokinetic properties

• Allosteric modulators of RecX:

  • Identify allosteric sites that affect RecX conformation

  • Design compounds that lock RecX in inactive conformations

  • Focus on regions that undergo conformational changes upon RecA binding

• RecX overexpression strategies:

  • Develop inducible systems to overexpress RecX in Acinetobacter

  • Create conditionally toxic RecX variants that disrupt DNA repair more completely

  • Design delivery systems (phage-based, nanoparticle) for RecX or its coding sequence

• Combination approaches:

  • Pair RecX-targeting strategies with conventional antibiotics

  • Identify synergistic combinations that prevent resistance development

  • Target multiple DNA repair pathways simultaneously

Table 4: Advantages and challenges of RecX-targeting approaches

To advance these approaches, structural studies of the RecX-RecA complex at atomic resolution are needed. Cryo-electron microscopy of RecA filaments with bound RecX could provide crucial insights into the precise interaction interface and guide rational drug design efforts.

How might studying RecX contribute to the development of novel genetic engineering tools for Acinetobacter species?

RecX research offers several promising avenues for developing genetic engineering tools:

• Recombineering efficiency enhancement:

  • Fine-tune RecX levels to optimize recombination frequencies

  • Develop RecX variants with modified activity for controlled recombination

  • Create inducible RecX systems to temporarily suppress recombination

• CRISPR-Cas9 system improvements:

  • Integrate RecX regulation with CRISPR-Cas9 for precise control of homology-directed repair

  • Develop RecX inhibitors to enhance homologous recombination when needed

  • Create fusion proteins combining RecX domains with Cas9 for novel functionalities

• Controlled mutagenesis systems:

  • Develop strains with tunable RecX expression for varying mutation rates

  • Create temperature-sensitive RecX variants for temporal control of genomic stability

  • Engineer RecX to respond to specific environmental signals for targeted evolution

• Novel cloning vectors:

  • Design Acinetobacter-specific vectors with RecX-based stability elements

  • Develop plasmids with RecX-regulated copy number control

  • Create specialized vectors for heterologous protein expression

• Biosensor development:

  • Engineer RecX-based biosensors for DNA damage detection

  • Create reporter systems using RecX-regulated promoters

  • Develop whole-cell biosensors for environmental monitoring

Table 5: RecX-based genetic tools for Acinetobacter bioengineering

These tools could significantly advance Acinetobacter research and potentially lead to biotechnological applications, such as engineered strains for bioremediation, bioproduction of valuable compounds, or specialized biosensors for environmental monitoring.