Recombinant Acinetobacter sp. Maf-like protein ACIAD1930 (ACIAD1930)

What is the ACIAD1930 protein and what is its role in Acinetobacter sp.?

ACIAD1930 is a Maf-like protein from Acinetobacter baylyi strain ATCC 33305/BD413/ADP1. Maf proteins represent a family of conserved proteins that have been implicated in cell division arrest. Research has demonstrated that Maf proteins, including ACIAD1930, exhibit nucleotide pyrophosphatase activity against both canonical nucleotides (dTTP, UTP, and CTP) and modified nucleotides (5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP) . These modified nucleotides represent the most abundant naturally occurring modifications found in all RNA species across all kingdoms of life.

The protein is 198 amino acids in length and belongs to the YhdE subfamily of Maf proteins, characterized by specific sequence motifs that distinguish them from the YceF subfamily .

What expression systems are most effective for producing recombinant ACIAD1930?

Recommended Protocol for E. coli Expression:

• Clone the ACIAD1930 gene into a pET expression vector (e.g., pET-28a(+)) to create a fusion protein with a His-tag for easier purification

• Transform competent E. coli BL21(DE3) cells with the recombinant plasmid

• Grow transformed cells in LB medium containing appropriate antibiotics (e.g., kanamycin at 100 μg/mL) at 37°C until OD600 reaches 0.6

• Induce protein expression with IPTG at a final concentration of 1 mmol/L

• Continue cultivation for 6-8 hours post-induction

• Harvest cells by centrifugation and process for protein purification

This approach has been successful for expressing other Maf proteins with yields sufficient for biochemical studies .

What is the optimal purification strategy for ACIAD1930?

Purification of His-tagged ACIAD1930 protein can be achieved using Ni-NTA affinity chromatography, a method that has been effective for other recombinant proteins with similar characteristics:

Purification Protocol:

• Resuspend bacterial pellet in lysis buffer (typically 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0)

• Disrupt cells using sonication or other mechanical methods

• Centrifuge lysate to separate soluble fraction containing the recombinant protein

• Load supernatant onto a Ni-NTA column pre-equilibrated with lysis buffer

• Wash column with wash buffer (typically 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8.0)

• Elute ACIAD1930 with elution buffer (typically 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0)

• Analyze purity by SDS-PAGE, aiming for >85% purity

If higher purity is required, additional chromatography steps such as gel filtration or ion exchange can be incorporated.

How should recombinant ACIAD1930 be stored to maintain optimal activity?

For optimal storage of recombinant ACIAD1930, the following recommendations are based on manufacturer guidelines and general protein storage principles :

Storage Recommendations:

• Short-term storage (up to 1 week): 4°C in appropriate buffer

• Medium-term storage (up to 6 months): -20°C/-80°C in liquid form

• Long-term storage (up to 12 months): -20°C/-80°C in lyophilized form

Buffer Composition:

• Add 5-50% glycerol (final concentration) to prevent freeze-thaw damage

• A default concentration of 50% glycerol is recommended for optimal stability

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL before adding glycerol

Avoid repeated freeze-thaw cycles as this can significantly reduce protein activity. If multiple uses are anticipated, prepare small working aliquots for storage .

How should experiments be designed to characterize ACIAD1930's enzymatic activity?

When designing experiments to characterize ACIAD1930's nucleotide pyrophosphatase activity, a methodical approach is essential:

Experimental Design Framework:

• Substrate Specificity Testing:

  • Test a panel of substrates including both canonical nucleotides (dTTP, UTP, CTP) and modified nucleotides (5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, 7-methyl-GTP)

  • Include controls from both YhdE and YceF subfamilies for comparison

  • Measure activity using a pyrophosphate detection assay or HPLC

• Kinetic Analysis:

  • Determine kinetic parameters (Km, Vmax, kcat) for each substrate

  • Use varying substrate concentrations (typically 0.01-2 mM range)

  • Plot Michaelis-Menten curves to determine enzyme kinetics

• Influence of Conditions:

  • Test activity across pH range (typically pH 6.0-9.0)

  • Examine temperature dependence (25-45°C)

  • Evaluate cation requirements (Mg²⁺, Mn²⁺, Ca²⁺)

These approaches have been effective in characterizing the enzymatic activities of other Maf family proteins and would be applicable to ACIAD1930 .

What controls are essential when studying ACIAD1930 activity?

Proper controls are critical for ensuring the reliability and interpretability of results when studying ACIAD1930:

Essential Controls for ACIAD1930 Activity Studies:

• Negative Controls:

  • Reaction mixture without enzyme

  • Heat-inactivated ACIAD1930 (typically 95°C for 10 minutes)

  • Reaction without substrate

  • Reaction with structurally similar but non-hydrolyzable substrate analogs

• Positive Controls:

  • Well-characterized Maf protein with known activity (e.g., E. coli YhdE)

  • Commercial nucleotide pyrophosphatase with established activity

• Specificity Controls:

  • YceF-subfamily Maf protein (known to have different substrate specificity)

  • Non-Maf nucleotide pyrophosphatase enzyme

• Technical Controls:

  • Internal standard for quantification

  • Standard curve for each product measured

  • Multiple time points to ensure linear reaction range

These control experiments will help distinguish ACIAD1930-specific activity from background reactions and provide benchmarks for comparing activity levels .

How can researchers measure the impact of mutations on ACIAD1930 function?

To evaluate how mutations affect ACIAD1930 function, a systematic approach combining structural analysis, site-directed mutagenesis, and functional assays is recommended:

Methodological Approach for Mutation Studies:

• Target Selection:

  • Focus on the signature Maf motif residues (S-R-E-K-D-K)

  • Include subfamily-specific motif residues (R-T-Q for YhdE-like proteins)

  • Select additional conserved residues based on structural analysis

  • Design control mutations in non-conserved regions

• Mutagenesis Technique:

  • Use site-directed mutagenesis via splicing PCR

  • For Acinetobacter, exploit its natural competence by direct transformation with PCR products

  • Design primers with 20 bp 5' extensions complementary to flanking regions

  • Verify successful mutations by sequencing

• Functional Characterization:

  • Express and purify each mutant protein using identical conditions

  • Compare enzymatic activity against wild-type ACIAD1930

  • Measure substrate specificity changes

  • Determine kinetic parameters for each mutant

  • Assess structural integrity via circular dichroism or thermal shift assays

• In vivo Studies:

  • Transform mutant constructs into Acinetobacter sp. ADP1

  • Assess physiological effects (growth rate, cell morphology)

  • Measure intracellular nucleotide levels

This approach leverages Acinetobacter sp. ADP1's natural competence and strong tendency towards homology-directed recombination, which allows for efficient genetic manipulation without specialized equipment .

How can ACIAD1930 be used to study horizontal gene transfer in Acinetobacter species?

ACIAD1930 can serve as an excellent marker for studying horizontal gene transfer (HGT) in Acinetobacter species due to the organism's exceptional natural competence properties:

Research Strategy for HGT Studies Using ACIAD1930:

• Tagged ACIAD1930 Construction:

  • Create fluorescently tagged or epitope-tagged versions of ACIAD1930

  • Insert these constructs into the chromosome via natural transformation

  • Verify integration via PCR and sequencing

• Transfer Experiments:

  • Mix labeled strain with recipient Acinetobacter strains under various conditions

  • Monitor transfer frequency through selection or fluorescence

  • Use different growth phases to optimize transfer (exponential growth is key)

• Environmental Variables Analysis:

  • Test the effect of different media compositions

  • Evaluate temperature effects (typically 30°C is optimal)

  • Measure transfer rates with varying cell densities

• Interbacterial Transfer Assessment:

  • Compare transfer rates between closely related and distant Acinetobacter species

  • Analyze recombination tract sizes (13-123 kb has been observed in clinical isolates)

  • Correlate with presence of specific competence genes (e.g., comP)

This approach takes advantage of Acinetobacter's unique ability to undergo natural transformation without specialized equipment, allowing simple addition of linear PCR products to growing cultures followed by selective plating .

What methodological approaches can be used to study ACIAD1930's role in nucleotide metabolism?

To investigate ACIAD1930's role in nucleotide metabolism, a combination of biochemical, genetic, and metabolomic approaches is recommended:

Comprehensive Methodology:

• Metabolomic Profiling:

  • Compare nucleotide pools in wild-type vs. ACIAD1930 knockout strains

  • Use targeted LC-MS/MS to quantify modified nucleotides

  • Perform pulse-chase experiments with labeled nucleotide precursors

  • Analyze downstream metabolites to identify pathways affected

• Genetic Approaches:

  • Create an ACIAD1930 deletion strain using natural transformation

  • Construct complementation strains with wild-type and mutant versions

  • Develop an inducible expression system to control ACIAD1930 levels

  • Perform genetic screens for synthetic lethality/sickness with ACIAD1930 mutants

• Biochemical Interaction Studies:

  • Identify protein interaction partners via pull-down assays

  • Use crosslinking coupled with mass spectrometry to capture transient interactions

  • Perform in vitro reconstitution of nucleotide metabolism pathways

  • Develop real-time assays for monitoring nucleotide turnover

• Stress Response Analysis:

  • Measure ACIAD1930 expression under various stress conditions

  • Test sensitivity of ACIAD1930 mutants to DNA-damaging agents

  • Evaluate mutator phenotypes in strains with altered ACIAD1930 levels

  • Assess impact on response to nucleotide pool imbalances

These approaches would provide comprehensive insights into how ACIAD1930 contributes to nucleotide homeostasis and cellular metabolism in Acinetobacter species.

How does ACIAD1930 compare with Maf proteins from other bacterial species?

Comparing ACIAD1930 with Maf proteins from other bacterial species reveals important evolutionary and functional relationships:

Comparative Analysis Framework:

• Sequence-Based Comparison:
  The Maf protein family divides into two main subfamilies: YhdE and YceF. ACIAD1930 belongs to the YhdE subfamily, sharing the characteristic R-T-Q motif rather than the W-Q-E motif found in YceF proteins .

  Subfamily	Signature Motif	Representative Members	ACIAD1930 Similarity
  YhdE	R-T-Q	E. coli YhdE, B. subtilis BSU28050, S. cerevisiae YOR111W	High (YhdE-like)
  YceF	W-Q-E	E. coli YceF, S. typhimurium STM1189	Low

• Functional Comparison:

  • YhdE-like Maf proteins (including ACIAD1930) show activity against both canonical nucleotides (dTTP, UTP, CTP) and modified nucleotides

  • YceF-like proteins show different substrate preferences, being less active against canonical nucleotides

  • Activity levels vary significantly between species (30-90 nmol/min per mg for YceF proteins vs. higher levels for YhdE-like proteins)

• Structural Analysis:

  • All Maf proteins contain the conserved S-R-E-K-D-K motif

  • Species-specific structural features can be identified through homology modeling

  • Active site residues are highly conserved across species, while peripheral regions show higher variability

• Genomic Context:

  • In many eukaryotes, Maf domains are fused to methyltransferase domains

  • In some archaea, Maf genes are associated with methyltransferase genes

  • Acinetobacter's genomic context may provide clues about ACIAD1930's species-specific functions

This comparative approach helps position ACIAD1930 within the broader evolutionary context of Maf proteins and can suggest potential functional specializations.

What are the common challenges in expressing recombinant ACIAD1930 and how can they be addressed?

Researchers working with recombinant ACIAD1930 may encounter several challenges during expression and purification. Here are methodological solutions:

Challenge 1: Low Expression Yield

• Problem: Poor expression levels in standard E. coli systems

• Solutions:

  • Optimize codon usage for the expression host

  • Try different expression vectors (pET vs. pGEX vs. pMAL)

  • Test various E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Adjust induction conditions (IPTG concentration, temperature, duration)

  • Consider autoinduction media for gentler induction

Challenge 2: Inclusion Body Formation

• Problem: ACIAD1930 forms insoluble aggregates

• Solutions:

  • Lower induction temperature (16-20°C overnight)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Add solubility-enhancing tags (SUMO, MBP)

  • If necessary, develop refolding protocols from inclusion bodies

Challenge 3: Protein Instability

• Problem: Rapid degradation during expression or purification

• Solutions:

  • Add protease inhibitors to all buffers

  • Use protease-deficient host strains

  • Keep samples at 4°C during all procedures

  • Add stabilizing agents (glycerol, specific ions)

  • Optimize buffer conditions (pH, salt concentration)

Challenge 4: Low Activity

• Problem: Purified protein shows minimal enzymatic activity

• Solutions:

  • Verify protein folding using circular dichroism

  • Test different buffer conditions for activity assays

  • Add potential cofactors (divalent metals, especially Mg²⁺)

  • Remove potential inhibitors via dialysis

  • Evaluate freeze-thaw effects on activity

These approaches have proven effective for expressing other challenging proteins and can be adapted for ACIAD1930 .

How can researchers analyze and interpret contradictory data about ACIAD1930 activity?

When faced with contradictory results regarding ACIAD1930 activity, a systematic approach to data analysis and experimental design is crucial:

Methodological Framework for Resolving Data Contradictions:

• Systematic Variation Analysis:

  • Identify all methodological variables between contradictory experiments

  • Systematically test each variable in isolation

  • Create a design of experiments (DOE) to evaluate multiple factors simultaneously

  • Quantify the effect size of each variable on the outcome

• Statistical Approach:

  • Apply appropriate statistical tests based on data distribution

  • Consider Bayesian analysis for incorporating prior knowledge

  • Perform power analysis to ensure sufficient sample size

  • Report effect sizes alongside p-values

• Technical Validation:

  • Use multiple independent methods to measure the same outcome

  • Verify reagent and sample quality with appropriate controls

  • Blind experimenters to conditions when possible

  • Have different researchers replicate key findings

• Data Integration Strategy:

  • Develop a mathematical model that can explain seemingly contradictory results

  • Consider concentration-dependent or context-dependent effects

  • Evaluate threshold effects or non-linear responses

  • Integrate results across different experimental scales (in vitro, cellular, in vivo)

These approaches align with best practices in experimental design and help resolve apparent contradictions by identifying underlying variables or mechanisms that explain disparate results .

What bioinformatic tools and approaches are most useful for studying ACIAD1930 and related Maf proteins?

For comprehensive analysis of ACIAD1930 and related Maf proteins, researchers should employ a suite of bioinformatic tools:

Recommended Bioinformatic Toolkit:

• Sequence Analysis:

  • Multiple sequence alignment (MUSCLE, Clustal Omega, MAFFT)

  • Phylogenetic tree construction (RAxML, MrBayes, IQ-TREE)

  • Motif identification (MEME, HMMER)

  • Conservation analysis (ConSurf, WebLogo)

• Structural Analysis:

  • Homology modeling (SWISS-MODEL, Phyre2, I-TASSER)

  • Molecular dynamics simulations (GROMACS, AMBER, NAMD)

  • Binding site prediction (CASTp, SiteMap)

  • Protein-protein interaction interfaces (HADDOCK, ClusPro)

• Genomic Context Analysis:

  • Synteny analysis (SynMap, Mauve)

  • Gene neighborhood conservation (MicrobesOnline, STRING)

  • Horizontal gene transfer detection (IslandViewer, Alien_Hunter)

  • Regulatory element prediction (MEME, RSAT)

• Functional Prediction:

  • Domain architecture analysis (InterProScan, SMART)

  • Substrate specificity prediction (3DLigandSite, COACH)

  • Enzyme classification (EFICAz, BRENDA)

  • Gene ontology enrichment (DAVID, GOrilla)

• Comparative Genomics:

  • Pan-genome analysis across Acinetobacter species

  • Identification of co-evolving gene families

  • Detection of lineage-specific adaptations

  • Mapping conservation across taxonomic groups

These tools provide complementary information that, when integrated, offers powerful insights into the evolutionary history, structural features, and potential functions of ACIAD1930 and related Maf proteins .

What are promising research directions for understanding ACIAD1930's role in Acinetobacter biology?

Several promising research directions could significantly advance our understanding of ACIAD1930's biological roles:

High-Priority Research Avenues:

• Cellular Response to Nucleotide Stress:

  • Investigate how ACIAD1930 expression changes under various nucleotide stress conditions

  • Determine if ACIAD1930 plays a role in preventing incorporation of modified or damaged nucleotides

  • Explore links between ACIAD1930 activity and DNA repair mechanisms

  • Assess impact on mutation rates and genome stability

• Metabolic Integration:

  • Map the metabolic pathways affected by ACIAD1930 activity

  • Determine how ACIAD1930 coordinates with other nucleotide metabolism enzymes

  • Investigate potential regulatory roles beyond enzymatic activity

  • Explore connections to RNA modification pathways

• Host-Pathogen Interactions:

  • Evaluate ACIAD1930's potential role in Acinetobacter pathogenesis

  • Determine if ACIAD1930 affects antibiotic resistance mechanisms

  • Investigate connections to virulence factor expression

  • Assess contributions to survival in host environments

• System-Level Integration:

  • Apply multi-omics approaches (transcriptomics, proteomics, metabolomics)

  • Develop mathematical models of nucleotide homeostasis incorporating ACIAD1930

  • Investigate emergent properties in complex cellular networks

  • Explore synthetic biology applications based on ACIAD1930 functions

These research directions would address fundamental gaps in our understanding while potentially revealing new strategies for addressing Acinetobacter infections .

How can ACIAD1930 research contribute to our understanding of antimicrobial resistance in Acinetobacter?

Research on ACIAD1930 could provide valuable insights into antimicrobial resistance mechanisms:

Methodological Approaches for Antimicrobial Resistance Studies:

• Natural Transformation and Resistance Gene Transfer:

  • Investigate if ACIAD1930 affects natural transformation efficiency

  • Determine roles in acquisition of resistance genes through horizontal transfer

  • Measure impact on recombination of large resistance islands (13-123 kb)

  • Develop methods to potentially inhibit this transfer process

• Stress Response and Antibiotic Tolerance:

  • Evaluate how ACIAD1930 activity changes during antibiotic exposure

  • Determine if it contributes to persister cell formation

  • Assess impacts on bacterial metabolism during antibiotic stress

  • Identify potential synergistic targets for combination therapy

• Evolutionary Dynamics:

  • Track ACIAD1930 sequence changes in evolving resistant populations

  • Compare variants between environmental and clinical isolates

  • Determine if specific variants correlate with resistance phenotypes

  • Assess horizontal gene transfer rates in the presence/absence of ACIAD1930

• Therapeutic Targeting:

  • Evaluate ACIAD1930 as a potential drug target

  • Screen for inhibitors of ACIAD1930 activity

  • Assess combination approaches targeting ACIAD1930 and conventional antibiotics

  • Develop assays for monitoring ACIAD1930 activity in clinical isolates

These approaches could reveal new mechanisms of resistance acquisition and potentially identify novel therapeutic strategies for combating multidrug-resistant Acinetobacter infections .