Recombinant Acinetobacter sp. Polyribonucleotide nucleotidyltransferase (pnp), partial

What is the basic function of Polyribonucleotide Nucleotidyltransferase in Acinetobacter species?

Polyribonucleotide nucleotidyltransferase (PNP) in Acinetobacter species functions as a 3'-5' exoribonuclease that participates in RNA degradation pathways. It catalyzes the phosphorolysis of RNA, removing nucleotides from the 3' end while releasing nucleoside diphosphates. Additionally, PNP can function in reverse as a polymerase, adding nucleotides to the 3' end of RNA molecules under certain conditions.

PNP plays crucial roles in:

• mRNA turnover and stability regulation

• Quality control of defective RNAs

• Cold adaptation responses

• Virulence factor expression

• Stress response mechanisms

When studying PNP in Acinetobacter, researchers should be aware that it often functions as part of a multiprotein complex called the degradosome, which can include other enzymes such as RNase E, RNA helicase, and enolase. The specific composition of this complex may vary between bacterial species and influence the function of PNP.

How can I express recombinant PNP from Acinetobacter sp. in laboratory conditions?

Expression of recombinant PNP from Acinetobacter species can be achieved using several expression systems, with selection depending on your specific experimental requirements:

Using Acinetobacter-E. coli shuttle vectors:

• The pVRL series plasmids (pVRL1 and pVRL2) offer an excellent system for expressing Acinetobacter genes, including pnp. These multipurpose shuttle plasmids can efficiently replicate in both Acinetobacter spp. and E. coli .

• Clone your pnp gene into the multilinker containing unique restriction sites available in these vectors. The pVRL1 plasmid combines the cryptic plasmid pWH1277 from Acinetobacter calcoaceticus (providing origin of replication for Acinetobacter), a ColE1-like origin of replication, antibiotic resistance cassettes, and a multilinker .

• If controlled expression is needed, use pVRL2, which contains an arabinose-inducible expression system (araC-PBAD) with negligible basal expression and high dynamic range of responsiveness to the inducer .

Methodological considerations:

• Select appropriate antibiotic markers based on the resistance profile of your Acinetobacter strain. pVRL vectors offer gentamicin or zeocin resistance markers that work effectively in multidrug-resistant (MDR) strains .

• These vectors have narrow host range, high copy number, and are stably maintained in Acinetobacter spp. due to a toxin-antitoxin system .

• Blue/white screening can be used to identify recombinant clones when using these vectors .

For optimal expression, culture conditions should be tailored to the specific Acinetobacter strain being used, with careful attention to growth temperature, media composition, and inducer concentration if using an inducible system.

What are the common challenges in purifying active PNP from recombinant Acinetobacter systems?

Purification of active PNP from recombinant Acinetobacter systems presents several challenges that researchers should anticipate and address:

Solubility issues:

• PNP may form inclusion bodies when overexpressed, requiring optimization of expression conditions (temperature, inducer concentration).

• Consider using the arabinose-inducible system in pVRL2, which allows fine-tuning of expression levels to maintain solubility while maximizing yield .

• Expression at lower temperatures (16-25°C) or co-expression with chaperones may increase the proportion of soluble protein.

Maintaining enzymatic activity:

• PNP activity is sensitive to oxidation of cysteine residues. Include reducing agents (DTT or β-mercaptoethanol) in purification buffers.

• The enzyme requires Mg²⁺ for activity, so include 5-10 mM MgCl₂ in purification and storage buffers.

• Avoid multiple freeze-thaw cycles that can lead to denaturation and activity loss.

Contamination with RNA:

• PNP binds RNA with high affinity, leading to co-purification of host RNA.

• RNase treatment during purification or high-salt washes (0.8-1.0 M NaCl) can help remove bound nucleic acids.

Purification strategy recommendations:

• Use affinity tags (His-tag or GST-tag) for initial capture, ensuring these tags don't interfere with activity.

• Implement size exclusion chromatography as a polishing step to separate monomeric protein from aggregates.

• Consider ion exchange chromatography to remove nucleic acid contaminants.

• Store purified protein in buffer containing 50% glycerol at -20°C for short-term or -80°C for long-term storage.

Test enzyme activity after each purification step to monitor recovery and identify steps that may compromise function.

How can I design experiments to investigate the role of PNP in Acinetobacter virulence and antibiotic resistance?

Investigating the role of PNP in Acinetobacter virulence and antibiotic resistance requires sophisticated experimental approaches that connect RNA metabolism to pathogenesis:

Genetic manipulation strategies:

• Gene knockout/knockdown: Utilize shuttle vectors like pVRL1/pVRL2 to express antisense RNA or CRISPR-Cas9 components targeting pnp . Complete knockout may be lethal, so consider conditional knockdown systems.

• Point mutations: Generate catalytically inactive variants by site-directed mutagenesis to distinguish between structural and enzymatic roles of PNP.

• Domain swapping: Replace domains with those from non-pathogenic bacteria to identify regions responsible for virulence-related functions.

Virulence phenotype assessment:

• Biofilm formation: Quantify changes in biofilm development using crystal violet staining and confocal microscopy.

• Adhesion assays: Measure attachment to epithelial cells or abiotic surfaces.

• Invasion assays: Determine the ability of mutants to invade host cells.

• Host survival models: Use Galleria mellonella or mouse infection models to assess in vivo virulence.

Antibiotic resistance connections:

• Examine if PNP affects expression of resistance genes through RNA stability mechanisms.

• Measure minimum inhibitory concentrations (MICs) across antibiotic classes in PNP-modified strains.

• Investigate PNP's potential role in stress responses that might indirectly affect antibiotic tolerance.

Transcriptome analysis:

• RNA-seq comparing wild-type and PNP-modified strains to identify differentially expressed transcripts.

• CLIP-seq (crosslinking immunoprecipitation) to identify direct RNA targets of PNP.

• Pulse-chase RNA labeling to measure transcript half-lives and determine which mRNAs are stabilized or destabilized by PNP.

When designing these experiments, consider that PNP's functions may overlap with other ribonucleases, requiring careful interpretation of results and potentially the creation of double mutants to reveal compensatory mechanisms. Integration of multiple approaches provides more robust evidence for PNP's role in virulence and antibiotic resistance phenotypes.

What are the methodological approaches for studying PNP interactions with the Acinetobacter RNA degradosome complex?

Studying PNP interactions within the Acinetobacter RNA degradosome complex requires specialized techniques to capture protein-protein and protein-RNA interactions:

Protein-protein interaction analysis:

• Co-immunoprecipitation (Co-IP):

  • Express tagged PNP in Acinetobacter using pVRL2 with arabinose-inducible expression

  • Cross-link protein complexes in vivo using formaldehyde (0.1-1%)

  • Immunoprecipitate using antibodies against the tag or native PNP

  • Identify interacting partners by mass spectrometry

• Bacterial two-hybrid screening:

  • Create fusion constructs between PNP domains and reporter fragments

  • Screen against a library of Acinetobacter proteins to identify novel interactions

  • Verify interactions with individual pull-down assays

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Purify native complexes from Acinetobacter

  • Determine molecular weight and stoichiometry of intact degradosome complexes

  • Compare complex composition under different stress conditions

Protein-RNA interaction methods:

• RNA immunoprecipitation (RIP):

  • Crosslink RNA-protein complexes in vivo

  • Immunoprecipitate PNP and associated RNAs

  • Identify bound RNAs by sequencing or RT-PCR

• CRAC (cross-linking and analysis of cDNAs) or CLIP-seq:

  • UV crosslink RNA-protein complexes

  • Purify PNP under denaturing conditions

  • Sequence associated RNAs to create genome-wide binding maps

Structural characterization:

• Cryo-electron microscopy of purified degradosome complexes

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Integrative structural modeling combining low-resolution data with computational predictions

Functional reconstitution:

• Express and purify individual components using the pVRL expression system

• Reconstitute minimal functional complexes in vitro

• Perform RNA degradation assays with defined substrates

• Test how complex composition affects substrate specificity and degradation kinetics

When performing these experiments, it's crucial to maintain physiologically relevant conditions. The degradosome composition can change dramatically depending on growth phase and stress conditions. Consider performing comparative studies under different environmental stresses (temperature, pH, oxidative stress) to understand the dynamic nature of these interactions.

How can contradictory results in PNP functional studies from different Acinetobacter strains be reconciled?

Reconciling contradictory results in PNP functional studies from different Acinetobacter strains requires systematic analysis of strain-specific factors and experimental conditions:

Strain-specific genetic context analysis:

• Comparative genomics approach:

  • Sequence the pnp gene and surrounding genomic regions from contradictory strains

  • Identify single nucleotide polymorphisms or structural variations that might affect function

  • Examine regulatory elements that could influence expression levels

  • Use shuttle vectors like pVRL1/pVRL2 to express variants in a common genetic background for direct comparison

• Transcriptome analysis:

  • Compare RNA-seq data from different strains to identify strain-specific gene expression patterns

  • Analyze potential compensatory mechanisms or redundant pathways that might mask PNP function in certain strains

Experimental design reconciliation:

• Standardize experimental conditions:

  • Use identical growth media, temperature, and growth phase for sampling

  • Ensure comparable expression levels when using recombinant systems

  • Standardize protein purification protocols and activity assay conditions

• Cross-validation with multiple techniques:

  • Apply both in vivo and in vitro approaches to validate findings

  • Use complementary methodologies to measure the same parameter

  • Consider the limitations of each assay and how they might influence results

Alternative hypothesis generation:

• Post-translational modifications:

  • Investigate strain-specific phosphorylation, acetylation, or other modifications

  • Use mass spectrometry to identify and quantify these modifications

  • Test how modifications affect enzyme activity and protein interactions

• Protein complex composition:

  • Determine if PNP associates with different partners in different strains

  • Examine if degradosome composition varies among strains

  • Test if complex formation alters substrate specificity or catalytic parameters

• Environmental adaptations:

  • Test PNP function under different stress conditions relevant to each strain's ecological niche

  • Examine temperature dependence, pH optima, and ion requirements across strains

Resolution strategies:

• Create chimeric proteins by domain swapping between contradictory strains to identify functional determinants

• Perform cross-complementation experiments using the pVRL vector system to express PNP variants in different strains

• Develop strain-specific models rather than attempting to force a universal model of PNP function

Understanding the environmental and evolutionary context of each strain can provide insights into why PNP may function differently. Consider adaptive advantages that might drive functional divergence in enzyme properties across the Acinetobacter genus.

What methodological approaches can be used to study the role of PNP in horizontal gene transfer and antibiotic resistance spread in Acinetobacter?

Investigating PNP's potential role in horizontal gene transfer (HGT) and antibiotic resistance spread requires specialized methodological approaches that connect RNA metabolism to genetic exchange mechanisms:

Conjugation and transformation efficiency studies:

• Construct pnp mutants using inducible systems in the pVRL2 vector to control expression levels

• Measure conjugation frequencies using antibiotic resistance markers

• Quantify natural transformation rates with genomic DNA containing selectable markers

• Assess uptake and stability of plasmids containing antibiotic resistance genes

RNA stability and regulatory effects:

• Examine if PNP affects stability of transcripts encoding conjugation machinery

• Investigate whether PNP influences expression of competence genes required for DNA uptake

• Determine if PNP-mediated RNA degradation affects translation of key components of mobile genetic elements

Integration with ICE (Integrative and Conjugative Elements) biology:

• Study whether PNP affects expression or excision of ICEs similar to ICETn43716385, which can carry antibiotic resistance genes

• Investigate if RNA processing by PNP influences ICE transfer frequency

• Examine co-localization of PNP with ICE-encoded proteins during conjugation events

Experimental design for specific mechanisms:

• For testing effects on conjugation:

  • Use filter mating assays with donor strains expressing different levels of PNP

  • Quantify transfer rates of plasmids carrying resistance genes like blaNDM-1 or msr(E)

  • Monitor expression of conjugation machinery using reporter fusions

• For natural transformation studies:

  • Prepare standardized genomic DNA or plasmid DNA containing resistance markers

  • Compare transformation frequencies between wild-type and PNP-altered strains

  • Use fluorescently labeled DNA to track uptake and processing

• For RNA-based regulatory effects:

  • Perform RNA stability assays on transcripts encoding transfer functions

  • Use ribosome profiling to assess translation efficiency of key components

  • Implement CLIP-seq to identify if PNP directly interacts with transcripts involved in HGT

Methodological considerations:

• Use appropriate controls for conjugation experiments, including DNase treatments to eliminate transformation

• Consider strain-specific differences in natural competence when designing transformation assays

• Account for growth rate differences between wild-type and PNP-altered strains when calculating transfer frequencies

• Include time-course experiments to distinguish between direct and indirect effects of PNP on gene transfer

This methodological framework allows researchers to systematically investigate whether PNP-mediated RNA processing affects the acquisition, maintenance, and spread of antibiotic resistance genes in Acinetobacter populations.

How can I design experiments to investigate the cold adaptation function of PNP in Acinetobacter species?

PNP plays a critical role in bacterial cold adaptation, particularly through selective RNA degradation mechanisms. Here's a comprehensive experimental design approach to investigate this function in Acinetobacter:

Growth and phenotypic characterization:

• Comparative growth analysis:

  • Construct conditional pnp mutants using the arabinose-inducible pVRL2 system

  • Compare growth curves at optimal temperature (37°C) versus cold conditions (15-20°C)

  • Measure lag phase duration, growth rate, and final cell density

  • Determine cold survival rates after prolonged exposure

• Physiological adaptations assessment:

  • Measure membrane fluidity changes using fluorescence anisotropy

  • Analyze fatty acid composition changes by gas chromatography

  • Compare protein synthesis rates using pulse-labeling with radioactive amino acids

  • Quantify ribosome profiles on sucrose gradients at different temperatures

Molecular mechanism investigation:

• Transcriptome analysis:

  • Perform RNA-seq comparing wild-type and PNP-deficient strains at optimal and cold temperatures

  • Conduct differential expression analysis to identify cold-induced transcripts affected by PNP

  • Measure RNA decay rates using rifampicin-based transcription inhibition followed by qRT-PCR

  • Identify specific RNA sequence motifs that might target transcripts for PNP-mediated degradation

• PNP enzyme kinetics:

  • Purify recombinant PNP expressed from pVRL vectors

  • Compare enzymatic activity at different temperatures (5-37°C)

  • Determine temperature-dependent changes in substrate specificity

  • Measure binding affinities for different RNA substrates as a function of temperature

Structural and interaction studies:

• PNP complex formation analysis:

  • Perform co-immunoprecipitation at different temperatures to identify temperature-dependent interaction partners

  • Use bacterial two-hybrid assays to test specific interactions at different temperatures

  • Analyze degradosome composition changes during cold adaptation

• Structural adaptations:

  • Conduct hydrogen-deuterium exchange mass spectrometry at different temperatures

  • Perform limited proteolysis to assess conformational changes

  • Use circular dichroism to measure secondary structure stability across temperatures

Experimental data table example:

Methodological controls and considerations:

• Include complementation controls using the pVRL1 vector expressing wild-type pnp

• Ensure that growth media composition remains constant across temperature conditions

• Account for growth phase effects by sampling at equivalent physiological states rather than at fixed time points

• Include multiple Acinetobacter species with different temperature ranges to identify conserved mechanisms

This experimental design provides a comprehensive framework for elucidating the specific molecular mechanisms by which PNP contributes to cold adaptation in Acinetobacter species.