Recombinant Synechocystis sp. DNA topoisomerase 4 subunit A (parC), partial

What is DNA topoisomerase IV and what is the function of the ParC subunit in Synechocystis sp.?

DNA topoisomerase IV is a type II topoisomerase that plays essential roles in DNA replication, chromosome segregation, and maintaining DNA topology. In bacteria, topoisomerase IV is typically composed of two subunits: ParC (the catalytic subunit) and ParE (the ATP-binding subunit), which form a heterotetrameric enzyme (ParC₂ParE₂).

In Synechocystis sp., as in other bacteria, the ParC subunit is responsible for DNA binding and the catalytic activity of breaking and rejoining DNA strands. Topoisomerase IV is particularly important for decatenating (unlinking) daughter chromosomes after DNA replication, which is essential for proper chromosome segregation before cell division .

The manipulation of topoisomerase expression in Synechocystis sp. PCC 6803 has shown that altering DNA topology affects cell division while allowing continued cell growth, suggesting topoisomerase IV plays a critical role in coordinating DNA replication and cell division in this cyanobacterium .

How does the ParC subunit of topoisomerase IV differ from gyrase subunits in structure and function?

• Sequence distinctions: DNA sequence analysis has identified a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and serves as a hallmark of the GyrA protein family, distinguishing it from ParC .

• Functional specificity: While DNA gyrase primarily introduces negative supercoils into DNA, topoisomerase IV excels at decatenation, separating interlinked DNA molecules during replication .

• Cellular localization: In bacteria like Caulobacter crescentus, the ParC subunit colocalizes with the replisome (DNA replication machinery) as it moves from the cell pole to the division plane during chromosome replication, whereas the ParE subunit is more dispersed throughout the cell .

These differences allow the two type II topoisomerases to perform complementary but distinct functions in managing DNA topology within bacterial cells.

What phenotypes result from ParC mutations or depletion in cyanobacteria?

Studies on topoisomerase IV mutations in cyanobacteria like Synechocystis sp. PCC 6803 have revealed distinct phenotypes:

When topoisomerase expression is manipulated in Synechocystis sp. PCC 6803:

These phenotypes differ from those observed in bacteria like E. coli and Salmonella typhimurium. In Caulobacter crescentus, Topo IV mutants show a "cell separation phenotype" where cells are highly pinched at multiple sites but do not produce anucleate cells, suggesting unique regulatory mechanisms coupling nucleoid partitioning and cell division in different bacterial species .

How does topoisomerase IV activity in Synechocystis sp. interact with DNA supercoiling to regulate gene expression?

DNA supercoiling in Synechocystis sp. represents a sophisticated regulatory mechanism that affects gene expression globally, with topoisomerase IV playing a crucial role in maintaining supercoiling homeostasis:

• Diurnal regulation: Supercoiling states in cyanobacteria fluctuate during day/night cycles, affecting transcription programs. Manipulating topoisomerases locks cells in transcriptional states reflecting dark/light transitions, suggesting topoisomerase IV contributes to circadian gene regulation through supercoiling maintenance .

• Promoter architecture sensitivity: Genes in Synechocystis sp. show distinct responses to supercoiling changes based on their promoter structures. Helically phased A-tracts (short repeats of A and T nucleotides at distances matching the DNA helix pitch) are particularly pronounced in polyploid cyanobacteria, making them sensitive to supercoiling changes .

• Twin-domain model application: Research supports the twin-domain model in Synechocystis, where transcription generates positive supercoiling ahead of RNA polymerase and negative supercoiling behind it. Topoisomerase IV helps resolve these topological stresses, affecting transcript abundances along transcription units, including rRNA genes .

• Stress response connection: The transcriptome response to altered supercoiling (e.g., through gyrase inhibition) overlaps significantly with responses to various stress conditions, suggesting supercoiling as a global regulatory mechanism that topoisomerase IV helps maintain .

What is the relationship between ParC and ParE subunits in topoisomerase IV, and how does this relationship affect enzyme function?

The ParC-ParE relationship in topoisomerase IV represents a complex functional partnership that is essential for enzyme activity:

• Structural interdependence: While ParC is the catalytic subunit and ParE is the ATP-binding subunit, both are required for complete enzyme function. Research has shown that an active ParE subunit is necessary for proper ParC localization to the replisome as it moves from the cell pole to the division plane during chromosome replication .

• Fusion possibility: Intriguingly, studies have demonstrated that a ParE-ParC fusion protein (creating a single polypeptide chain containing both subunits) is a functional topoisomerase capable of catalyzing both decatenation and relaxation reactions. This artificial fusion protein can even complement temperature-sensitive growth in both parC and parE mutant strains, indicating it can substitute for topoisomerase IV in vivo .

• Evolutionary implications: The ability to create functional fusion proteins reinforces the evolutionary relationship between prokaryotic heterotetrameric type II topoisomerases and eukaryotic homodimeric type II topoisomerases .

• Localization patterns: In some bacteria, the ParC subunit colocalizes with the replisome during DNA replication, while the ParE subunit is dispersed throughout the cell .

How does topoisomerase IV function differ between unicellular cyanobacteria like Synechocystis sp. and other bacteria?

Topoisomerase IV function shows notable variations across bacterial species with different cell division mechanisms:

• Cell division phenotypes: Topo IV mutants in Synechocystis sp. and Caulobacter crescentus differ markedly from those in E. coli and Salmonella typhimurium. While E. coli Topo IV mutants form long filamentous cells with defective nucleoid segregation and frequently produce anucleate cells, C. crescentus Topo IV mutants are highly pinched at multiple sites ("cell separation phenotype") but do not produce DNA-less cells .

• Nucleoid segregation effects: In C. crescentus, abnormal nucleoid segregation in parE mutants becomes apparent only in specific genetic backgrounds (e.g., with conditional mutations in the ftsA cell division gene), whereas in E. coli, segregation defects are more readily observable .

• Polyploidy considerations: Synechocystis sp. is polyploid (contains multiple genome copies), which may allow it to tolerate topological problems that would be lethal in monoploid bacteria .

• Environmental adaptation: Cyanobacteria like Synechocystis sp. undergo significant metabolic shifts between day and night, requiring specialized regulatory mechanisms involving DNA supercoiling and topoisomerase activity .

What are the most effective approaches for expressing and purifying recombinant Synechocystis sp. DNA topoisomerase IV ParC?

Expressing and purifying recombinant Synechocystis sp. ParC requires careful consideration of several methodological aspects:

• Expression system selection:

  • E. coli BL21(DE3) or similar strains typically provide good expression levels for recombinant bacterial proteins

  • Codon optimization may be necessary, as cyanobacterial codon usage differs from E. coli

  • Consider using solubility tags (e.g., MBP, SUMO, or TrxA) if initial expression yields insoluble protein

• Construct design:

  • Include a 6xHis or similar affinity tag for purification

  • Consider expressing a ParE-ParC fusion protein, which has been shown to be functional in other bacterial systems

  • Design constructs with precision: for partial ParC constructs, careful analysis of domain boundaries is critical

• Expression conditions:

  • Lower induction temperatures (16-20°C) often improve solubility

  • IPTG concentration optimization (typically 0.1-0.5 mM)

  • Extended expression times (overnight) at lower temperatures

• Purification strategy:

  • Two-step purification combining affinity chromatography (Ni-NTA) followed by ion exchange or size exclusion chromatography

  • Include ATP, Mg²⁺, and reducing agents in purification buffers to maintain protein stability

• Quality control:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Activity assays using supercoiled plasmid DNA to confirm functionality

  • Dynamic light scattering to assess homogeneity and aggregation state

What assays are most appropriate for evaluating topoisomerase IV activity in Synechocystis sp.?

Several assays can effectively measure topoisomerase IV activity, each with specific advantages:

• DNA decatenation assay:

  • The gold standard for topoisomerase IV, as decatenation is its primary biological function

  • Use kinetoplast DNA (kDNA) as substrate - a network of interlocked DNA minicircles

  • Optimization: Adjust enzyme concentration, reaction time, and ATP/Mg²⁺ concentrations

  • Readout: Conversion of kDNA networks to individual minicircles

  • Advantages: Directly measures the physiologically relevant activity of topoisomerase IV

• DNA relaxation assay:

  • Measures conversion of negatively supercoiled plasmid DNA to relaxed topoisomers

  • Optimization: Use chloroquine-containing gels to distinguish between different topoisomers

  • Readout: Progressive conversion of supercoiled plasmid to a ladder of relaxed topoisomers

  • Advantages: Technically simpler than decatenation assays; can detect partial activity

• DNA unknotting assay:

  • Uses site-specific recombination systems (e.g., bacteriophage λ Int system) to generate defined DNA knots

  • Measures the removal of these recombinant knots, which topoisomerase IV excels at resolving

  • Readout: Conversion of knotted DNA to unknotted forms, separated by gel electrophoresis

  • Advantages: High specificity for topoisomerase IV over other topoisomerases

• In vivo assays:

  • Use temperature-sensitive topoisomerase IV mutants complemented with recombinant ParC constructs

  • Measure rescue of growth defects, cell morphology, and plasmid supercoiling state

  • Readout: Growth rate, cell morphology by microscopy, plasmid supercoiling by chloroquine gel electrophoresis

How can CRISPR-Cas9 technologies be effectively employed to study topoisomerase IV function in Synechocystis sp.?

CRISPR-Cas9 technologies offer powerful approaches to study topoisomerase IV function in Synechocystis sp.:

• CRISPRi-based knockdown:

  • CRISPRi (CRISPR interference) has been successfully employed to knock down gyrase subunits in Synechocystis sp. PCC 6803

  • Design: Use catalytically inactive Cas9 (dCas9) with guide RNAs targeting parC

  • Advantage: Allows tunable, partial repression rather than complete knockout, which may be lethal

  • Implementation: Place dCas9 under an inducible promoter for temporal control

• Base editing and prime editing:

  • Design: Use CRISPR base editors or prime editors to introduce specific point mutations in parC

  • Target: Create mutations analogous to known quinolone-resistance mutations to study structure-function relationships

  • Advantage: Creates precise changes without double-strand breaks or homology-directed repair

• Endogenous tagging:

  • Design: Use CRISPR-Cas9 with homology-directed repair to add fluorescent or affinity tags to the endogenous parC gene

  • Applications: Monitor ParC localization during cell cycle or purify native protein complexes

  • Optimization: Consider linker design and tag position to maintain protein function

• Multiplexed CRISPR approaches:

  • Simultaneous manipulation of multiple genes in topoisomerase pathways

  • Example: Combined knockdown of parC and overexpression of topA to study interactions between different topoisomerases

  • Design: Use orthogonal CRISPR systems or multiple guide RNAs

  • Analysis: Employ systems biology approaches to understand network effects

How should researchers analyze and interpret changes in DNA supercoiling in Synechocystis sp. when topoisomerase IV function is altered?

Analyzing DNA supercoiling changes requires sophisticated approaches to capture both global and local effects:

• Gel-based supercoiling analysis:

  • Two-dimensional gel electrophoresis with chloroquine in the second dimension effectively separates topoisomers

  • Quantification method: Calculate the distribution of topoisomers and determine the superhelical density (σ)

  • Interpretation: When topoisomerase IV is inhibited, DNA typically becomes more negatively supercoiled, while inhibition of gyrase leads to DNA relaxation

• Plasmid reporter systems:

  • Use small reporter plasmids to monitor supercoiling changes

  • Analysis method: Extract plasmid DNA from cells with altered topoisomerase IV function and analyze supercoiling state

  • Interpretation: In Synechocystis, small endogenous plasmids may show transient relaxation followed by increased supercoiling when topoisomerase balance is disrupted

• RNA-seq analysis for supercoiling-sensitive gene expression:

  • Global transcriptome analysis following topoisomerase IV alteration

  • Analysis approach: Identify genes with expression patterns that correlate with supercoiling changes

  • Key patterns: Look for 5'/3' gradients along transcription units, which are indicative of supercoiling effects on transcription elongation

  • Data interpretation: Group genes based on similar expression responses - in Synechocystis, genes often group into categories that overlap with diurnally co-expressed groups

• Promoter structure analysis:

  • Examine how supercoiling affects different promoter architectures

  • Analysis method: Correlate promoter sequence features (e.g., A-tract patterns, G+C content) with expression changes

  • Interpretation: A+T-rich and G+C-rich genes often respond differently to supercoiling changes

What experimental controls are essential when studying the effects of recombinant ParC on DNA topology in Synechocystis sp.?

Robust experimental design for studying recombinant ParC requires several essential controls:

• Enzyme activity controls:

  • Positive control: Commercial E. coli topoisomerase IV to validate assay conditions

  • Negative control: Reaction without enzyme or with heat-inactivated enzyme

  • Inhibitor control: Reaction with topoisomerase IV inhibitor (e.g., norfloxacin)

  • ATP dependence: Reactions with and without ATP to confirm type II topoisomerase activity

• Recombinant protein controls:

  • Wild-type ParC: For comparison with mutant or partial constructs

  • Catalytic site mutant: To demonstrate specificity of observed effects

  • ParE co-expression: To assess activity of complete topoisomerase IV complex

  • ParE-ParC fusion: As alternative functional form of the enzyme

• In vivo complementation controls:

  • Empty vector control: When testing complementation in mutant strains

  • Wild-type strain: Baseline for normal phenotype and DNA topology

  • Temperature-sensitive controls: When using conditional mutants

  • Drug-resistant ParC: To distinguish direct from indirect effects when using topoisomerase inhibitors

• DNA topology measurement controls:

  • Supercoiled and relaxed plasmid standards: For gel-based assays

  • Time-course sampling: To capture both immediate and adaptive responses

  • Different DNA substrates: Plasmids of various sizes and catenated DNA

  • Normalization controls: To account for variations in DNA extraction efficiency

• Gene expression analysis controls:

  • Constitutively expressed genes: To normalize expression data

  • Genes known to be supercoiling-sensitive: As positive controls for supercoiling effects

  • Genes known to be supercoiling-insensitive: As negative controls

  • Time-matched wild-type samples: To account for growth phase differences

These controls ensure that observed effects are specifically attributable to recombinant ParC activity rather than experimental artifacts or indirect effects.