Recombinant Bacillus subtilis DNA topoisomerase 4 subunit B (parE), partial

Basic Research Questions

• What is the structure and function of DNA topoisomerase IV in Bacillus subtilis?

Bacillus subtilis DNA topoisomerase IV is a type II topoisomerase composed of two subunits: ParC (subunit A) and ParE (subunit B). When purified together, these subunits appear as two predominant bands on SDS-PAGE with molecular weights of approximately 93 kDa (ParC) and 75 kDa (ParE), as predicted by their respective sequences .

Functionally, topoisomerase IV acts by creating a transient double-stranded break in one DNA segment (G-segment) and passing another intact segment (T-segment) through the break . During this process, the enzyme covalently attaches to the 5'-terminus of each DNA strand, forming a "cleavage complex" that is essential for maintaining genome integrity .

Topoisomerase IV's primary activities include:

• Relaxing both positive and negative supercoils

• Removing DNA knots and tangles (decatenation)

• Critical role in chromosome segregation after DNA replication

Unlike DNA gyrase, topoisomerase IV cannot introduce negative supercoils into DNA, as it uses a "canonical" strand passage mechanism that captures existing DNA crossovers .

• How does topoisomerase IV differ from other topoisomerases in B. subtilis?

B. subtilis possesses multiple topoisomerases with distinct functions:

The key difference is that gyrase decreases DNA linking number below that of relaxed DNA (creating negative supercoils), while topoisomerase IV primarily relaxes supercoils and untangles DNA . Research has shown that gyrase removes positive supercoils more rapidly and processively than topoisomerase IV, while maintaining lower levels of cleavage complexes with positively supercoiled DNA, making it the safer enzyme to work ahead of replication forks .

• What experimental evidence demonstrates the essentiality of parE in B. subtilis?

The essentiality of parE in B. subtilis has been conclusively demonstrated through several experimental approaches:

• Gene disruption studies have shown that disruption of either parE or parC results in failure of nucleoid segregation .

• Conditional inactivation of parE leads to a strong decrease in cell viability and yields a subpopulation of elongated cells containing large, asymmetrically located nucleoids .

• Immunofluorescence microscopy and green fluorescence protein fusion studies have revealed that ParC is localized at the poles of bacteria in rapidly growing cultures, and this localization requires functional ParE, suggesting that topoisomerase IV activity is crucial for proper cellular function .

The essentiality of these genes is related to their role in chromosome segregation. Without functional ParE, cells cannot properly decatenate daughter chromosomes following DNA replication, leading to segregation defects and eventual cell death .

• What is the subcellular localization of ParE and ParC in B. subtilis cells?

Research using immunofluorescence microscopy and direct visualization with green fluorescence protein fusions has revealed distinct localization patterns for the topoisomerase IV subunits:

• ParC is localized at the poles of the bacteria in rapidly growing cultures, showing a bipolar distribution pattern .

• ParE is distributed uniformly throughout the cell, without specific localization .

• The bipolar localization of ParC requires functional ParE, suggesting that topoisomerase IV activity is necessary for proper localization .

This subcellular distribution differs from DNA gyrase, where both GyrA and GyrB subunits are associated with the nucleoid . These distinct localization patterns provide physiological evidence for the different roles of these enzymes, with topoisomerase IV potentially being part of the bacterial segregation machinery positioned at cell poles .

Advanced Research Questions

• What methods are most effective for expressing and purifying recombinant B. subtilis ParE?

Based on published research, the following protocol has proven effective for expressing and purifying recombinant B. subtilis ParE:

• Cloning Strategy:

  • Clone the parE gene from B. subtilis with a hexahistidine (his6) tag

  • Express the recombinant protein in Escherichia coli expression systems

• Expression Conditions:

  • Use E. coli strains optimized for protein expression (BL21(DE3) or similar)

  • Induce expression with IPTG at optimal concentration (typically 0.5-1.0 mM)

  • Grow at lower temperatures (25-30°C) after induction to enhance solubility

• Purification Strategy:

  • Lyse cells using sonication or French press in buffer containing protease inhibitors

  • Perform metal affinity chromatography using Ni-NTA resin to capture His-tagged ParE

  • Include a second purification step such as ion exchange or gel filtration chromatography

  • For topoisomerase IV complex formation, co-purify ParE with ParC

When ParE and ParC subunits are purified together, they result in two predominant bands at approximately 93 kDa (ParC) and 75 kDa (ParE) on SDS-PAGE . The purity and activity of the recombinant protein should be assessed using standard quality control methods including SDS-PAGE and functional assays.

• How can researchers assess the functional activity of recombinant ParE in vitro?

The functional activity of recombinant ParE can be assessed using the following assays:

• ATP-dependent Decatenation/Supercoiling Assay:

  • Combine purified ParE with its partner subunit ParC to form active topoisomerase IV

  • Use kinetoplast DNA (kDNA) as a substrate, which consists of interlocked DNA circles

  • Incubate enzyme with kDNA in the presence of ATP and appropriate buffer conditions

  • Analyze reaction products by agarose gel electrophoresis to observe decatenation

  • Expected result: conversion of kDNA network to individual minicircles

• DNA Relaxation Assay:

  • Use supercoiled plasmid DNA (e.g., pBR322) as a substrate

  • Standard conditions: 10 nM topoisomerase IV, 5 nM supercoiled DNA, 1 mM ATP in appropriate buffer (e.g., 40 mM HEPES pH 7.6, 100 mM KGlu, 10 mM Mg(OAc)₂, 50 mM NaCl)

  • Analyze reaction products by agarose gel electrophoresis to observe conversion of supercoiled DNA to relaxed forms

• Cleavage Complex Formation Assay:

  • Incubate enzyme with DNA in the presence of quinolone antibiotics

  • Add SDS to trap cleavage complexes

  • Analyze by agarose gel electrophoresis after proteinase K treatment

  • Compare cleavage complex formation with positively versus negatively supercoiled DNA

The functional complementation between subunits can also be tested using these assays to verify that recombinant ParE can form an active enzyme with its partner ParC .

• What is the relationship between topoisomerase IV and other DNA repair/maintenance systems in B. subtilis?

Topoisomerase IV functions within a complex network of DNA repair and maintenance systems in B. subtilis:

• Interaction with SOS Response:

  • B. subtilis has an SOS system regulated by RecA and LexA (DinR) similar to E. coli, but with different gene composition

  • While topoisomerase IV is not directly part of the SOS regulon, its function in chromosome segregation is essential for proper DNA repair

• Relationship with Site-Specific Recombination:

  • The RipX protein (similar to XerC/XerD in E. coli) is involved in resolving chromosome dimers

  • Topoisomerase IV (ParC/ParE) removes catenation nodes from replicated chromosomes, while RipX resolves chromosome dimers that result from an odd number of crossovers

  • These systems work in concert for proper chromosome partitioning

• Chromosome Segregation and DNA Repair Interplay:

  • ParA regulates DNA replication initiation by interacting with DnaA

  • ParB can inhibit ParA dimerization, affecting chromosome partitioning

  • In some bacteria like Streptomyces coelicolor, topoisomerase I (TopA) is recruited to ParB complexes, and its activity is required to resolve segregating chromosomes

• Functional Redundancy with Type I Topoisomerases:

  • Overexpression of topoisomerase IV can compensate for the loss of topoisomerase I (TopA) in B. subtilis

  • A strain with increased parEC expression can survive without both type I topoisomerases (TopA and TopB)

  • This suggests functional overlap between type I and type II topoisomerases in managing DNA topology

This network of interactions highlights the complex interplay between topoisomerase IV and other DNA maintenance systems in ensuring genomic stability.

• How do mutations in parE affect chromosome segregation and cell viability in B. subtilis?

Mutations in parE have profound effects on chromosome segregation and cell viability in B. subtilis:

• Segregation Defects:

  • Disruption of parE results in failure of nucleoid segregation

  • Cells show characteristic elongation and contain large, asymmetrically located nucleoids

  • Anucleate cells are frequently observed in parE mutants due to improper chromosome partitioning

• Cell Division Abnormalities:

  • Analysis of germinated, outgrowing spores shows that placement of FtsZ rings and septa is affected in parE mutant strains by the first division after germination

  • This indicates that proper chromosome segregation by topoisomerase IV is necessary for normal cell division

• Experimental Evidence from Point Mutations:

  • A study on experimental evolution of B. subtilis identified a mutation in parC that was associated with increased fitness at low pressure (5 kPa)

  • This suggests that topoisomerase IV function can be adaptively modified to suit different environmental conditions

• Genetic Interactions:

  • In some cases, defects caused by parE mutations cannot be suppressed by inactivating recombination (recA mutation), unlike in E. coli where xerC/xerD chromosome partitioning defects are suppressed by recA mutations

  • This indicates potentially different mechanisms or additional roles for topoisomerase IV in B. subtilis compared to E. coli

These observations highlight the essential nature of ParE and its role in chromosome segregation, with mutations leading to severe defects in cell division and viability.

Methodological Questions

• What genetic approaches can be used to study parE function in B. subtilis?

Several genetic approaches have been developed to study parE function in B. subtilis:

• Traditional Gene Disruption:

  • PCR-based methods using antibiotic resistance cassettes

  • Requires ~500 bp of homology flanking the target region for efficient recombination

  • Cannot be directly applied to essential genes like parE without conditional systems

• Conditional Expression Systems:

  • IPTG-inducible or xylose-inducible promoters to control parE expression

  • Allow depletion studies by growing cells in the absence of inducer

  • Useful for studying the effects of ParE depletion on cell physiology

• CRISPR-Cas9 Gene Editing:

  • Allows for precise genome modifications including point mutations

  • Protocol includes:
    a. Designing proto-spacers adjacent to PAM sequences (NGG) near the target site
    b. Creating a plasmid with the proto-spacer for CRISPR/Cas9 alteration
    c. Constructing an editing plasmid with the desired mutation
    d. Transformation and screening of transformants

• CRISPRi for Partial Knockdown:

  • Single guide RNA (sgRNA) targeting causes RNA polymerase stalling

  • Allows titration of parE expression levels without complete elimination

  • Useful for studying essential genes like parE

• Gene Amplification Reporters:

  • Systems such as a GFP-ermC cassette inserted downstream of parEC can detect genomic amplifications

  • Used to study compensatory mechanisms, as seen when topoisomerase I (topA) is deleted

These approaches provide researchers with tools to study parE function through various genetic manipulations, from complete knockout (when combined with suppressors) to subtle alterations in expression or protein structure.

• What are the optimal buffer conditions and assay parameters for measuring ParE activity in vitro?

Based on published research, the following buffer conditions and assay parameters are optimal for measuring B. subtilis ParE activity in vitro:

Standard Reaction Buffer Components:

• 40 mM HEPES (pH 7.6)

• 100 mM potassium glutamate (KGlu)

• 10 mM magnesium acetate (Mg(OAc)₂)

• 50 mM NaCl

• 1 mM ATP (required for activity)

DNA Relaxation Assay Parameters:

• Enzyme concentration: 10 nM topoisomerase IV (ParC + ParE complex)

• Substrate: 5 nM supercoiled plasmid DNA (e.g., pBR322)

• Reaction volume: 20 μl

• Temperature: 37°C

• Incubation time: 30 minutes (for endpoint assays) or various time points (for kinetic studies)

Decatenation Assay Parameters:

• Enzyme concentration: 5-20 nM topoisomerase IV

• Substrate: 200-400 ng kDNA

• ATP: 1 mM

• Temperature: 37°C

• Incubation time: 30-60 minutes

Controls and Analysis:

• Positive control: Commercial E. coli topoisomerase IV

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

• Analysis method: Agarose gel electrophoresis (0.8-1.0% gels)

• Detection: Ethidium bromide staining or SYBR Green

For inhibition studies with fluoroquinolones, the concentration range typically used is 0.1-100 μM, with IC₅₀ values determined from dose-response curves .

These conditions have been shown to produce reliable and reproducible results in published studies examining B. subtilis topoisomerase IV activity.

• How can fluorescent protein fusions be designed to study ParE localization without affecting function?

Designing effective fluorescent protein fusions to study ParE localization requires careful consideration of several factors:

• Fusion Orientation and Linker Design:

  • N-terminal fusions are generally preferred for ParE, as C-terminal modifications may interfere with interactions with ParC

  • Include a flexible linker sequence (e.g., GGGGS repeats) between ParE and the fluorescent protein to minimize structural interference

  • Linker length of 5-15 amino acids is typically optimal to provide sufficient separation

• Choice of Fluorescent Protein:

  • monomeric versions of fluorescent proteins (e.g., mGFP, mYFP) prevent artificial aggregation

  • Superfolder GFP (sfGFP) may be preferred due to its rapid folding and stability

  • Avoid using large fluorescent proteins that might interfere with ParE function

• Expression Level Control:

  • Use native promoter expression when possible to maintain physiological levels

  • For higher expression, inducible promoters with tight regulation (e.g., Pspac) can be used

  • Avoid overexpression, which can lead to artifactual localization patterns

• Integration Strategy:

  • Chromosomal integration at the native locus maintains proper regulation

  • Integration can be performed at ectopic loci (amyE or lacA) if native locus integration disrupts function

  • Confirm integration using PCR with primers:

    • For amyE: 5′-TTCTTCGCTTGGCTGAAAAT-3′ (forward), 5′-CACCAGGTTTTTGGTTTGCT-3′ (reverse)

    • For lacA: 5′-TAGACGAAAGCGCCAAGATT-3′ (forward), 5′-CTGGCGTTTTCCGTTTGTAT-3′ (reverse)

• Functional Validation:

  • Verify that the fusion protein complements a ParE depletion strain

  • Compare growth rates and cell morphology between wild-type and fusion-expressing strains

  • Perform in vitro activity assays with purified fusion protein to confirm enzymatic function

Successful examples from the literature include GFP-ParC fusions that have revealed bipolar localization in B. subtilis cells while maintaining functionality .

• What approaches can be used to study the interplay between topoisomerase IV and other DNA maintenance systems?

Several approaches can be used to investigate the interplay between topoisomerase IV and other DNA maintenance systems:

• Genetic Interaction Analysis:

  • Construct double or triple mutants combining parE/parC mutations with mutations in other DNA repair pathways

  • Synthetic lethality or synthetic sickness indicates functional relationships

  • Example: Combining topA deletions with parEC amplifications revealed that topoisomerase IV can functionally replace type 1A topoisomerases

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify physical interactions between ParE/ParC and other proteins

  • Bacterial two-hybrid assays to screen for interaction partners

  • Tandem affinity purification (TAP) tag approaches to isolate protein complexes

  • Cross-linking mass spectrometry to capture transient interactions

• Fluorescence Microscopy Colocalization:

  • Dual-color fluorescence microscopy using different fluorescent protein fusions

  • Quantify colocalization between ParE/ParC and other DNA maintenance proteins

  • Example: Studies in other bacteria have shown recruitment of TopA to ParB complexes

• ChIP-Seq Analysis:

  • Chromatin immunoprecipitation followed by sequencing to map ParE/ParC binding sites

  • Compare with binding profiles of other DNA maintenance proteins

  • Identify genomic regions where multiple systems converge

• Conditional Depletion Systems:

  • Deplete ParE/ParC and examine effects on other DNA maintenance pathways

  • Use CRISPRi to simultaneously knock down multiple components

  • Measure DNA damage responses during depletion

• Specific Activity Assays:

  • Measure topoisomerase IV activity in extracts from strains with mutations in various DNA repair pathways

  • Study whether purified DNA repair proteins affect topoisomerase IV activity in vitro