Recombinant Bacteroides fragilis Transposase for insertion sequence element IS21-like (tnpA), partial

What is the Bacteroides fragilis IS21-like transposase and what is its genomic context?

The Bacteroides fragilis IS21-like transposase (tnpA) is an enzyme that catalyzes the movement of DNA segments within the bacterial genome. It is specifically associated with the IS21 insertion sequence element found at the termini of conjugative transposons (CTns) in B. fragilis. This transposase is part of a newly described family of CTns that differ significantly from previously characterized transposons in Bacteroides species. The IS21 transposases are located at both ends of CTn9343, a 64,229 bp genetic element containing 61 potential open reading frames identified in B. fragilis strain NCTC 9343 . The IS21-like transposase shows 100% sequence identity across its 548 amino acid length in B. fragilis strains, indicating strong conservation of this element . This high conservation suggests its essential functional role in the biology of these mobile genetic elements.

How does the IS21-like transposase differ from other transposases in Bacteroides species?

The IS21-like transposase in B. fragilis is part of a distinct family of conjugative transposons that show several key differences from previously described CTns in Bacteroides species. Unlike other CTns such as CTnDOT, these new elements (CTn86 and CTn9343) do not carry the tetQ gene, which is commonly found in other Bacteroides transposons . Additionally, the excision from the chromosome to form a circular intermediate is not regulated by tetracycline, which is a distinctive regulatory characteristic of many other Bacteroides CTns . The IS21-like transposases are predicted to employ a different mechanism of transposition compared to other known CTns, and their sequences have very limited similarity with elements like CTnDOT . These unique features suggest that the IS21-like transposase is part of a novel mobile genetic system within the Bacteroides genus.

What is the relationship between the IS21-like transposase and pathogenicity in B. fragilis?

The IS21-like transposase plays a significant role in the pathogenicity of B. fragilis through its association with the Bacteroides fragilis pathogenicity island (BfPAI). In enterotoxigenic B. fragilis (ETBF) strain 86-5443-2-2, the BfPAI is integrated within the conjugative transposon CTn86 between the mob region and the bfmC gene . This genetic arrangement suggests that the IS21-like transposase may facilitate the mobilization and horizontal transfer of virulence factors, including the bft gene encoding the B. fragilis toxin. Research indicates that the presence of these mobilizable elements could enable the transfer of virulence factors from enterotoxigenic strains to non-toxigenic B. fragilis strains, thereby expanding the population of potentially pathogenic bacteria . Additionally, the increased expression of genes associated with these mobile elements has been linked to the success of B. fragilis during inflammatory conditions, suggesting a potential role in adaptation to host inflammatory responses .

What methods are commonly used to express and purify recombinant B. fragilis IS21-like transposase?

Recombinant B. fragilis IS21-like transposase can be expressed using several host systems, with E. coli being the most commonly employed for initial characterization studies. The protein is typically expressed with a purification tag (such as His-tag or GST) to facilitate isolation. For expression in E. coli, the tnpA gene is first amplified from B. fragilis genomic DNA using PCR with primers designed based on the known sequence of the IS21-like element . The amplified gene is then cloned into an appropriate expression vector containing a strong promoter (such as T7) and transformed into an E. coli expression strain.

For protein purification, standard chromatographic techniques are employed following cell lysis. These typically include:

• Affinity chromatography (using Ni-NTA for His-tagged proteins)

• Ion exchange chromatography

• Size exclusion chromatography for final polishing

The purified recombinant protein typically achieves greater than 85% purity as determined by SDS-PAGE analysis . For functional studies, in vitro activity assays using labeled DNA substrates are performed to assess the transposition activity of the purified enzyme. Alternative expression systems including yeast, baculovirus, or mammalian cells can be employed when proper folding or post-translational modifications are concerns .

How do the linear and circular forms of IS21-containing transposons differ functionally in B. fragilis?

Recent research has revealed that IS21-containing transposons in B. fragilis can exist simultaneously in two distinct conformations within the same genome: linear chromosomally-integrated forms and circular intermediates . The linear form contains a unique genomic insert in the region encoding mobilization machinery, while the circular form represents an excised intermediate that may be capable of transfer or reintegration . This dual-form existence has significant functional implications:

This novel finding regarding the dual existence of these elements has significant implications for understanding horizontal gene transfer mechanisms. The relationship between these two forms appears to be dynamic and potentially responsive to environmental conditions, particularly inflammatory states . The cellular controls governing the balance between these forms and the molecular triggers that may shift this equilibrium represent important areas for future research, with implications for both pathogenicity and antibiotic resistance spread.

What are the optimal conditions for studying IS21-like transposase activity in vitro and in vivo?

Studying IS21-like transposase activity requires carefully optimized conditions for both in vitro biochemical analysis and in vivo functional characterization.

In vitro studies:
For biochemical characterization, purified recombinant IS21-like transposase requires specific buffer conditions:

• pH range: 7.0-8.0 (typically HEPES or Tris-based buffers)

• Divalent cations: Mg²⁺ or Mn²⁺ (usually 5-10 mM)

• Reducing agents: DTT or β-mercaptoethanol (1-5 mM)

• DNA substrates: Supercoiled plasmids containing the specific target sequences

• Temperature: 30-37°C

In vivo studies in B. fragilis:
For genetic manipulation and functional studies in B. fragilis, electroporation conditions must be optimized:

• Growth phase: For simple plasmid transformation, cells harvested at 48 hours yield highest numbers of transformants; for homologous recombination, early exponential phase (OD₆₆₀ ~0.2) is optimal

• Electric field strength: Transformation efficiency increases linearly with field strength from 5.0 to 12.5 kV/cm

• Post-pulse incubation: At least 3 hours is required for maximum transformation efficiency for replicative plasmids; 12 hours for suicide vector integration

• Selectable markers: Cefoxitin resistance (Cfx^r) provides significantly higher transformation efficiency compared to erythromycin or chloramphenicol resistance markers

For studying transposition events in vivo, induction conditions that might trigger enhanced transposon mobility should be considered, including inflammatory mediators or stress conditions, as these have been associated with increased expression of genes within these mobile elements .

What techniques can be employed to track horizontal transfer of IS21-containing genetic elements between bacterial populations?

Tracking horizontal transfer of IS21-containing genetic elements between bacterial populations requires sophisticated experimental approaches. The following methodologies can be employed:

• Marker-based detection systems:

  • Engineering selectable markers (antibiotic resistance genes) into the mobile element

  • Using fluorescent protein genes as visual reporters of transfer events

  • Employing counter-selectable markers to detect rare transfer events

• PCR-based detection methods:

  • Quantitative PCR to measure copy number variations of specific transposon regions

  • Long-range PCR to detect specific integration events and junctions

  • Digital PCR for absolute quantification of transfer events in mixed populations

• Advanced sequencing approaches:

  • Whole genome sequencing to identify integration sites and structural variations

  • Oxford Nanopore long-read sequencing to resolve complex transposon structures

  • Single-cell sequencing to detect heterogeneity in bacterial populations

• Functional genomics approaches:

  • RNA-seq to measure expression of transposon genes under different conditions

  • ChIP-seq to study protein-DNA interactions involved in transposition

  • Tn-seq to assess the impact of transposon insertions on bacterial fitness

• Imaging techniques:

  • Fluorescence in situ hybridization (FISH) to visualize specific DNA elements

  • Live-cell imaging using fluorescent reporters to track transfer events in real-time

For B. fragilis specifically, colony blot hybridization, PCR, and sequence analysis have been successfully used to determine structural features of IS21-containing transposons and to detect their presence across different strains . The ability to efficiently introduce foreign DNA through electroporation using optimized parameters (as described in 2.2) enables genetic manipulation for tracking purposes .

How does the molecular mechanism of IS21-like transposase differ from that of other transposition systems?

The IS21-like transposase in B. fragilis employs a distinct molecular mechanism compared to other transposition systems. While detailed biochemical characterization is still emerging, comparative analysis with related systems reveals several key differences:

The IS21-like transposase in B. fragilis shares some characteristics with the IS21 family of insertion sequences found in other bacteria, but the complete transposition unit in B. fragilis has evolved distinct properties related to its role in larger conjugative transposons . Unlike many transposition systems that require specific environmental triggers (like antibiotics), the IS21-containing elements in B. fragilis appear to have a more complex regulatory system that may respond to inflammatory conditions or other host factors . The structural organization of the transposase within the larger conjugative element represents an evolutionary innovation that facilitates the dissemination of pathogenicity islands and potentially antibiotic resistance genes .

What are the critical factors for successful genetic manipulation of B. fragilis using IS21-like transposase systems?

Successful genetic manipulation of B. fragilis using IS21-like transposase systems requires careful consideration of several critical factors:

• Plasmid selection and preparation:

  • The choice of shuttle vector significantly impacts transformation efficiency

  • Plasmids containing the cefoxitin resistance marker (Cfx^r) yield substantially higher transformation rates than those with erythromycin or chloramphenicol resistance genes

  • Plasmid pLYL05 has been shown to generate approximately 10^4 transformants per μg DNA, which is 2- to 900-fold more transformants than other tested plasmids

  • In vivo methylation of plasmid DNA in B. fragilis prior to extraction greatly improves integration efficiency for suicide vectors

• Cell preparation conditions:

  • For simple plasmid transformation, cells harvested from 48-hour cultures yield optimal results

  • For gene deletion via homologous recombination, early exponential phase cells (OD₆₆₀ ~0.2) are required

  • Competent cells can be prepared and stored frozen in 10% glycerol with no loss of transformation efficiency

• Electroporation parameters:

  • Electric field strength shows a linear relationship with transformation efficiency in the range of 5.0 to 12.5 kV/cm

  • Post-pulse incubation time is critical: at least 3 hours for replicative plasmids, but 12 hours for suicide vector integration

• Homologous recombination strategy:

  • For targeted gene disruption, suicide vectors containing ~2 kb homologous sequences flanking the target region are most effective

  • Two-step recombination processes (integration followed by resolution) require careful screening to identify desired recombinants

  • The efficiency of targeted integration is substantially improved when using plasmids prepared from B. fragilis rather than from E. coli (48.7% vs. 0% success rate in tested conditions)

These parameters have been experimentally validated for genetic manipulation in B. fragilis strains NCTC9343 and YCH46, and similar approaches can be applied to other Bacteroides species with appropriate optimization .

How can researchers design experiments to study the role of IS21-like transposases in virulence gene transfer?

To study the role of IS21-like transposases in virulence gene transfer, researchers should consider the following experimental design approaches:

• Genetic modification of transposase elements:

  • Create point mutations or deletions in the IS21-like transposase genes to generate non-functional variants

  • Engineer tagged versions of the transposase for protein localization and interaction studies

  • Develop inducible expression systems to control transposase activity temporally

• Tracking pathogenicity island transfer:

  • Label the Bacteroides fragilis pathogenicity island (BfPAI) with reporter genes

  • Create co-culture systems with donor (ETBF) and recipient (non-toxigenic) B. fragilis strains

  • Utilize selective media and molecular detection methods to identify transfer events

  • Quantify transfer rates under various conditions (inflammation, antibiotic stress, etc.)

• In vitro transposition assays:

  • Develop cell-free systems using purified recombinant IS21-like transposase

  • Use fluorescently labeled DNA substrates containing the BfPAI and flanking regions

  • Measure transposition activity through gel-shift assays and DNA footprinting

• Animal model experiments:

  • Establish gnotobiotic mouse models colonized with genetically marked strains

  • Induce inflammation through chemical agents (DSS, TNBS) or pathogen challenge

  • Monitor transfer of virulence factors between bacterial populations in vivo

  • Assess the impact of transposase activity on disease progression

• Structural and functional analysis:

  • Determine the crystal structure of the IS21-like transposase to identify functional domains

  • Perform mutagenesis of key residues to map the active site and DNA binding regions

  • Conduct protein-protein interaction studies to identify cofactors required for transposition

An experimental workflow might begin with the construction of reporter systems, followed by in vitro characterization of transposition activity, validation in laboratory co-culture systems, and ultimately testing in relevant animal models of intestinal inflammation. Throughout these experiments, researchers should employ appropriate controls, including transposase mutants and non-inflammatory conditions, to establish causality between transposase activity and virulence gene transfer .

What are the challenges in distinguishing between IS21-like transposase-mediated gene transfer and other horizontal gene transfer mechanisms?

Distinguishing between IS21-like transposase-mediated gene transfer and other horizontal gene transfer mechanisms presents several significant challenges:

• Overlapping molecular signatures:

  • Mobile genetic elements often share similar features, making it difficult to attribute transfer events to specific mechanisms

  • The presence of multiple transfer systems within the same bacterial strain complicates attribution

  • IS21-like elements may work in conjunction with other transfer mechanisms rather than independently

• Technical limitations in detection methods:

  • Standard PCR-based approaches may not distinguish between different transfer mechanisms

  • Short-read sequencing technologies struggle to resolve repetitive regions common in transposable elements

  • Culture-based methods may introduce selection bias that obscures natural transfer dynamics

• Temporal dynamics of transfer events:

  • Transfer events are often rare and stochastic, requiring sensitive detection methods

  • Different transfer mechanisms may dominate under different environmental conditions

  • Laboratory conditions may not accurately reproduce the complex environments where transfers naturally occur

• Experimental controls and validation challenges:

  • Creating truly isogenic strains differing only in specific transposase activity is technically demanding

  • Distinguishing cause from correlation requires careful genetic complementation studies

  • Confirming the functional role of IS21-like transposases requires both loss-of-function and gain-of-function approaches

To address these challenges, researchers should employ multiple complementary approaches:

• Use long-read sequencing technologies (PacBio, Oxford Nanopore) to resolve complex genomic arrangements

• Develop specific molecular markers that uniquely identify IS21-mediated events

• Design experiments with appropriate genetic controls (transposase mutants, catalytically inactive variants)

• Employ mathematical modeling to distinguish between different transfer mechanisms based on population dynamics

• Use advanced imaging techniques to visualize transfer events at the single-cell level

By combining these approaches, researchers can more confidently attribute gene transfer events to IS21-like transposase activity versus other mechanisms such as conjugation, natural transformation, or phage-mediated transduction that may operate simultaneously in bacterial populations .

What bioinformatic approaches can be used to identify and characterize novel IS21-like transposases in bacterial genomes?

Identifying and characterizing novel IS21-like transposases in bacterial genomes requires sophisticated bioinformatic approaches:

• Sequence-based identification strategies:

  • Profile Hidden Markov Models (HMMs) based on known IS21-like transposase sequences

  • Position-Specific Scoring Matrices (PSSMs) to detect conserved domains

  • BLAST-based searches using known IS21-like transposases as queries

  • Pattern recognition algorithms to identify terminal inverted repeats characteristic of IS elements

• Structural feature detection:

  • Identification of terminal inverted repeats (TIRs) flanking potential transposase genes

  • Detection of target site duplications (TSDs) that often result from transposition events

  • Recognition of conserved catalytic motifs (DDE/DDD domains) characteristic of transposases

  • Prediction of DNA-binding domains specific to IS21 family transposases

• Genomic context analysis:

  • Examination of flanking sequences to identify potential mobilizable regions

  • Detection of integration hotspots or preference patterns

  • Identification of associated genes commonly found in IS21-containing transposons

  • Comparative analysis of synteny disruptions indicating insertion events

• Evolutionary analysis tools:

  • Phylogenetic analysis to classify novel transposases within the IS21 family

  • Selection pressure analysis to identify functionally important residues

  • Horizontal gene transfer detection algorithms to identify recent acquisition events

  • Comparative genomics to track transposon dissemination across bacterial lineages

• Functional annotation approaches:

  • Protein domain prediction to identify catalytic and regulatory regions

  • Structural modeling based on homology to characterized transposases

  • Gene expression correlation analysis to identify co-regulated gene clusters

  • Network analysis to identify functional associations with other cellular systems

An integrated bioinformatic workflow might begin with genome-wide scanning for IS21-like sequences, followed by structural feature validation, genomic context analysis, evolutionary classification, and finally functional annotation. This approach has successfully identified novel IS21-like transposases in various bacterial genomes, including the characterization of CTn86 and CTn9343 in B. fragilis, which contain IS21 transposases at their termini .