IS200/IS605 Family-Associated TnpB Increases Transposon Activity and Retention
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eLife assessment
This important study uses an innovative set of reporter assays to probe the role of the TnpB protein in IS608 transposition. The work provides independent support for the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase. The overall approach is solid, but the authors should consider how the activity of the TnpB protein used, or the levels of ωRNA, impact their model.
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Abstract
The IS200/IS605 family of insertion sequences are abundant mobile elements associated with one of the most numerous genes found in nature, tnpB 1–3 . Previous studies suggest that TnpB protein may be an evolutionary precursor to CRISPR Cas enzymes, and TnpB has received renewed interest having itself been shown to function as a Cas-like RNA-guided DNA endonuclease 3,4 . However, interpretation of the fundamental role of TnpB in transposition and how it contributes to genome dynamics 5 remains controversial without direct, real-time measurement in live cells. Here, using a suite of fluorescent reporters coupled to transposition in live Escherichia coli , we show that IS608-TnpB causes increased transposon activity, and assists in preventing transposon loss from host genomes. Analyzing our results through a mathematical model of transposon dynamics, we discuss the multifaceted roles it may play in transposon regulation. The mutually beneficial transposon-TnpB interaction may explain the prevalence of tnpB , creating conditions for the appropriation of TnpB’s RNA-guided endonuclease activity for adaptive immunity.Phylogenetic evidence suggests that tnpB , one of the most numerous genes found in nature, is the ancestral form of CRISPR-Cas enzymes and played a critical role in the evolution of adaptive immunity. However, the role TnpB plays in transposition that has contributed to its wide distribution remains unclear. Here, we use a unique approach that couples fluorescent reporters to transposition to non-perturbatively quantify transpositional dynamics in live cells. In contrast to previous indirect methods suggesting that TnpB suppresses transposition, our results instead clearly demonstrate that TnpB significantly increases transposition rates and enhances transposon retention within the host genome, resulting in a mutually beneficial interaction between transposons and TnpB that can account for its wide distribution.
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eLife assessment
This important study uses an innovative set of reporter assays to probe the role of the TnpB protein in IS608 transposition. The work provides independent support for the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase. The overall approach is solid, but the authors should consider how the activity of the TnpB protein used, or the levels of ωRNA, impact their model.
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Reviewer #1 (Public Review):
Summary:
The nuclease protein TnpB is ubiquitous across microorganisms. It is associated with the stabilization of transposable genetic elements and is a putative precursor of the RNA-guided endonuclease Cas9 from the CRISPR system. Despite its potential as a gene-editing tool, TnpB is not well understood. In this study, the authors use a fluorescence-based construct to individually quantify the transposable-element excision rate and the abundance of the two ancillary proteins TnpA and TnpB. They develop a mathematical model describing the role of TnpB in transposable-element stabilization.Strengths:
The methodology (with schematic shown in Figure 1A) is powerful and sophisticated. The authors are able to de-convolve excision events from the individual abundances of TnpA and TnpB.Weaknesses:
The claim that …Reviewer #1 (Public Review):
Summary:
The nuclease protein TnpB is ubiquitous across microorganisms. It is associated with the stabilization of transposable genetic elements and is a putative precursor of the RNA-guided endonuclease Cas9 from the CRISPR system. Despite its potential as a gene-editing tool, TnpB is not well understood. In this study, the authors use a fluorescence-based construct to individually quantify the transposable-element excision rate and the abundance of the two ancillary proteins TnpA and TnpB. They develop a mathematical model describing the role of TnpB in transposable-element stabilization.Strengths:
The methodology (with schematic shown in Figure 1A) is powerful and sophisticated. The authors are able to de-convolve excision events from the individual abundances of TnpA and TnpB.Weaknesses:
The claim that TnpB expression level correlates positively (and significantly) with the probability of a growth-disrupting integration (called 'b') is not well-supported by the data shown in Fig. 3D. The modelling of results shown in Figure 4 are not tied directly to the experimental data shown in Figures 1-3. -
Reviewer #2 (Public Review):
Summary:
Dr. Khulman and colleagues present a very interesting experimental and mathematical modeling work on IS608 transposition. The system has a number of unique advantages that create experimental possibilities utilized here to investigate transposition dynamics. This kind of approach is badly missing from the field and I believe the experiments and modeling work shown here and the type of results that can be derived are groundbreaking and certainly deserve high visibility.Strengths:
The attempt to measure and model transposition dynamics in cells.Weaknesses:
- Lack of controls using an active site mutant of TnpB.
- Lack of control in RecA- cells as transposon restoration is proposed to be dependent on homologous recombination.
- Lack of consideration of the levels of ωRNA present. -
Reviewer #3 (Public Review):
The authors use a set of reporter assays to probe the impact of TnpB on IS605 transposition. This is an innovative approach to studying transposition that will be of interest to other groups. The authors conclude that TnpB likely has two activities: (ii) promoting "homing", where the transposon is restored following excision, and (ii) promoting the transposition activity of the transposase TnpA. The paper provides an independent validation of the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase.
Strengths
- The innovative use of reporter assays is an excellent way to infer the details of transposition, making a convincing case that TnpB plays two distinct …Reviewer #3 (Public Review):
The authors use a set of reporter assays to probe the impact of TnpB on IS605 transposition. This is an innovative approach to studying transposition that will be of interest to other groups. The authors conclude that TnpB likely has two activities: (ii) promoting "homing", where the transposon is restored following excision, and (ii) promoting the transposition activity of the transposase TnpA. The paper provides an independent validation of the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase.
Strengths
- The innovative use of reporter assays is an excellent way to infer the details of transposition, making a convincing case that TnpB plays two distinct roles.Weaknesses
- The authors need to discuss their conclusions in light of a very relevant recent paper from the Sternberg group demonstrating a role for TnpB in homing.
- There doesn't appear to be an assessment of how well the model fits the experimental data, or any attempt to test how accurately the model can predict the effects of perturbing the system.
- The figures could be presented more clearly. -
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