Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity

Summary

A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells.

Abstract

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.

Introduction

Transposable elements (TEs) are genetic elements that mobilize within their host genomes by excision or catalyze copying followed by genomic reintegration. TEs exist in all domains of life, and transposition restructures the host genome, mutating coding and control regions¹. This generates mutations and diversity that play an important role in evolution^2,3, development^4,5, and several human diseases⁶, including cancer⁷.

Using novel genetic constructs that couple aspects of transpositional activity to fluorescent reporters, our previous work described the development of an experimental system based on the bacterial TE IS608, a representative of the widespread IS200/IS605 family of TEs, that allows for the real-time visualization of transposition in individual live cells⁸ (Figure 1). The TE system is displayed in Figure 1A. The TE comprises the transposase coding sequence, tnpA, flanked by Left End (LE) and Right End (RE) imperfect palindromic repeats (IPs), which are the recognition and excision sites for TnpA. tnpA is expressed using the promoter P_LTetO1, which is repressed by the tet repressor and is inducible with anhydrotetracycline (aTc)⁹. The TE splits the -10 and -35 sequences of a constitutive P_lacIQ1 promoter¹⁰ for the blue reporter mCerulean3¹¹. As shown in Figure 1C, when the production of tnpA is induced, the TE can be excised, leading to promoter reconstitution. The produced cell expresses mCerulean3 and fluoresces blue. The N-terminus of TnpA is fused to the yellow reporter Venus¹², allowing measurement of the TnpA levels by yellow fluorescence.

IS608 and other members of the IS200/IS605 family of transposons also typically encode a second gene of the thus far unknown function, tnpB¹³. The TnpB proteins are a tremendously abundant but imperfectly characterized family of nucleases encoded by several bacterial and archaeal TEs^14,15, which often consist of only tnpB¹⁶. Furthermore, recent studies have renewed interest in TnpB by finding that TnpB functions as a CRISPR/Cas-like programmable RNA-guided endonuclease that will yield either dsDNA or ssDNA breaks under diverse conditions^17,18. However, it remains unclear what role TnpB may play in regulating transposition. To perform real-time visualization of the effects of TnpB on IS608 transposition, a version of the transposon, including the coding region of TnpB with an N-terminal fusion to the red fluorescent protein mCherry, was created.

Complementing more detailed bulk-level studies performed by the Kuhlman lab¹⁹, it is shown here how real-time imaging of transposon activity can quantitatively reveal the impact of TnpB or any other accessory proteins on transpositional dynamics. By fusing TnpB to mCherry, the individual transpositional events are identified by blue fluorescence and correlated with expression levels of TnpA (yellow fluorescence) and TnpB (red fluorescence).

Protocol

1. Preparation of bacterial cultures

1. Grow E. coli strain MG1655 with plasmid transposon constructs (previously described in Kim et al.⁸) overnight in LB with the appropriate antibiotics (25 µg/mL of kanamycin, see Table of Materials) at 37 °C.
   NOTE: The sequences of the constructs used and the related sequences are available as GenBank²⁰ accession numbers OP581959, OP581957, OP581958, OP717084, and OP717085.
2. To achieve steady-state exponential growth, dilute cultures ≥100 fold into the M63 medium (100 mM KH₂PO₄, 1 mM MgSO₄, 1.8 µM FeSO₄, 15 mM [NH₄]₂SO₄, 0.5 µg/mL thiamine [vitamin B1]) supplemented with a carbon source (0.5% w/v glucose here) and appropriate antibiotics (see Table of Materials).
3. Grow cultures at 37 °C until the optical density at 600 nm (OD600) reaches ~0.2. The cultures are ready for use.

2. Slide preparation

1. Prepare a slide by boiling M63 with 0.5% w/v glucose and 1.5% w/v agarose in the microwave to melt the agarose and ensure that it is completely molten and well mixed.
2. Allow the mixture to cool to ~55 °C before adding antibiotics and inducers (25 µg/mL Kanamycin and 10 ng/µL anhydrotetracycline [aTc], see Table of Materials).
3. Place a microscope slide on the workbench. Stack two more slides perpendicular to the first and place another on top, parallel to the bottom slide. Ensure that there is a gap equal to one slide thickness between the bottom and top slides. Pipette ~1 mL of the M63 agarose mixture into this gap between the slides slowly to create a small gel square.
4. Once the gel has solidified (~10-15 min), slide the top slide to remove it. Trim the agarose pad with a razor blade or knife. Then pipette 2.5 µL of the culture (step 1.3) and put the coverslip on top.
5. Seal the space between the slide and the coverslip with epoxy (see Table of Materials). Allow the epoxy to dry and the cells to settle onto the agarose pad for at least 1 h at 37 °C.

3. Timelapse fluorescence microscopy

1. Place the prepared sample (step 1.3) on a fluorescence microscope (see Table of Materials) in an environment heated and maintained at 37 °C.
   1. Set the exposure times appropriate for the camera used for image acquisition. Adjust the illumination intensity to minimize photobleaching.
      NOTE: An exposure time of 2 s for each wavelength was used for the present study.
   2. For each wavelength, find a Field of View (FOV) containing minimal fluorescence. Acquire images to use during the analysis for background subtraction.
2. Set up a protocol to acquire images in a grid at different wavelengths and at regular time intervals.
   1. Encode timelapse photography into the protocol. Set the acquisition frequency to the desired time interval (20 min here) and the total timelapse duration to the desired length (24 h).
   2. Encode appropriate wavelengths into the protocol (depending upon the construct used).
      NOTE: The mCherry excitation peak is at 587 nm and the emission peak at 610 nm²¹; mVenus is at 515 nm and 527 nm¹², while mCerulean3 is at 433 nm and 475 nm¹¹.
   3. Set the grid size to capture between the desired number of FOVs.
      ​NOTE: The representative data shown here used 8 x 8 FOVs.

4. Image analysis

1. Perform background subtraction on each color channel by using the respective background images acquired in step 3.1.2. For all the analysis steps, we use standard modules in the open-source platform Fiji²² (see Table of Materials).
2. Approximate the total population at each point in time by thresholding the mCerulean channel and dividing the threshold area by the average cell area.
3. To count the unique excision events, take the time derivative of the mCerulean3 channel. Perform this by subtracting successive images in the mCerulean3 channel. The excision events will be detected in the time derivative as a bright flash of fluorescence.
   1. Threshold the stack of excision events to eliminate unwanted fluorescence. Note that this process will threshold out parts of the excisions themselves. To fix this, dilate the images to restore the excisions to their original sizes.
      NOTE: Analyses using similar thresholding and image analysis techniques can be performed on the other fluorescence channels too (e.g., correlate excision events with levels of transposase TnpA [yellow Venus fluorescence] and TnpB [red mCherry fluorescence].

Representative Results

This method of visualizing transposon activity in live cells by fluorescence microscopy, while having lower throughput than bulk fluorescence measurements, allows direct visualization of transposon activity in individual live cells. Transposon excision events result in the reconstitution of the promoter for mCerulean3 (Figure 1), allowing identification of cells undergoing transposon activity by bright blue fluorescence (Figure 2, TnpB+: Supplementary Movie 1 and TnpB-: Supplementary Movie 2).

It is found that cells expressing the accessory protein TnpB (Figure 3, orange) experience 4-5 times higher levels of transposon activity compared to those that do not (Figure 3, blue), consistent with the more detailed bulk-level studies¹⁹. This is particularly notable as the inclusion of the coding sequence of mCherry-tnpB increases the length of the transposon by ~2,000 bp, while previous studies have found that IS608 transposon excision is an exponentially decreasing function of transposon length²³.

An advantage of real-time imaging is that once identified, cells undergoing transpositional events can be further tracked and analyzed to determine other characteristic parameters, such as growth rate, to determine the distribution of fitness effects or the expression level of accessory proteins to determine their impact on transpositional activity. For example, in TnpB+ cells, cells undergoing transposon excision events have higher expression levels of mCherry-TnpB than the general population (Figure 4A). Moreover, for cells undergoing excision events (Figure 4B, dark yellow), TnpB+ cells (Figure 4B, bottom) express only marginally higher levels of Venus-TnpA transposase than TnpB- cells (Figure 4B, top) (TnpB- 158.3 ± 68.2 AU, TnpB+: 193 ± 79.9 AU), which is higher than the yellow fluorescence of the general population (Figure 4B, light yellow). Taken together, these data suggest that TnpB protein is responsible for the observed higher levels of transpositional activity.

Figure 1: Genetic constructs for imaging of real-time transposon dynamics. (A) The mCerulean3 promoter is disrupted by the TE, the ends of which are flanked by the left end and right end faulty palindromic sequences (LE IP and RE IP). The transposase, tnpA (gray), is expressed from P_LtetO1, which is regulated by the tet repressor (gray) and is inducible with anhydrotetracycline (aTc). The sequences of the Promoter/TE junction and promoter -10 and -35 sequences (red boxes), and TnpA cleavage sites are shown by arrows. (B) The TnpB+ construct is where mCherry-tnpB has been transcriptionally fused to venus-tnpA such that both are transcribed as a polycistronic mRNA, mimicking the natural configuration of IS608. (C) Upon excision, the mCerulean3 promoter is repaired, and the cell exhibits blue fluorescence. The reconstituted promoter sequence is displayed below the diagram. Please click here to view a larger version of this figure.

Figure 2: Visualization of transposon excision events. The example field of view of TnpB+ with cells (A) immediately before and (B) after detecting transposon excision events by blue fluorescence. White arrows indicate excision events. The time difference between the two frames is 20 min. Scale bar = 5 µm. Please click here to view a larger version of this figure.

Figure 3: TnpB enhances transposon excision rate. The excision rate for TnpB+ cells (orange) and TnpB- (blue) cells. The mean rate from three replicates is shown as points with shaded regions with a 95% confidence interval. The data are aligned so that cells begin excising at t = 0. The maximum measured rate for TnpB+ cells was 5.1 ± 2.4 x 10^-2 events per cell per hour, while for TnpB- was 1.4 ± 0.48 x 10^-2 events per cell per hour. The average rate over the whole interval shown was 2.6 ± 1.8 x 10^-2 events per cell per hour for TnpB+ cells and 5.3 ± 2.9 x 10^-3 events per cell per hour for TnpB- cells. Please click here to view a larger version of this figure.

Figure 4: Protein expression statistics for excising cells versus total cell population. Each frame was divided into 64 equal blocks, and fluorescence was measured for cells excising within the block and for all cells contained within the block regardless of the excision activity. Probability, as plotted on the y-axis, is measured as the number of pixels of the indicated intensity in each cell type divided by the total number of pixels. The block size for each frame was set to 445 x 445 pixels. (A) The cells that undergo excision events (dark red) express more TnpB than the general population (light red). The average red fluorescence for excising cells was 51.3 ± 15.4 AU (dark red), while that for all cells was 42.5 ± 7.4 AU (light red). (B) Venus-TnpA transposase levels are similar in TnpB- (top) and TnpB+ (bottom) cells. The data sets are normalized so that the mean yellow fluorescences of the total cell population (light yellow) are equal for TnpB+/- at 105.7 AU. The cells that have been identified as undergoing transposon excision events (dark yellow) exhibit higher yellow fluorescence than the general population, with similar distributions for TnpB- (top) and TnpB+ (bottom). Mean yellow fluorescences of the excising populations are TnpB-: 158.3 ± 68.2 AU and TnpB+: 193 ± 79.9 AU. Please click here to view a larger version of this figure.

Supplementary Movie 1: Real-time dynamics of IS608 excision with TnpB expression. A movie showing a 5 h segment of real-time IS608 dynamics; a frame is captured every 20 min. IS608 excision is visualized as the development of bright blue fluorescence. The cells shown in this movie include the expression of TnpB. Please click here to download this File.

Supplementary Movie 2: Real-time dynamics of IS608 excision without TnpB expression. A movie showing a 5 h segment of real-time IS608 dynamics; a frame is captured every 20 min. IS608 excision is visualized as the development of bright blue fluorescence. The cells shown in this movie do not include the expression of TnpB. Please click here to download this File.

Discussion

The unique method presented here for real-time imaging of transposable element activity in live cells is a sensitive assay that can directly detect transposition in live cells and in real-time and correlate this activity with the expression of accessory proteins. While the throughput is lower than can be accomplished by bulk methods, this method achieves detailed measurements of TE activity and protein expression in individual living cells.

A variety of tools and techniques can be employed to grow cells directly on the microscope for real-time imaging. The method used here of cell growth on agarose pads has the advantage of being fast, cheap, and easy to perform. A possible disadvantage, depending on the cellular growth state of interest, is that resources available to support cell growth in the agarose pad are limited, and hence cells will naturally exhaust these resources and stop growing after a relatively short period of time (12-24 h). Consequently, care must be taken to prepare the cells in steady state growth and inoculate the pad at a low enough density to give ample time for measurement. Microfluidics can be employed to maintain cells in steady state exponential growth for extended periods of time²⁴, although these methods require additional expertise, equipment, and setup to be effective.

Complementing more detailed work from the Kuhlman lab¹⁹, it is illustrated here that the IS200/IS605 TE family-associated protein TnpB increases the rate of IS608 excision by up to five-fold, and that increased excision is directly correlated with higher expression levels of TnpB. These methods are one example of improved assay techniques that may help shed light on transposon activity and its impact on mutational and evolutionary dynamics.

Materials

Name	Company	Catalog Number	Comments
2 Ton Clear Epoxy	Devcon	31345
Agarose	Sigma-Aldrich	5066
Ammonium sulfate	Sigma-Aldrich	AX1385-1
Anhydrotetracycline hydrochloride	Sigma-Aldrich	37919
Argon Laser	Melles Griot	35-IMA-840-015
Blue Filter Cube	Chroma	Ex: Z457/10X, Em: ET485/30M
D(+)Glucose	Sigma-Aldrich	G7021
Eclipse Ti-E Microscope	Nikon		Discontinued
Eppendorf epTIPS Boxes and Refill Trays, Volume: 0.1 to 10 µL, Length: 3.4 cm, 1.33 in., PP (Polypropylene)	Eppendorf North America Biotools	22491504
Eppendorf epTIPS Boxes and Refill Trays, Volume: 50 to 1000 µL, Length: 7.1 cm, 2.79 in., PP (Polypropylene)	Eppendorf North America Biotools	22491555
Ferrous Sulfate Acs 500 g	Fisher Scientific	706834
Fiji			Fiji (imagej.net)
Fisher BioReagents LB Broth, Miller (Granulated)	Fisher Scientific	BP9723-2
Glass Cover Slide	Fisher Scientific	12-542B
Kanamycin Sulfate	Sigma-Aldrich	1355006
Magnesium sulfate Cert Ac	Fisher Scientific	XXM63SP3KG
Microscope Heater	World Precision Instruments	96810-1
Potassium Phosphate Monobasic	Fisher Scientific	17001H
ProScan III Stage	Prior
Red Filter Cube	Chroma	Ex: ET560/40X, Em: ET645/75M
Sapphire 561 LP Laser	Coherent	1170412
Slide, Microscope	Fisher Scientific	125535B
Thiamine Hydrochloride	Sigma-Aldrich (SIAL)	T1270-100G
Ti-LU4 Laser Launch	Nikon
Yellow Filter Cube	Chroma	Ex: Z514/10X, Em: ET535/30M