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].

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 Mo...

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 ...

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