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.
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.
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 evolution2,3, development4,5, 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 PLTetO1, 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 PlacIQ1 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 TEs14,15, 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 conditions17,18. 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).
1. Preparation of bacterial cultures
2. Slide preparation
3. Timelapse fluorescence microscopy
4. Image analysis
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 PLtetO1, 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.
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.
The authors have nothing to disclose.
Financial support for this research was provided by startup funds from the University of California.
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 |