Physics of Living Systems

IS200/IS605 Family-Associated TnpB Increases Transposon Activity and Retention

Davneet Kaur
Thomas E. Kuhlman author has email address

Department of Physics, University of Illinois Urbana Champaign, Urbana, IL, USA
Department of Physics and Astronomy, University of California Riverside, Riverside, CA, USA

https://doi.org/10.7554/eLife.92919.1

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Figures and data

Design
(A) Genetic Constructs for Inducible Transposon Quantification: Transposons constructed for experiments. (i) TnpB introduced in trans with Tn4rev, MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB and (ii) in cis, MG1655 ∆lac pJK14-P_Ltet-01-tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR. (B) Venus fluorescence data as a function of aTc concentration for when TnpB is introduced in trans with Tn4rev for different [IPTG] [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. (C) (i) Immobilized transposon, no IPTG (purple, ◊), (ii) Immobilized transposon, with 2000μM IPTG (lizard green, +), (iii) Immobilized transposon with TnpB, no IPTG (fuchsia, ◯) and (iv) Immobilized transposon with TnpB, with 2000μM IPTG (arctic, X). (B-C) Points are fit to a Hill function (solid lines, same color; see Supplementary Information SI.i). Quantitative features of the response are extracted and included in Supplementary Table S1 and plotted in D-F with corresponding color and symbols. Also included is the Tn4rev without TnpB strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯) (D) TnpB induction results in higher saturating TnpA-Venus (H_max). Peak Venus fluorescence is a relative measurement of the population-averaged number of transposons per cell. (E) Increased TnpB induction results in earlier TnpA-Venus expression [inflection point of curves in (B), x*]. (F) TnpB induction results in increased TnpA-Venus sensitivity to aTc [slope at inflection point S(x*)].

Design
(A) Genetic Constructs for Inducible Transposon Quantification: Transposons constructed for experiments. (i) TnpB introduced in trans with Tn4rev, MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB and (ii) in cis, MG1655 ∆lac pJK14-P_Ltet-01-tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR. (B) Venus fluorescence data as a function of aTc concentration for when TnpB is introduced in trans with Tn4rev for different [IPTG] [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. (C) (i) Immobilized transposon, no IPTG (purple, ◊), (ii) Immobilized transposon, with 2000μM IPTG (lizard green, +), (iii) Immobilized transposon with TnpB, no IPTG (fuchsia, ◯) and (iv) Immobilized transposon with TnpB, with 2000μM IPTG (arctic, X). (B-C) Points are fit to a Hill function (solid lines, same color; see Supplementary Information SI.i). Quantitative features of the response are extracted and included in Supplementary Table S1 and plotted in D-F with corresponding color and symbols. Also included is the Tn4rev without TnpB strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯) (D) TnpB induction results in higher saturating TnpA-Venus (H_max). Peak Venus fluorescence is a relative measurement of the population-averaged number of transposons per cell. (E) Increased TnpB induction results in earlier TnpA-Venus expression [inflection point of curves in (B), x*]. (F) TnpB induction results in increased TnpA-Venus sensitivity to aTc [slope at inflection point S(x*)].

Excision as a function of TnpA
Transposon Activity and Retention in Response to TnpB. (A) Cumulative Cerulean fluorescence data as a function of aTc concentration (0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL) for TnpB in trans with Tn4rev for different IPTG concentrations [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. Cumulative Cerulean fluorescences are fit to a Hill function (solid lines, same color; see Supplementary Information SI.i). Quantitative features of the responses are extracted and included in C-E and Supplementary Table S2. (B) Number of excision events is proportional to transposase concentration: Cumulative Cerulean fluorescence vs. Venus-transposase concentration is plotted for Tn4rev only strain (red, ◯), TnpB introduced in trans with Tn4rev for all concentrations of IPTG (same colors and symbols as in A), and TnpB introduced in cis with Tn4rev (orange, X). The data collapse to a single curve and a linear regression is fit to the data (solid line). (C) TnpB induction results in higher saturating Cumulative Cerulean levels (H_max). (D) Increased TnpB induction results in earlier Cumulative Cerulean-excision [inflection point of curves in A, x*]. (E) TnpB induction results in increase excision sensitivity [slope at inflection point of curves in A S(x*)] to aTc concentration. (F-H) qPCR reactions determining relative numbers for (F) plasmid, (G) total transposon, and (H) unexcised plasmid count. P-values quantifying the significance of differences are given in Supplementary Tables S4 and S5. See Supplementary Table S7 for primer sequences.

Excision as a function of TnpA
Transposon Activity and Retention in Response to TnpB. (A) Cumulative Cerulean fluorescence data as a function of aTc concentration (0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL) for TnpB in trans with Tn4rev for different IPTG concentrations [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. Cumulative Cerulean fluorescences are fit to a Hill function (solid lines, same color; see Supplementary Information SI.i). Quantitative features of the responses are extracted and included in C-E and Supplementary Table S2. (B) Number of excision events is proportional to transposase concentration: Cumulative Cerulean fluorescence vs. Venus-transposase concentration is plotted for Tn4rev only strain (red, ◯), TnpB introduced in trans with Tn4rev for all concentrations of IPTG (same colors and symbols as in A), and TnpB introduced in cis with Tn4rev (orange, X). The data collapse to a single curve and a linear regression is fit to the data (solid line). (C) TnpB induction results in higher saturating Cumulative Cerulean levels (H_max). (D) Increased TnpB induction results in earlier Cumulative Cerulean-excision [inflection point of curves in A, x*]. (E) TnpB induction results in increase excision sensitivity [slope at inflection point of curves in A S(x*)] to aTc concentration. (F-H) qPCR reactions determining relative numbers for (F) plasmid, (G) total transposon, and (H) unexcised plasmid count. P-values quantifying the significance of differences are given in Supplementary Tables S4 and S5. See Supplementary Table S7 for primer sequences.

Effects of TnpB on growth
Effects of TnpB on Bacterial Growth: (A) TnpB expression in the presence of: (i) the active TE (blue) and (ii) a negative control plasmid without the active TE (red). Induction range is identical for both, [IPTG] = {0μM, 10μM, 20μM, 50μM, 100μM, 200μM, 2000μM}. However, the mCherry-TnpB response to induction is different as TnpB is unstable in the absence of other TE features (red). (B) Growth rate of immobilized strain without TnpB and no IPTG (purple), without TnpB and with IPTG (green), with TnpB and no IPTG (magenta), and with TnpB and with IPTG (light blue) versus Venus fluorescence per cell for all concentrations of aTc. Immobilized strains with the same concentrations of TnpA-Venus have a greater growth defect when TnpB is coexpressed compared to when only TnpA is expressed. (C) Growth rate is fit to an exponential decay function of Cumulative Cerulean determining b as described in Supplementary Information SI.viii for Tn4rev only (red, ◯), TnpB with Tn4rev in trans for different IPTG concentrations [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)] and for TnpB in cis with Tn4rev (orange, X). (D) b increases with TnpB until it saturates. The outliers to this pattern correspond to the curves with R² values < 0.9 (Supplementary Table S6). Data point for strain with TnpB introduced in cis to Tn4rev is plotted at zero point for mCherry-TnpB because it has multiple values along the fitted curve.

Effects of TnpB on growth
Effects of TnpB on Bacterial Growth: (A) TnpB expression in the presence of: (i) the active TE (blue) and (ii) a negative control plasmid without the active TE (red). Induction range is identical for both, [IPTG] = {0μM, 10μM, 20μM, 50μM, 100μM, 200μM, 2000μM}. However, the mCherry-TnpB response to induction is different as TnpB is unstable in the absence of other TE features (red). (B) Growth rate of immobilized strain without TnpB and no IPTG (purple), without TnpB and with IPTG (green), with TnpB and no IPTG (magenta), and with TnpB and with IPTG (light blue) versus Venus fluorescence per cell for all concentrations of aTc. Immobilized strains with the same concentrations of TnpA-Venus have a greater growth defect when TnpB is coexpressed compared to when only TnpA is expressed. (C) Growth rate is fit to an exponential decay function of Cumulative Cerulean determining b as described in Supplementary Information SI.viii for Tn4rev only (red, ◯), TnpB with Tn4rev in trans for different IPTG concentrations [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)] and for TnpB in cis with Tn4rev (orange, X). (D) b increases with TnpB until it saturates. The outliers to this pattern correspond to the curves with R² values < 0.9 (Supplementary Table S6). Data point for strain with TnpB introduced in cis to Tn4rev is plotted at zero point for mCherry-TnpB because it has multiple values along the fitted curve.

Model of transposon dynamics
Modeling of Transposon and TnpB Dynamics: (A) Schematic of Mathematical Model: The rate equations described in Supplementary SII.i are evolved over time to generate the total number of transposons, number of plasmids with and without transposons in their original sites, the total number of excision events (Cerulean) and a measure of the relative transposase molecules (Venus). (B-E) The coupled differential equations were evolved over three generations to mimic experimental conditions and the above phase diagrams were generated. Phase diagrams were generated with the y-axis and x-axis measuring the induction quantities with aTc and IPTG respectively. Here, the induced excision rate per TE is a function of the aTc concentration as it induces TnpA, which executes excision. Tuning of the TnpB concentration from zero to one in the model accounts for increased peak values and earlier initiation of expression that we observe experimentally for (B) total transposon number (qPCR data), (C) transposase concentration (Venus-TnpA fluorescence data), (D) number of excision events (cumulative Cerulean fluorescence data) and (E) total excised plasmids (qPCR data).

(A) Genetic Constructs for Inducible and Trackable Transposons:
All transposons constructed for experiments. The strain which has Tn4rev only (i), MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR. TnpB introduced to the transposon in trans (ii), MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB and in cis (iii), MG1655 ∆lac pJK14-P_Ltet-01-tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR. A version of the transposon (iv) without the excision sites, rendering it immobile. To the immobile transposon, we introduce, in trans, both TnpB: CZ071 pJK14-P_Ltet-01-tnpA & pZA31-P_LlacOid-mCherry-tnpB, and its negative control plasmid: CZ071 pJK14-P_Ltet-01-tnpA & pZA31-P_LlacOid-SmR. A control for the mCherry fusion to TnpB is also constructed (v), MG1655 ∆lac pJK14-P_Ltet-01-tn4rev-tnpB & pZA31-P_LlacOid-SmR. (B) TnpB is translationally fused to mCherry, introduced to the transposon in trans and individually induced with IPTG (0 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM & 2000 μM). (C) TnpB is translationally fused to mCherry and introduced to the transposon in cis. mCherry-TnpB and Venus-TnpA are induced simultaneously with aTc (0 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL, 5 ng/mL, 9 ng/mL, 20 ng/mL, 30 ng/mL, 50 ng/mL, 80 ng/mL, 100 ng/mL and 200 ng/mL).

Quantification of Venus-Transposase Concentration to aTc Induction:
Venus fluorescence data as a function of aTc concentration (0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL) for (A) (i) Tn4rev only strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯), (ii) TnpB introduced in trans with Tn4rev at [IPTG] = 0: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB (light green, ∆), (iii) TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR (orange, X).

Titration of Excision Rate with Anhydrotetracycline (aTc):
(A,B) Cerulean fluorescence data for [aTc] = {0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL} for (A) (i) Tn4rev only strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯), (ii) TnpB introduced in trans with Tn4rev, [IPTG] = 0: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB (light green, ∆), (iii) TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-P_Ltet-01-01-Tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR (orange, X), and (B) TnpB is introduced in trans with Tn4rev, MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev pZA31-P_LlacOid-mCherry-tnpB for different concentrations of IPTG [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. Cerulean fluorescence data sets are each fit to a Hill function (same color as data points for each) of the form of eq. (S3.1.2) and quantitative features of the responses are extracted and included in C-E and Supplementary Table S2 for the strain with TnpB in cis with Tn4rev as it does not have a singular mCherry-TnpB value. (C) Higher Cerulean levels with TnpB induction when TnpA is maximally expressed (H_max). (D) Earlier initiation of Cerulean expression (inflection point, x*) for lower aTc concentrations with increased TnpB induction. (E) Indiscernible pattern of excision sensitivity to aTc concentration (slope at inflection point S(x*)) with increased TnpB concentration.

Titration of Excision Rate with Anhydrotetracycline (aTc):
(A,B) Cerulean fluorescence data for [aTc] = {0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL} for (A) (i) Tn4rev only strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯), (ii) TnpB introduced in trans with Tn4rev, [IPTG] = 0: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB (light green, ∆), (iii) TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-P_Ltet-01-01-Tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR (orange, X), and (B) TnpB is introduced in trans with Tn4rev, MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev pZA31-P_LlacOid-mCherry-tnpB for different concentrations of IPTG [0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)]. Cerulean fluorescence data sets are each fit to a Hill function (same color as data points for each) of the form of eq. (S3.1.2) and quantitative features of the responses are extracted and included in C-E and Supplementary Table S2 for the strain with TnpB in cis with Tn4rev as it does not have a singular mCherry-TnpB value. (C) Higher Cerulean levels with TnpB induction when TnpA is maximally expressed (H_max). (D) Earlier initiation of Cerulean expression (inflection point, x*) for lower aTc concentrations with increased TnpB induction. (E) Indiscernible pattern of excision sensitivity to aTc concentration (slope at inflection point S(x*)) with increased TnpB concentration.

Titration of Excision Event Number with Anhydrotetracycline (aTc):
Cumulative Cerulean fluorescence data as a function of inducer concentration for (0ng/mL, 1ng/mL, 2ng/mL, 3ng/mL, 5ng/mL, 9ng/mL, 20ng/mL, 30ng/mL, 50ng/mL, 80ng/mL, 100ng/mL and 200ng/mL) for (i) Tn4rev only strain: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-SmR (red, ◯), (ii) TnpB introduced in trans with Tn4rev: MG1655 ∆lac pJK14-P_Ltet-01-Tn4rev & pZA31-P_LlacOid-mCherry-tnpB (light green, ∆), and (iii) TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-P_Ltet-01-01-Tn4rev-mCherry-tnpB & pZA31-P_LlacOid-SmR (orange, X).

Excision Rate Increases and Peaks with Transposase Concentration:
Excision rate (Cerulean fluorescence per cell) vs. transposase number (Venus fluorescence per cell) is plotted for: Tn4rev only strain: MG1655 ∆lac pJK14-PLtet-01-Tn4rev & pZA31-PLlacOid-SmR (red, ◯), TnpB introduced in trans with Tn4rev: MG1655 ∆lac pJK14-PLtet-01-Tn4rev pZA31-PLlacOid-mCherry-tnpB for different concentrations of IPTG (0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)), and TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-PLtet-01-Tn4rev-mCherry-tnpB & pZA31-PLlacOid-SmR (orange, X). Both Venus-TnpA concentration and the excision rate of TEs from plasmids increase with TnpB. As the concentration of transposase increases, the rate of excision events increases proportionally until it reaches its peak value.

Excision Rate Increases and Peaks with Transposase Concentration:
Excision rate (Cerulean fluorescence per cell) vs. transposase number (Venus fluorescence per cell) is plotted for: Tn4rev only strain: MG1655 ∆lac pJK14-PLtet-01-Tn4rev & pZA31-PLlacOid-SmR (red, ◯), TnpB introduced in trans with Tn4rev: MG1655 ∆lac pJK14-PLtet-01-Tn4rev pZA31-PLlacOid-mCherry-tnpB for different concentrations of IPTG (0μM (yellow, ∆), 10μM (green, □), 20μM (turquoise, ✳), 50μM (teal, ▽), 100μM (navy, ◁), 200μM (magenta, ) & 2000μM (violet, ✵)), and TnpB introduced in cis with Tn4rev: MG1655 ∆lac pJK14-PLtet-01-Tn4rev-mCherry-tnpB & pZA31-PLlacOid-SmR (orange, X). Both Venus-TnpA concentration and the excision rate of TEs from plasmids increase with TnpB. As the concentration of transposase increases, the rate of excision events increases proportionally until it reaches its peak value.

Decay of Number of Transpose Molecules per cell over time:
The decay coefficients of the transposase concentration over time, βg, for the strain with introduction of TnpB in trans with Tn4rev are normalized to values for the negative control strain and plotted for all concentrations of TnpA and TnpB are plotted. For lower concentrations of both proteins, the number of transposase molecules decays at a higher rate over time. Increase in TnpA and TnpB both result in maintenance of transposase concentration due to a lower decay rate over time.

Excision Rate Can Have Bimodal Slope During Exponential Growth.
Determination of average excision rate per cell as the slope of mCerulean3 fluorescence versus optical density. For intermediate IPTG concentrations (e.g., 20 μM and 100 μM shown here), the response can appear with bimodal slope. In such cases, the integral of fluorescence versus OD (Cumulative Cerulean) is calculated as given in eq. S3.2.3, with m₁ and m₂ determined as the slopes of the red and yellow lines, respectively.

Quantitative Features of Venus-TnpA Induction Response

Quantitative Features of Excision Rate Response

Quantitative Features of Excision Number Response

P-values for Changes in Total Transposon Number

P-values for Changes in Plasmid-Transposon Number

Quality of Fit of Exponential Decay of Growth Rate

Primer Sequences for qPCR

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