Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Abstract

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.

Figure 1.

Construction of functional fluorescently-labeled Esp1396I restriction-modification system. (A) Genetic organization of the wild-type Esp1396I and Esp1396I_Fluo encoding fusions of the restriction endonuclease and methyltransferase genes to, respectively, mCherry and Venus fluorescent proteins genes is schematically shown. Individual genes are represented by arrows whose directions indicate the direction of transcription. Blue arrows indicate C-protein genes. The three sites of binding of C-protein dimers are shown as light blue rectangles labeled “C". (B) The titers of λ_vir phage lysate determined on lawns of E. coli cells with or without indicated plasmids are shown. Mean results from three independent measurements with standard deviations are presented. (C) Aliquots of whole-cell lysates corresponding to indicated numbers of E. coli cells transformed with pEsp1396I_Fluo were separated by SDS-PAGE and blotted with anti-Venus or anti-mCherry antibodies. As controls, known amounts of purified Venus and mCherry proteins were loaded on the same gel.

Figure 2.

Determining amount of Esp1396I enzymes in individual E. coli cells. (A–C) Fluorescence images of bacteria harboring pEsp1396I_Fluo plasmid in Venus (A) and mCherry (B) channels and their overlay (C). (D) A representative intensity profile in Venus (black line) and mCherry (red line) channels through the dashed line of enlarged images of cells from A and B (shown to the left) demonstrating different localization of M.Esp1396I::Venus and R.Esp1396I::mCherry. (E) Histograms of R.Esp1396I::mCherry and M.Esp1396I::Venus molecule numbers per cell (N = 2069). (F) Correlation plot for R.Esp1396I::mCherry and M.Esp1396I::Venus concentrations in individual E. coli cells analyzed in E. The correlation coefficient between R.Esp1396I::mCherry and M.Esp1396I::Venus concentrations is r = 0.67.

Figure 3.

Dynamics of Esp1396I enzymes accumulation in individual transformed E. coli cells. (A and B) A representative kinetic series of images showing Venus (green) and mCherry (magenta) fluorescence in a microcolony growing from a single transformed cell. The initial part of the sequence is shown in A, in B, the full duration of experiment is shown. A nonlinear contrast was used in B to allow simultaneous visualization of bright and dim signals (see Materials and Methods). White arrows in A show transformed cell at early stages before Venus accumulation is clearly visible. Another kinetic series is shown in Supplementary Figure S1. (C) Atypical cells in growing microcolonies transformed with pEsp1396I_Fluo. See text for details. Atypical cells are indicated by white arrows. In each panel, two different examples of identified atypical cell types are shown. (D) Quantification of a representative kinetic series showing changes in Venus and mCherry fluorescence intensities per individual cell in microcolony growing from a single transformed cell over time. Dashed lines at infinity show mean stationary R.Esp1396I::mCherry and M.Esp1396I::Venus levels in cells from exponentially growing cultures.

Figure 4.

Dynamic modeling of Esp1396I enzymes accumulation in individual transformed E. coli cells. (A) Logarithm of the experimentally measured number of cells, as a function of time. The first and the second time interval, characterized respectively by the fast and slow cell division rate, are clearly visible. As the time dependences in these two intervals are nearly linear, the respective linear fits are indicated, with the slopes corresponding to the cell division rate. (B and C) Model vs. experiment for the expression dynamics of Esp1396I restriction endonuclease (B) and methyltransferase (C) as a function of time. Circles correspond to experimentally measured concentrations of protein fusions (expressed in relative units), while the full lines correspond to the model predictions. Note that the time is set to zero at the point of the first available measurement.