A novel system of bacterial cell division arrest implicated in horizontal transmission of an integrative and conjugative element

Abstract

Integrative and conjugative elements (ICEs) are widespread mobile DNA elements in the prokaryotic world. ICEs are usually retained within the bacterial chromosome, but can be excised and transferred from a donor to a new recipient cell, even of another species. Horizontal transmission of ICEclc, a prevalent ICE in proteobacteria, only occurs from developed specialized transfer competent (tc) cells in the donor population. tc cells become entirely dedicated to the ICE transmission at the cost of cell proliferation. The cell growth impairment is mediated by two ICEclc located genes, parA and shi, but the mechanistic and dynamic details of this process are unknown. To better understand the function of ParA and Shi, we followed their intracellular behavior from fluorescent protein fusions, and studied host cell division at single-cell level. Superresolution imaging revealed that ParA-mCherry colocalized with the host nucleoid while Shi-GFP was enriched at the membrane during the growth impairment. Despite being enriched at different cellular locations, the two proteins showed in vivo interactions, and mutations in the Walker A motif of ParA dislocalized both ParA and Shi. In addition, ParA mutations in the ATPase motif abolished the growth arrest on the host cell. Time-lapse microscopy revealed that ParA and Shi initially delay cell division, suggesting an extension of the S phase of cells, but eventually completely inhibit cell elongation. The parA-shi locus is highly conserved in other ICEclc-related elements, and expressing ParA-Shi from ICEclc in other proteobacterial species caused similar growth arrest, suggesting that the system functions similarly across hosts. The results of our study provide mechanistic insight into the novel and unique system on ICEs and help to understand such epistatic interaction between ICE genes and host physiology that entails efficient horizontal gene transfer.

Fig 1. The parA-shi region of ICEclc.

(A) Location of parA-shi gene cluster on ICEclc nearby the attL end. Positions and directions of primers used for 1st (150201) and 2nd (140705) PCRs to amplify 5’RACE products are indicated as small arrows. Lower part shows the sequence of a 1.4-kb 5’RACE product which determined the transcription starting site (adenine in red) of the parA-shi cluster. The boundary between the 5’RACE product and the cloning vector used is illustrated. (B) Agarose gel electrophoresis of the 2nd PCR product in 5’RACE. A specific 1.4-kb product is indicated by an arrow head. Presence or absence of the reverse transcriptase in each cDNA synthesis reaction is shown by ‘+’ or ‘-‘, respectively. (C) Representative micrographs and average fluorescence intensities of P. putida single cells carrying ICEclc and single insertions of P_alp-egfp and P_inR-echerry. Images show eGFP (green), eCherry (red), and merged with phase–contrast channels. Scale bar indicates 10 μm. Scatter plot showing correlation between single cell eCherry versus eGFP fluorescence values (circles) in stationary phase 3CBA-grown cultures. Grey zones indicate cells below cutoff values of the fluorescence expressions (for calculation, see S2 Fig). The inset in the scatter plot shows the number (and proportion) of cells separated by the cutoff values. Green and red lines indicate eGFP and eCherry cutoff values, respectively. Note that 4.7% of the 1,107 cells tested express both fluorescence proteins.

Fig 2. Subcellular localization and interaction of ParA and Shi proteins in P. putida without ICEclc.

(A) Population growth of cells carrying pME6032 derivatives. Cells are cultured with IPTG, and their turbidity is measured. Genetic information expressed from P_tac promoter on the vector is indicated. Error bars represent standard deviation (SD) from the mean in triplicate assays. Vector, pME6032 (empty). (B) Representative micrographs and fluorescence profiles of single cells expressing either ParA-mCherry or Shi-eGFP, or both. Phase contrast (PhC) and fluorescence (eGFP, mCherry, and Hoechst33342) images are acquired at 4h after IPTG induction. Scale bar indicates 2 μm. Fluorescence and transmission light intensities of representative single cells are measured longitudinally (dotted arrows). In the right plots, the intensities are shown in grey (transmission light), green (eGFP), red (mCherry), and blue (Hoechst33342) lines along the long axes of the cells. (C) Representative superresolution images of cells expressing Shi-eGFP with either ParA(wild-type)-mCherry, ParA(K15E)-mCherry or ParA(K15Q)-mCherry, or without ParA-mCherry. Fluorescence images are acquired at 4h after IPTG induction. Aberrant foci are indicated by white triangles. Scale bar indicates 1 μm. (D) Co-immunoprecipitation of ParA-mCherry with Shi-eGFP using GFP affinity beads. Cell extracts were prepared from strains used in (B). Whole cell (W), flow-through (F), and bead (B) fractions were analyzed by immunoblot using anti-RFP and anti-GFP antibodies. Control, cells expressing ParA and Shi without fluorescence fusions.

Fig 3. Dynamics of cellular growth and ParA and Shi expression at single-cell level of P. putida.

(A) Cell length changes in two representative lineages with IPTG (leading to expression of ParA and Shi, blue squares) or without (grey circles). Cellular lengths measured at each time point connected for visibility with a line. Arrows indicate cell division events. (B) Box plots of doubling times in cells with or without IPTG induction. P-value in Wilcoxon rank test is indicated. (C) Average fluorescence intensity changes from Shi-eGFP (green) and ParA-mCherry (red) in individual cells in two representative lineages with (square) or without IPTG (circle). Same cell lineages as in panel A. Time indicates the duration from the start of the incubation.

Fig 4. Experimental test of two working hypotheses for ParA-Shi-mediated cell growth inhibition.

(A) Schematic illustrations of two scenarios of growth inhibition: division blocking (Hypothesis 1) or elongation blocking (Hypothesis 2). (B) Average cell length changes in individual cells of two representative micro colonies with IPTG (leading to ParA and Shi induction, blue circles) or without (grey circles). Average cell length at each time point calculated from all cells in the microcolony at that point. Error bars indicate standard deviations. (C) Maximum cell lengths across all cells incubated in presence or absence of IPTG. Cells which did not divide until the end of the experiments are eliminated from analysis. P-value in Wilcoxon rank test is indicated. (D) Correlation between maximum cell length and doubling time with (right panel) or without IPTG (left panel), plotted for all individual cells. Same datasets as presented in Fig 3B are used. Spearman’s correlation coefficient (ρ) and the number of cells used for analysis (n) are indicated. (E) Box plots of cellular elongation rate in the presence or absence of IPTG. Elongation rate of each cell is calculated by dividing the difference between start and final cell length with elapsed time. Cells which existed less than 4 frames (60 minutes) or did not divide until the end of the experiments are eliminated from analysis. P-value in Wilcoxon rank test is indicated. (F) Same as (D), but between elongation rate and doubling time.

Fig 5. Effect of ParA and Shi on P. putida chromosome replication and segregation.

(A) Schematic illustration of cell cycle of P. putida. S phase, elongation of newly emerged daughter cells, and chromosome replication and segregation (nucleoid shown as red oval). G phase is defined from nucleoid segregation to cell division. Time required for S and G phases are denoted as T_S and T_G, respectively. Hence, the doubling time (T_D) of the cell is the sum of T_S and T_G. (B) Box plots of T_S (left) and T_G (right) of cells emerged in different time periods in the presence of IPTG. Here we regarded the cells pre-existing at the start of the time-lapse observation as the first generation. The doubling time of cells is not significantly changed by IPTG induction until the second division (S4 Fig), and thus we compared cells from the first and second generation with later offspring (>2 generations). P-value in Wilcoxon rank test is indicated. (C) Correlations of T_S or T_G with doubling time (T_D) in the presence of IPTG. T_S (orange circle) and T_G (brown triangle) of 80 cells as (B) are plotted as function of their doubling time (T_D). The linear regressions are estimated by MATLAB polyfit functions (T_S = 14.5 + 0.647T_D, T_G = −14.5 + 0.353T_D). The statistical significance of two slopes were tested by analysis of covariance using MATLAB function (p<0.001). (D) Representative micrographs (left panels) and box plots of cellular length (right) of single-nucleoid cells with or without IPTG. Merged images of phase-contrast and fluorescence (Hoechst33342) channels were acquired at 16h after IPTG induction. Scale bar indicates 5 μm. Asterisk indicates significance of difference (P<0.005) in Wilcoxon test.

Fig 6. Cellular elongation rate depending on the birth time of cells.

(A) Correlation between cellular elongation rate and birth time in the presence (blue) or absence (gray) of IPTG. (B) Box plots of cellular elongation rates in the different birth time of cells with (blue) or without IPTG (gray). Letters above plots show significance group based on Kruskal-Wallis test followed by Dwass-Steele-Critchlow-Fligner post hoc test (p<0.005).

Fig 7. Phylogenetic analysis of ParA and effect of parA-shi expression in different bacteria.

(A) Schematic illustration of parA-shi locus on various ICEs. ICE names (if named), species names and accession numbers are shown. Genes are indicated as in the respective genome accession. Coloration is based on predicted functions. (B) Maximum-likelihood (ML) tree based on the amino acid sequences of Walker ATPase family proteins. The 18 protein sequences are aligned and used for construction of the ML tree by using the Jones-Taylor-Thornton model. MinD is used as an outgroup. The bootstrap values (100 resampling) are shown on each branch. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Proteins of which functions are experimentally demonstrated are denoted by asterisks. Accession numbers are shown in brackets. (C) Population growth of C. necator H16 (upper) and E. coli MG1655 (lower) cells carrying either pME6032 or pME-parAshi. Cells are cultured with IPTG, and their culture turbidity is measured. Error bars represent SD from the mean in triplicate assays.