A long-term epigenetic memory switch controls bacterial virulence bimodality
- The Hebrew University of Jerusalem, Israel
Figures
Figure 1 with 1 supplement
EPEC displays the bimodal colony size after virulence activation.
Bacterial cultures of EPEC or E. coli K-12 were grown overnight in LB media (Non-Activation) or in DMEM for 3 hr at 37°C to OD600 ~0.3 (Activation). Cultures were then diluted, plated on LB agar, …
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Figure 1—source data 1
This Source Data file contains appearance time histogram raw data for Figure 1A and C (activated and non-activated EPEC cultures) from ScanLag experiments.
Data collected from four experiments. The data were collected by the ScanningManager software application and analyzed by TimeLapse analysis software application http://bio-site.phys.huji.ac.il/Materials. For figures histogram was fitted to total 100% bacteria. Data columns marked with * and ** are used for creating Figure 1A and C respectively.
- https://doi.org/10.7554/eLife.19599.004
Figure 1—figure supplement 1
EPEC growth on LB measured by ScanLag and time-lapse microscopy.
Static cultures of EPEC were grown in DMEM for 3 hr at 37°C to OD600 ~0.3 (activating conditions). Cultures were then diluted, plated on LB agar, and incubated at 32°C (non-activating conditions). (A…
Figure 2
Memory and reset of colony-size bimodality.
(A) Scheme of the experimental procedure for measuring the stability of colony morphotypes: Colonies of EPEC grown on LB agar were picked 1000 min after plating, resuspended, re-plated on LB agar, …
Figure 3
Model and measurements of bimodal switching rates.
(A) Scheme and equations of a bimodal switching model. The two morphotypes, BIG and SMALL, are characterized by different growth rates, µB and µS, respectively, and different switching rates a and b.…
Figure 4 with 1 supplement
Per operon is essential for establishing colony-size bimodality.
(A) Scheme of key regulatory genes of the EPEC virulence machinery. Ler is the T3SS master regulator and its expression is induced by two redundant positive regulators, PerC and GrlA (Bustamante et …
Figure 4—figure supplement 1
ScanLag colony appearance phenotype of EPEC virulence pathway mutants.
A histogram of the fraction of colonies detected at each time point for bacteria taken from indicated activated cultures. Deletions in genes ler, grlRA, bfpA, and perC, encoding key regulators of …
Figure 5 with 2 supplements
Co-expression of PerA and PerB results in colony-size bimodality.
(A) A histogram of the fraction of colonies detected at each time point for bacteria taken from indicated cultures of EPEC ΔperA mutant transformed with pPerA-GFP (ΔperA/pPerA-GFP), pPerB-GFP (ΔperA/…
Figure 5—figure supplement 1
Expression control of pPerB-GFP in wild type EPEC.
(A) Histogram of the fraction of colonies wild type EPEC transformed with pPerB-GFP plasmid (wt/pPerB-GFP) and grown as in Figure 5. This experiment was repeated in at least two independent …
Figure 5—figure supplement 2
Co-expression of PerA and PerB causes bimodality of colony growth in E. Coli K-12 MG1655.
A histogram of colony area growth time, i.e. the time taken to increase the colony area from 20 to 80 pixels for bacteria taken from indicated activated cultures of MG1655 E. Coli K-12 bacteria …
Figure 6 with 1 supplement
Bimodality of perABC expression during activation underlies colony-size bimodality.
EPEC ΔperA containing the plasmid pPerABC-GFP was grown under activating conditions. (A) Time-lapse microscopy of the activated ΔperA/ pPerABC-GFP under non-activating conditions (i.e. on LB-agar …
Figure 6—figure supplement 1
Expression of perABC in wild-type EPEC during activation.
(A) Expression of perABC in a wild-type EPEC population grown in activating conditions was analyzed using a transcriptional GFP reporter for perABC (wt/pPerABC-GFP). Activated culture was placed on …
Figure 7 with 1 supplement
Ler expression is unimodal during activation but becomes bimodal when cells are shifted to non-activating conditions.
EPEC ler-gfp was grown under activating conditions. (A) Flow cytometry analysis (t = 0 min) shows unimodal GFP (Ler-ON state) expression (n = 10000). (B) Time-lapse microscopy during growth under …
Figure 7—figure supplement 1
Ler is differentially expressed in BIG and SMALL colony morphotypes.
(A) EPEC containing a chromosomal ler-gfp fusion was grown for 3 hr in DMEM at 37°C and plated on LB agar at 32°C. ScanLag analysis and colony appearance time was monitored and plotted. (B) BIG and …
Figure 8 with 1 supplement
PerABC maintains long-term memory through a hysteretic switch.
(A) Scheme of the hysteretic switch in wild-type EPEC. Subjecting a culture to activating conditions for several hours results in a majority of SMALL bacteria (green) (State 1) (Figure 3B). Even …
Figure 8—figure supplement 1
Loading control of proteins for Western blot analysis.
Total protein analysis of Stain-Free Precast Gels (Bio-Rad) was used as loading control. Same gel was used for protein transfer to membrane and following Western blot procedure (Figure 8). Lane (−) …
Figure 9 with 3 supplements
Bimodal perABC expression correlates with bimodality in microcolony formation and host cell attachment.
(A) Phase-contrast images of the dynamics of self-aggregation observed by time-lapse microscopy of bacteria from BIG or SMALL colonies. Scale bar: 15 μm. See also Videos 1 and 2. (B) Time-lapse …
Figure 9—figure supplement 1
Bacteria from SMALL colonies have enhanced self-aggregation properties in liquid culture and increased formation of microcolonies on host cells.
(A) BFP-related aggregation observed directly in EPEC cultures. EPEC bacteria from different conditions were diluted in LB to ~107 bacteria/ml and grown under non-activating conditions. Images were …
Figure 9—figure supplement 2
Bacteria from SMALL colonies induce massive pedestal formation during HeLa infection.
HeLa cells were infected for 2 hr with different inoculums of ler-GFP EPEC (green). The following cultures were used: EPEC culture grown in DMEM for 3 hr at 37°C (activated), and bacteria from …
Figure 9—figure supplement 3
Invasion of HeLa cells by EPEC from SMALL and BIG colonies.
(A) Schematic illustration of gentamicin protection assay of HeLa cells infected with a 1:1 mixture of wild-type EPEC from SMALL and BIG colonies tagged with GFP and mCherry, respectively. (B) Plot …
Videos
Video 1
Dynamics of self-aggregation observed by time-lapse microscopy in BIG bacteria.
Bacteria were resuspended from a BIG colony and placed on a wet LB agarose pad for imaging bacteria in suspension. Bacteria divide and remain mostly planktonic.
Video 2
The dynamics of self-aggregation observed by time-lapse microscopy in SMALL bacteria.
Bacteria were resuspended from a SMALL colony and placed on a wet LB agarose pad for imaging bacteria in suspension. Bacteria divide and aggregate continuously until they reach stationary phase, …
Video 3
Dynamics of infection by SMALL (green) and BIG (red) bacteria on HeLa cells.
The SMALL bacteria form microcolonies attached to the HeLa cells, whereas the BIG bacteria remain mostly planktonic (same as Figure 9C).
Video 4
Dynamics of infection by SMALL (red) and BIG (green) bacteria on HeLa cells.
The SMALL bacteria form microcolonies attached to the HeLa cells, whereas the BIG bacteria remain mostly planktonic (same as Video 3 but with fluorescent markers opposite tagging).
Additional files
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Supplementary file 1
Whole genome sequencing of the BIG and SMALL morphotypes.
The relative copy number was obtained by coverage analysis of the whole genome sequencing data for BIG and SMALL bacteria (Goldberg et al., 2014). The WGS analysis reveals differences in EAF plasmid copy number only and the raw data is available as NCBI BioProject PRJNA255355 (Accessions: SRX757584 and SRX757585 for SMALL and BIG respectively).
- https://doi.org/10.7554/eLife.19599.027
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Supplementary file 2
List of strains and plasmids.
- https://doi.org/10.7554/eLife.19599.028
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Supplementary file 3
List of primers used in this study.
- https://doi.org/10.7554/eLife.19599.029
