Open Access Highly Accessed Research article

Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

Tobias Bergmiller1*, Rafael Peña-Miller2, Alexander Boehm3 and Martin Ackermann1

Author Affiliations

1 Department of Environmental Sciences, ETH Zurich, Switzerland, and Department of Environmental Microbiology, Eawag, Switzerland

2 Biosciences, University of Exeter, Exeter, UK

3 Institut für Molekulare Infektionsbiologie, University of Wuerzburg, Germany

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BMC Microbiology 2011, 11:118  doi:10.1186/1471-2180-11-118

Published: 27 May 2011

Additional files

Additional File 1:

Movie 1. TB80 (ppGpp+) growing on LB agar with 0.1% L-arabinose. 100 frames (one frame per two minutes) were compressed into 10 seconds. The scale bar is 5 μm in size (same in all movies hereafter).

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Additional File 2:

Movie 2: MG1655 growing on LB agar with 0.4% glucose. 100 frames (one frame per two minutes) were compressed into 10 seconds.

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Additional File 3:

Figure S1: MG1655 expressing GFP from Para shifted from LB arabinose 0.01% to LB glucose 0.4%. This experiment was performed with the wild type strain MG1655 carrying a plasmid encoding a transcriptional fusion of gfp to Para [29]. The strain was grown in 0.01% arabinose, analogously to the depletion experiments with TB80 and TB84, washed in LB supplemented with glucose and transferred onto an agar pad consisting of LB agar with 0.4% glucose. The level of GFP fluorescence decreased rapidly and approached the level of background fluorescence when cells reached generation 4.

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Additional File 4:

Movie 3. TB80 (ppGpp+) growing on LB agar with 0.4% glucose. 150 frames (one frame per two minutes) were compressed into 15 seconds. This movie was used to extract the growth dynamics shown in Figure 2 and 3.

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Additional File 5:

Figure S2: Lineage trees of microcolonies of A) MG1655 growth and B) YgjD depletion. After tracking of individual cells across recorded images with "Schnitzcell", the lineage structure of a microcolony can be derived. In such a lineage tree, the branch length corresponds to the time interval between divisions, and division events occur at branching points. The different colors depict the color code used for cells from different generations throughout all figures. The dots at the end of individual branches represent the time points where individual physiological measurements (cell size and fluorescent intensity) were derived from.

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Additional File 6:

Figure S3: Depletion of essential genes induces unique phenotypes. Time-lapse experiments of cells depleting for fldA, ffh and dnaT (see Additional Files 7, 8 and 9 - movies 4, 5 and 6) were tracked, and the cell size at division over consecutive divisions was plotted.

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Additional File 7:

movie 4: Depletion of FldA from growing cells. A Para-fldA conditional lethal mutant was shifted from 0.1% arabinose to an agar pad with 0.4% glucose. FldA is essential for isoprenoid biosynthesis [44], and as the movie shows, depletion of FldA leads to lysis of cells. 80 frames (one frame per four minutes) were compressed into 8 seconds.

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Additional File 8:

movie 5: Depletion of Ffh from growing cells. A Para-ffh conditional lethal mutant was shifted from 0.1% arabinose to an agar pad with 0.4% glucose. Ffh protein is part of the signal recognition particle translocation system, that cotranslationaly sequesters proteins into or across the cytoplasmic membrane [45]. Depletion resulted in visible intracellular aggregates, followed by elongation and cell lysis. 120 frames (one frame per two minutes) were compressed into 12 seconds.

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Additional File 9:

movie 6: Depletion of DnaT from growing cells. A Para-dnaT conditional lethal mutant was shifted from 0.01% arabinose to a 0.4% glucose containing agar pad. Depletion resulted in filament formation, which is in agreement with "unbalanced" growth upon abrogation of DNA replication. dnaT (and the following gene dnaC) is part of the "primosome" and is crucial for initiation of DNA replication. 100 frames (one frame per four minutes) were compressed into 10 seconds.

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Additional File 10:

Figure S4: Effects of minimum inhibitory concentrations (MIC) of chloramphenicol and kanamycin on growth of E. coli MG1655. Recorded image series of E.coli MG1655 growing on MIC concentrations of chloramphenicol (2.5 μg/ml) and kanamycin (5 μg/ml) (see Additional Files 11 and 12 - movies 7 and 8) were tracked, and the cell size over consecutive division was plotted.

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Additional File 11:

movie 7: Growth of E. coli MG1655 on 2.5 μg/ml chloramphenicol. E. coli MG1655 was precultured in LB medium and transferred to an agar pad containing 2.5 μg/ml chloramphenicol. 100 frames (one frame per four minutes) were compressed into 10 seconds,.

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Additional File 12:

movie 8: Growth of E. coli MG1655 on 5 μg/ml kanamycin. E. coli MG1655 was precultured in LB medium and transferred to an agar pad containing 5 μg/ml kanamycin. 60 frames (one frame per four minutes) were compressed into 6 seconds.

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Additional File 13:

Figure S5: Coupling of cell elongation rate and interval between division across multiple experiments. The pattern observed in Figure 3 is repeatable and consistent across independent experiments. Non-parametric correlation analysis for the differences between sisters in these two traits was performed for seven independent microcolonies (YgjD depletion in TB80), and the median and the range of the correlation coefficients is reported; the median correlation coefficients are negative from generation 3 on, indicating a coupling between cell elongation rate and the interval between two divisions.

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Additional File 14:

Movie 9. TB84 (ppGpp0) growing on LB agar with 0.4% glucose. 200 frames (one frame per two minutes) were compressed into 20 seconds.

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Additional File 15:

Figure S6: YgjD is also essential in absence of (p)ppGpp. Data of cell numbers versus time from three independent experiments; each experiment is based on a microcolony that was initiated with a single cell of strain TB84 (ppGpp0), and grown in the presence of glucose, leading to YgjD depletion. Cell division terminates after about five to six divisions.

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Additional File 16:

Figure S7: Control movies of Papt and Prsd expression of TB80 grown with 0.1% L-arabinose. Single cell measurements of cell elongation rate and GFP fluorescence of two strains with transcriptional reporters for Papt (A and B) and Prsd (B and C), analogous to Figure 5 in the main manuscript.

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Additional File 17:

Figure S8: DNA staining of cells with and without YgjD in TB80 (ppGpp+) and TB84 (ppGpp0). Cells were grown for two hours in liquid culture, and stained with 1 μg/ml DAPI (4',6-diamidino-2-phenylindole) to visualize DNA. Scale bars are 5 μm. A) TB80 grown with 0.1% arabinose to induce YgjD expression. B) TB80 grown with 0.4% glucose, leading to YgjD depletion. Cells are small, and the DNA stain occupies a large fraction of the cell area. C) TB84 grown with 0.4% glucose, leading to YgjD depletion. Cells are elongated, and the DNA stain only occupies a small fraction of the cell area.

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