Inducible asymmetric cell division and cell differentiation in a bacterium

Abstract

Multicellular organisms achieve greater complexity through cell divisions that generate different cell types. We engineered a simple genetic circuit that induces asymmetric cell division and subsequent cell differentiation in Escherichia coli. The circuit involves a scaffolding protein, PopZ, that is stably maintained at a single cell pole over multiple asymmetric cell divisions. PopZ was functionalized to degrade the signaling molecule, c-di-GMP. By regulating synthesis of functionalized PopZ via small molecules or light, we can chemically or optogenetically control the relative abundance of two distinct cell types, characterized by either low or high c-di-GMP levels. Differences in c-di-GMP levels can be transformed into genetically programmable differences in protein complex assembly or gene expression, which in turn produce differential behavior or biosynthetic activities. This study shows emergence of complex biological phenomena from a simple genetic circuit and adds programmable bacterial cell differentiation to the genetic toolbox of synthetic biology and biotechnology.

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Fig. 1: A method for using polar asymmetry to generate two distinct cell types.
Fig. 2: Direct visualization of c-di-GMP levels in asymmetrically dividing cells using a split-GFP reporter.
Fig. 3: Differential gene expression in asymmetrically dividing cells.
Fig. 4: Control of asymmetric cell division by light.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

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Acknowledgements

The authors would like to thank J. Tabor for providing plasmids related to the light-inducible transcription activation system. N. Ward provided reagents and equipment. K. Helm and C. Childs, from the Flow Cytometry Shared Resource at the University of Colorado Cancer Center, were supported by the Cancer Center Support Grant No. P30CA046934. This work was supported by School of Energy Resources at the University of Wyoming and by the National Institutes of Health under award numbers 2P20GM103432 and R01GM118792.

Author information

N.V.M. conceived and performed experiments, collected and analyzed data and contributed to the writing of the manuscript. A.F. had a similar role relating to the development of the c-di-GMP biosensor. M.G. guided research strategies and edited the manuscript. G.R.B. conceived experiments, guided research strategies and wrote the manuscript.

Correspondence to Grant R. Bowman.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Table 1, Supplementary Figures 1–10, Supplementary Note 1

Reporting Summary

Supplementary Video 1

Asymmetric cell division of YhjH-mChy-PopZ positive cells bearing plasmids cells bearing plasmid pBad-YmP. Same conditions as Fig. 1c. This experiment was performed three times and produced similar results.

Supplementary Videos 2

Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.

Supplementary Videos 3

Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.

Supplementary Video 4

Phase contrast movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed more than three times and produced similar results.

Supplementary Video 5

mChy fluorescence movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed three times and produced similar results.

Supplementary Video 6

Phase contrast movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, right panels. This experiment was performed more than three times and produced similar results.

Supplementary Video 7

mChy fluorescence movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Figure 4f,g, right panels. This experiment was performed three times and produced similar results.

Supplementary Video 8

Phase contrast movie of wild-type (MG1655) cells. This experiment was performed more than three times and produced similar results.

Supplementary Video 9

Phase contrast movie of ∆motA-motB strain. This experiment was performed more than three times and produced similar results.

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