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.

Controlling gene expression with light: a multidisciplinary endeavour

“…Chemical modification of nucleic acids with photocages has been used to create conductive pathways in synthetic tissues [ 37 , 38 ] and control the development of zebrafish [ 7 , 55 , 60 , 134 , 135 ] and cancer therapeutics [ 75 , 76 , 79 , 137 ] with light. While naturally light-sensitive proteins have been used to control cell division [ 99 ], bioreactors [ 129 ], and genome activation in zebrafish and mice [ 146 , 151 , 153 ]. Identification of new applications is vital to realising the full potential of light-controlled gene expression.…”

“…In their most basic forms, these systems were used in bacterial edge detection algorithms [ 94 ] and for dual-colour control over gene expression [ 95 ]. More efficient versions were later developed by genetic refactoring and mutagenesis [ 96 , 97 ] and have been used to tightly control metabolic flux [ 98 ], cell division [ 99 ] and feedback loops in bacteria [ 100 ].…”

Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria

“…In the presence of c-di-GMP, FimX and PilZ interact leading to a close proximity between GFP 11 and GFP 10 which reconstitute with GFP 1-9 to form a fluorescent GFP. The fluorescence intensity reflects the c-di-GMP concentration and heterogeneity in c-di-GMP levels in a population can be analysed by microscopy or flow cytometry (Mushnikov et al 2019).…”

Construction of intracellular asymmetry and asymmetric division in Escherichia coli

“…An asymmetric division of the PopZ scaffold in E. coli has been reported since its first identification 21 . Only recently did a study begin to directly couple PopZ to downstream signaling for a synthetic cell differentiation mechanism in E. coli 52 . Specifically, a c-di-GMP-degrading phosphodiesterase was fused with PopZ to differentiate c-di-GMP levels, and differential gene expression was achieved via the asymmetric inheritance of this key enzyme 52 .…”

“…Only recently did a study begin to directly couple PopZ to downstream signaling for a synthetic cell differentiation mechanism in E. coli 52 . Specifically, a c-di-GMP-degrading phosphodiesterase was fused with PopZ to differentiate c-di-GMP levels, and differential gene expression was achieved via the asymmetric inheritance of this key enzyme 52 . Note that in the study, differentiation (i.e., the difference in fluorescent reporter levels) was observed at the second or later divisions 52 .…”