Construction of intracellular asymmetry and asymmetric division in Escherichia coli

Abstract

The design principle of establishing an intracellular protein gradient for asymmetric cell division is a long-standing fundamental question. While the major molecular players and their interactions have been elucidated via genetic approaches, the diversity and redundancy of natural systems complicate the extraction of critical underlying features. Here, we take a synthetic cell biology approach to construct intracellular asymmetry and asymmetric division in Escherichia coli, in which division is normally symmetric. We demonstrate that the oligomeric PopZ from Caulobacter crescentus can serve as a robust polarized scaffold to functionalize RNA polymerase. Furthermore, by using another oligomeric pole-targeting DivIVA from Bacillus subtilis, the newly synthesized protein can be constrained to further establish intracellular asymmetry, leading to asymmetric division and differentiation. Our findings suggest that the coupled oligomerization and restriction in diffusion may be a strategy for generating a spatial gradient for asymmetric cell division.

Localized Proteolysis for the Construction of Intracellular Asymmetry in Escherichia coli

“…The reporter was constitutively expressed in the cell; however, the level would be low due to destabilization via the C-end degron. PopZ, an oligomeric protein upstream in the C. crescentus polarity pathway, , was used as the polarized scaffold to reconstitute enzymatic functions; SpmXΔC, a fragment of the stalk-positioning factor that preserves a direct binding to PopZ in E. coli , , was used as the adaptor for recruitment. Both N (1–118) and C (119–242) halves of the TEV proteases were designed to fuse with the SpmXΔC adaptor.…”

“…When PopZ is present, TEV is reassembled and becomes functional at the PopZ pole, enabling a localized cleavage of the C-terminal degron and thus a polarized stabilization of the reporter (Figure b). We previously demonstrated that, in order to establish an intracellular asymmetry at the length scale of an E. coli cell, diffusion must be actively countered . Given this prior knowledge, DivIVA, a Bacillus subtilis oligomeric protein with a propensity to target negative membrane curvature, , was fused to GFP and used as the reporter in the design.…”

“…Given this prior knowledge, DivIVA, a Bacillus subtilis oligomeric protein with a propensity to target negative membrane curvature, , was fused to GFP and used as the reporter in the design. When DivIVA-GFP is stabilized at the PopZ pole, the cooperative pole-targeting capability can compete with the diffusion toward the midcell . Also, owing to its oligomeric nature, the diffusion coefficient of DivIVA-GFP is 20-fold smaller than regular GFP (0.32 ± 0.1 and 7.23 ± 3.05 μm 2 s –1 for DivIVA-GFP and freely diffusing GFP, respectively), rendering it more likely to be immobilized at the pole.…”

“…Given the aforementioned protein-based tools, we reasoned that we could design a synthetic circuit that performs local degradation to artificially generate intracellular asymmetry in Escherichia coli . In a recent study, we demonstrated that the oligomeric protein PopZ from Caulobacter crescentus could serve as a robust synthetic polarity basis to reconstitute split T7 TNA polymerase and activate gene expression asymmetrically in E. coli . Here, we reported that a phenotypically similar intracellular asymmetry can also be achieved in E. coli with a complementary design that utilizes split tobacco etch virus (TEV) protease to locally stabilize an unstable protein, again through a reconstitution via the PopZ scaffold.…”

Agrochemical control of gene expression using evolved split RNA polymerase

“…Self-assembly of the evolved T7 RNAP system (eRNAP) has been largely eliminated through series of directed evolution ( Pu, Zinkus-Boltz & Dickinson, 2017 ; Pu, Kentala & Dickinson, 2018 ). This eRNAP platform enables detecting protein interaction ( Dewey & Dickinson, 2020 ; Lin et al, 2021 ) and manipulating strength of interaction ( Zinkus-Boltz, DeValk & Dickinson, 2019 ; Dewey et al, 2021 ). ABA inducible CRISPR/Cas9 gene editing in mammalian cells has been demonstrated using this evolved split T7 RNAP system ( Pu, Kentala & Dickinson, 2018 ).…”