Asymmetric cell division is one of the principal mechanisms to accomplish pattern formation and cell specialization. In metazoans, stem cells have the ability to either generate two identical copies of themselves or to divide asymmetrically and thereby initiate the production of a differentiated progeny. Cellular differentiation induces changes at the epigenetic level and substantially alters the transcriptional landscape of the daughter cell. Altogether, these changes make de-differentiation a rare event. Torecapitulate cellular differentiation in a population of bacteria, biologists implemented various synthetic circuits based on co-repressive toggles. Despite being functional and sensitive to stimuli, these systems are usually difficult to tune and differentiation is only occurring transiently. In this study, in order to achieve cellular differentiation in a reliable manner, Molinari and colleagues engineer an inducible system for the asymmetric segregation of plasmids.
In order to engineer a synthetic E. coli strain able to asymmetrically divide upon stimulation, Molinari and colleagues exploit the chromosome partitioning (par) system of the bacterium C. crescentus. The underlying mechanisms of the par system are commonly used by different organisms for segregating low copy number plasmids and rely on the pairing of a cis-acting centromere-like sequence (the parS region) to a centromere-binding protein (parB-like protein). The parS-parB interaction results in the formation of a large nucleoprotein complex that, if needed, can be shuttled to the opposite poles of a diving cell by dedicated motor proteins. The asymmetric plasmid partition (APP) system presented in the paper is based on two plasmids. The “target plasmid” contains the centromere-like sequence (parS), along with a constitutively-expressed red fluorescent protein (mRFP). The second plasmid, called “regulatory plasmid”, encodes for the parB protein fused to a yellow fluorescent protein (sfYFP) under control of an arabinose-inducible promoter. When cells are grown under normal conditions, both plasmids are symmetrically segregated and are present in all the cell of a population. In presence of arabinose, the parB-sfYFP protein is expressed and binds to the parS region of the target plasmid. The formation of bulky nucleoprotein complexes results in the retention of the target plasmids in one of the two daughter cells. Loss of the target plasmid in the second daughter cell is then seen as its terminal differentiation.
Live-cell imaging of arabinose-treated cells impressively shows how, once reaching a considerable size, the yellow parS-containing nucleoprotein complexes are retained in the undifferentiated cell. Despite initially being present in both daughter cells, the red fluorescence is quickly lost and is never observed to be recovered in the differentiated cells. To further test their system, the authors grew the progenitor cells without selection and in the absence of arabinose. Under these conditions, APP does not occur and cells can be interchangeably plated with or without selection. Differently, if cells are initially grown in presence of arabinose, only the undifferentiated cells -which retained the nucleoprotein complexes- form colonies in selective plates. By picking one of these colonies and repeating the above-described procedure, the authors obtained strikingly reproducible results, indicating that progenitor cells are able to fully recover from a round of APP.
The pMB origin of replication present in the original target plasmid confers a low number of ~10-20 copies/cell. The authors investigate if the APP system is equally capable of dealing with a higher number of target plasmids (~300-500 copies/cell) or a very low number of plasmids (~5 copies/cell) which, in normal conditions, are actively partitioned by E. coli cells. To do this, the pMB origin present in the original target plasmid was either substituted with the pUC or the pSC101 origins of replication. Afterwards, the three versions of the target plasmid were tested with various amounts of inducer to explore the tunability of the APP systems. These experiments showed that the loss of progenitor cells in the population is dose dependent. Additionally, the performance of the APP system is not affected by changes in copy number or the presence on the plasmid of an actively-segregated origin of replication.
At last, by repurposing the SopC/SopB elements of E. coli’s fertility plasmid the authors implemented a second, independent APP circuit. Shortly, the parS sequence in the target plasmid was exchanged with SopC, whereas the parB gene in the regulatory plasmid was substituted with SopB. Similar to what was observed for the parS/ParB system, increasing amounts of arabinose cause a decrease in the fraction of progenitor cells. Interestingly, parB only recognizes and binds ParS, whereas SopB exclusively binds SopC. This feature makes the two APP systems orthogonal to each other and opens the possibility of combining them in the same cell. Next, the authors explore this opportunity by generating a three plasmids APP circuit, comprising a regulatory plasmid carrying the parB and the sopB genes and two target plasmids including either the parS or the sopC sequence. In order to be responsive to independent stimuli, parB expression is driven by an arabinose promoter and SopB is controlled by an IPTG promoter. Using the assays already described above, Molinari and colleagues show that progenitor cells carrying this new circuit can partially differentiate, if only treated with one inducer, or terminally differentiate when exposed to both inducers. Notably, terminal differentiation can be achieved by both contemporary and sequential addition of the two inducers and in all experiments some of the progenitor cells remain in the culture, ready to reseed the population after induction has ceased.
What I like about this work
Asymmetric cell division is a process commonly observed in a variety of organisms to produce two different daughter cells. The authors of this study aimed at engineering an E. coli strain able to asymmetrically segregate a reporter plasmid upon induction. I like how Molinari and colleagues repurpose a naturally occurring mechanism -usually used by bacteria for symmetrically segregating low copy number plasmids- to achieve asymmetric segregation at will in a synthetic cell. In my opinion the manuscript is presenting a neat and robust synthetic circuit enabling cellular differentiation.
How long do the progenitor cells need to be exposed to a transient stimulus in order to initiate differentiation?
In figure 1d, the nucleoprotein complex is localizing to the cell’s pole. Is this pattern stochastic? Or is parB actively retained at this location?
Since ParB and SopB are not interacting, the relative nucleoprotein complexes could independently segregate to the two daughter cells upon double induction. Do you see this happening and if yes, how often?
Do you reckon the system could be expanded to three, or more, orthogonal circuits?
Posted on: 17th February 2019