Our results suggest further that YgjD depletion has two (possibly linked) effects: first, depletion triggers (p)ppGpp synthesis. Second, it leads to termination of cell division. To gain insights in which phase of the cell cycle YgjD-depleted cells are arrested we visualized the DNA-content of individual
cells with DNA-staining and subsequent fluorescence microscopy (Additional File 17 – Figure S8). After YgjD depletion in (p)ppGpp+ cells (TB80), DNA was localized at midcell and filled large areas of the cell (Additional File 17 – Figure S8 b), possibly indicating that cells were unable to carry out additional cell divisions due to “”nucleoid occlusion”" [31]. This mechanism prevents premature cell division before chromosomes
www.selleckchem.com/products/elacridar-gf120918.html have been distributed to opposite cell halves. However, termination of cell division also manifests in a (p)ppGpp0 strain (Additional File 17 – Figure S8 c): depleted cells were elongated, 3-deazaneplanocin A chemical structure and only a small fraction of the cell volume was filled with DNA. Thus, in the (p)ppGpp0 background, nucleoid occlusion alone cannot be responsible for termination of cell division. The elongated phenotype of YgjD depleted (p)ppGpp0 cells resembles filamentous cells blocked in cell division. However, since abrogating cell division is not inhibiting DNA replication or DNA segregation [32] it appears unlikely that YgjD directly affects cell division. Conclusions Our results show that single cell experiments coupled with statistical analysis can uncover phenotypic transitions that come about when an essential gene is depleted. We captured phenotypic changes with high temporal resolution across several cell generations. Cell tracking techniques allowed us to build
lineages of cells, and to BYL719 analyze correlations between phenotypic traits at the level of sister cells emerging from the same division. This information can be used to describe growth transitions on the cellular level. We found that YgjD depletion has two, possibly linked, effects: a decrease in cell size that is accompanied by accumulation of (p)ppGpp, and the arrest of cell division. The involvement of (p)ppGpp in the alteration of cell size homeostasis under YgjD depletion conditions might explain the discrepancies between two studies ([3] and [17]) that observed Glutathione peroxidase opposite effects on cell size upon YgjD depletion. Katz et al. [17] used a relA + spoT + strain that is very similar to the ppGpp+ strain TB80 used here, and – consistent with our findings – observed shorter cells upon YgjD depletion. In contrast the MC4100 derivative that was used by Handford and colleagues [3] carries a relA1 allele. This allele is known to cause reduced cellular (p)ppGpp levels under certain growth conditions [26, 33]. Thus, their finding of elongated cells upon YgjD depletion might be similar to what we observed with the ppGpp0 strain TB84.