The 3D-structure of the Escherichia coli nucleoid through one division

Abstract

The three-dimensional (3D) architecture of both eukaryotic and prokaryotic genomes acts to reflect its functional state, but also plays a role in its regulation in terms of replication, recombination, chromosome segregation, and cell division (Dame, Kalmykowa et al. 2011; Thanbichler, Wang et al. 2005). It has become increasingly evident that chromosomal interactions with both each other and other nucleoid components play a critical role within this dynamic relationship between nucleoid organization and genome function in bacteria (Thanbichler, Wang et al. 2005). In addition to its obvious role as a the container for the genetic and biochemical material essential for life, the question remains as to whether bacterial cell shape has a functional contribution in regards to cellular functions including growth, adaptation and division. Previous work achieved an unbiased change in Escherichia coli (E. coli) cell shape by confining single cells within microchambers and forcing them to grow into the corresponding shape of the chamber (Takeuchi, Diluzio et al. 2005). The aim of the original project was to produce both circular and rod shaped E. coli cells in the absence of any genetic mutation based on the method by Takeuchi et al (2005), and subsequently perform Genome Conformation Capture (GCC) and RNA isolation. This would have allowed isolation of the effects of cell shape and concentration effects on both nucleoid structure and genome function. However, unexpected negative results meant that the original aims of this project could not be achieved. Previous works used a mixture of synchronized and non-synchronized prokaryotic cells to study genome organization (Cagliero, Grand et al. 2013; Umbarger, Toro et al. 2011). They both identified linkages between structure and functional outcomes, in particular regarding replication and transcription. However, neither addressed the issue of how structural nucleoid changes are linked to temporal progression through growth stages. Therefore, the aim of the second project was to identify any changes in the global nucleoid structure of an E. coli cell during progression from stationary phase through to cell division, and if these changes relate to transcription. To address this question, a population of E. coli cells was synchronized by passage through stationary phase (adapted from Cutler and Evans 1966), and were subsequently allowed to grow through one generation time. I used GCC and RNA-sequencing to isolate network connectivity maps and the transcriptome of the nucleoid at fixed points; the initial point being when cells were in stationary phase and the final point being just prior to cell division. The results from this experiment provide an understanding of the nucleoid geometry at the micron scale, consistent with the view that the nucleoid has a linearly arranged sausage-shaped structure with a temporally-conserved “well-selfattached core” (Viollier, Thanbichler et al. 2004; Toro and Shapiro 2010). Moreover, the detection of both long and short distance interactions is consistent with previous work involving empirical measures and modeling which indicated that intra-nucleoid interactions play an important role in the E. coli nucleoid structure (Wiggins, Cheveralls et al. 2010).