Self-organised segregation of bacterial 1 chromosomal origins 2

12 The chromosomal replication origin region (ori) of characterized bacteria is dynamically positioned throughout the cell 13 cycle. In slowly growing Escherichia coli, ori is maintained at mid-cell from birth until its replication, after which newly 14 replicated sister oris move to opposite quarter positions. Here, we provide an explanation for ori positioning based on the 15 self-organisation of the Structural Maintenance of Chromosomes complex, MukBEF, which forms dynamically 16 positioned clusters on the chromosome. We propose that a non-trivial feedback between the self-organising gradient of 17 MukBEF complexes and the oris leads to accurate ori positioning. We find excellent agreement with quantitative 18 experimental measurements and confirm key predictions. Specifically, we show that oris exhibit biased motion towards 19 MukBEF clusters, rather than mid-cell. Our findings suggest that MukBEF and oris act together as a self-organising 20 system in chromosome organisation-segregation and introduces protein self-organisation as an important consideration 21 for future studies of chromosome dynamics. 22 Introduction 23 The faithful and timely segregation of genetic material is essential for all cellular life. In eukaryotes the responsibility for 24 chromosome segregation lies with a well-understood macromolecular machine, the mitotic spindle. In contrast, the 25 mechanisms underlying bacterial chromosome segregation are much less understood mechanistically, but are just as 26 critical for cellular proliferation (Badrinarayanan et al., 2015). The starting point for bidirectional chromosomal 27 replication, the origin (ori), has a crucial role in chromosome organisation and segregation. Not only is it duplicated and 28 segregated first but its dynamic genomic position defines the position of other chromosomal regions with respect to the 29 cell (Duigou and Boccard, 2017; Junier et al., 2014). 30 In new-born Escherichia coli cells growing under relatively slow growing conditions in which initiation of replication 31 and its completion occur within a single cell generation, the ‘home’ position of the origin (henceforth and in the model, 32 ori) is at mid-cell (Nielsen et al., 2006b; Niki et al., 2000; Wang et al., 2006). After replication, and consequent 10-15 33 2 min of ‘cohesion’, arising at least in part from interlinking of the two daughter chromosomes (precatenation) (Joshi et al., 34 2011; Nicolas et al., 2014; Nielsen et al., 2006a; Nolivos et al., 2016; Reyes-Lamothe et al., 2008; Wang et al., 2008), 35 duplicated origins migrate rapidly to opposite quarter positions, which become the new home positions for the remainder 36 of the cell cycle (Cass et al., 2016; Kuwada et al., 2013). Other genomic loci migrate sequentially with similar dynamics 37 (Cass et al., 2016). 38 The mechanisms that underlie ori positioning and direct newly replicated sisters to opposite cell halves remain unclear 39 (Badrinarayanan et al., 2015). This is particularly the case in E. coli and its relatives, which do not carry ParABS systems 40 that facilitate the segregation of low copy plasmids and some other bacterial chromosomes (Livny et al., 2007). However, 41 MukBEF, a functional homolog of ubiquitous Structural Maintenance of Chromosomes (SMC) complexes (Nolivos and 42 Sherratt, 2014; Rybenkov et al., 2014), plays a role in E. coli chromosome organisation-segregation. One of its functions 43 is to recruit the type II topoisomerase Topo IV (Li et al., 2010; Nicolas et al., 2014), which is required for the timely 44 removal of catenanes from newly replicated sister chromosomes (Wang et al., 2008). Under slow growth conditions, 45 MukBEF forms a small number of dynamic clusters (visualised as fluorescent foci) located at the middle or quarter 46 positions (Den Blaauwen et al., 2001; Ohsumi et al., 2001), in close association with ori (Danilova et al., 2007), and the 47 splitting and movement of these foci occurs concurrently with the segregation of oris to the quarter positions (Danilova et 48 al., 2007; Nicolas et al., 2014). Foci consist of on average 16 dimeric slowly-diffusing MukBEF complexes 49 (Badrinarayanan et al., 2012b). The colocalisation with ori is not required for either MukBEF foci formation or 50 positioning: depletion of Topo IV results in cells with multiple catenated oris forming a single focus at mid-cell but with 51 multiple MukBEF clusters positioned throughout the nucleoid (Nicolas et al., 2014). Thus MukBEF clusters are not 52 necessarily assembled at or bound to ori, consistent with the lack of any sequence specificity (Nolivos et al., 2016). 53 Furthermore, restoration of Topo IV activity leads to the decatenated oris moving to the MukBEF clusters suggesting that 54 MukBEF recruits or positions ori (Nicolas et al., 2014). Consistent with this hypothesis, depletion of functional MukBEF 55 results in ori mis-positioning that is subsequently restored upon repletion (Badrinarayanan et al., 2012a). 56 If MukBEF clusters position oris, what positions MukBEF clusters? Given that molecules in the clusters turnover 57 continuously with a timescale of about one minute (Badrinarayanan et al., 2012b) and that MukBEF binds DNA non58 specifically, how does it even form clusters? We have proposed that a self-positioning stochastic Turing pattern can 59 explain the positioning of MukBEF clusters (Murray and Sourjik, 2017) (see Figure 2, Figure 2–figure supplement 1 and 60 the methods section for a review). A Turing pattern is a spatial pattern in the concentration of a reactant in a reaction61 diffusion system that arises spontaneously due to a diffusion-driven instability (Koch and Meinhardt, 1994; Turing, 62 1952). Put simply, diffusion, rather than having a homogenising effect can actually, in combination with chemical 63 reactions, create a spatially varying concentration profile. Such patterns are examples of self-organisation, a more general 64 term that describes any dissipative non-equilibrium energy-dependent order that arises as a result of collective non-linear 65 interactions (Halley and Winkler, 2008). We used the Turing mechanism to explain the positioning of MukBEF foci and 66 showed that a flux-balance mechanism and stochasticity work together to ensure that a specific Turing pattern is selected: 67 short cells consistently have a single centre-positioned peak in the MukBEF concentration, while longer cells have 68 quarter positioned peaks. 69 With this model in hand, we now investigate how MukBEF clusters could position chromosomal origins. In particular, 70 we address whether the self-organising MukBEF gradient proposed in our model has the correct properties to act as an 71 attracting gradient for ori. Additionally, it is critical that each newly replicated sister ori is recruited to a different 72 3 MukBEF focus, a non-trivial requirement. We find that a self-organising MukBEF gradient can indeed accurately 73 reproduce the observed ori dynamics, apparent diffusion constant and drift rate. A proposed preferential loading of 74 MukBEF within ori introduces a non-trivial interaction between MukBEF foci and oris that leads to accurate and stable 75 partitioning as an emergent property of the system. Importantly, the model does not contain any actual directed force. 76 MukBEF requires energy in the form of ATP to establish a self-organised gradient but it is not pulled to the middle or 77 quarter positions by any active force. Similarly, the attraction of ori up the MukBEF gradient may be due to energetic 78 considerations and the elastic nature of the chromosome (a DNA-relay) resulting on the macro scale in an effective 79 (rectification) force and directed motion. 80