Analysis of ParAB dynamics in mycobacteria shows active movement of ParB and differential inheritance of ParA

Abstract

Correct chromosomal segregation, coordinated with cell division, is crucial for bacterial survival, but despite extensive studies, the mechanisms underlying this remain incompletely understood in mycobacteria. We report a detailed investigation of the dynamic interactions between ParA and ParB partitioning proteins in Mycobacterium smegmatis using microfluidics and time-lapse fluorescence microscopy to observe both proteins simultaneously. During growth and division, ParB presents as a focused fluorescent spot that subsequently splits in two. One focus moves towards a higher concentration of ParA at the new pole, while the other moves towards the old pole. We show ParB movement is in part an active process that does not rely on passive movement associated with cell growth. In some cells, another round of ParB segregation starts before cell division is complete, consistent with initiation of a second round of chromosome replication. ParA fluorescence distribution correlates with cell size, and in sister cells, the larger cell inherits a local peak of concentrated ParA, while the smaller sister inherits more homogeneously distributed protein. Cells which inherit more ParA grow faster than their sister cell, raising the question of whether inheritance of a local concentration of ParA provides a growth advantage. Alterations in levels of ParA and ParB were also found to disturb cell growth.

Competition between DivIVA and the nucleoid for ParA binding promotes segrosome separation and modulates mycobacterial cell elongation

“…A careful analysis of ParB positioning during the cell cycle revealed that the disruption of the ParA interaction with DivIVA altered the movement of segrosomes. It was previously shown that the ParB complex that migrates towards the new cell pole exhibits higher velocity than the one migrating towards the old cell pole (Ginda et al , ; Uhía et al ., ). Moreover, a recent visualisation of the nucleoid in relation to the localisation of ParB complexes revealed that the nucleoid is asymmetrically positioned and shifted towards the new pole and that the old pole segrosome is located close to the edge of the nucleoid throughout most of the cell cycle (Hołówka et al , ).…”

“…Although the control of the mycobacterial cell cycle is crucial for pathogenicity, the orchestration of its key events remains unexplored. While previous studies have described the main components of the mycobacterial chromosome segregation machinery, the ParA and ParB proteins, and have reported ParA interaction with a polar growth determinant DivIVA homologue (named Wag31 in mycobacteria) (Jakimowicz et al, 2007;Ginda et al, 2013;Uhía et al, 2018), the exact mechanism of asymmetric chromosome segregation and the biological role of the ParA-DivIVA interaction have not been investigated. In mycobacteria, as in most studied bacterial species (Bacillus subtilis, Caulobacter crescentus, Vibrio cholerae, Myxococcus xanthus, Corynebacterium glutamicum, and Streptomyces coelicolor, but not Escherichia coli and some other γ-proteobacteria), origin of chromosomal replication (oriC) regions are organised into complexes named segrosomes by ParB binding to oriC-proximal parS sites (Chaudhuri and Dean, 2011;Wang et al, 2013;Graham et al, 2014;Zhang and Schumacher, 2017).…”

“…Usually immediately (but in some species after some cohesion time) following the initiation of replication, ParB complexes duplicate, and then one or both complexes are moved to their target cell locations. The separation of segrosomes and their movement are dependent on the action of ParA (Fogel and Waldor, 2006;Toro et al, 2008;Gerdes et al, 2010;Ptacin et al, 2010;Lutkenhaus, 2012;Yamaichi et al, 2012;Lim et al, 2014); however, the generation of the force that moves segregating ParB complexes was under debate for a long time and only recently the widely accepted model emerged (Leonard et al, 2005;Gerdes et al, 2010;Lutkenhaus, 2012;Funnell, 2014;Lim et al, 2014;Vecchiarelli et al, 2014;Badrinarayanan et al, 2015;Le Gall et al, 2016;Surovtsev et al, 2016). ParA homologues bind ATP, dimerise, and, as ATP-bound dimers, non-specifically interact with the nucleoid (Leonard et al, 2005;Hester and Lutkenhaus, 2007).…”

“…According to diffusion-based models of ParB movement, ParA binding to nucleoid is crucial for the transport of ParB complexes. The interaction with the ParB complex enhances ATP hydrolysis and triggers ParA release from the nucleoid, generating a gradient of nucleoid-bound ParA and providing the driving force for segrosome movement (Lim et al, 2014;Vecchiarelli et al, 2014;Surovtsev et al, 2016). Markedly, for most of the cell cycle, ParB complexes are precisely localised in bacterial cells in genus-specific positions, e.g.…”

Spatial control over near-critical-point operation ensures fidelity of ParABS-mediated bacterial genome segregation

“…As such, the ParABS system can work as a Brownian ratchet: the ParB-bound cargo "self-drives" by both creating and following a ParA gradient over the DNA. This protein gradient-based Brownian ratchet model provides a conceptual framework that allowed us to explain -for the first time in a coherent manner -the diverse motility patterns of PCs evidenced in vivo (Hu et al 2017a) and starts to gain support from in vivo experiments (Debaugny et al 2018;Le Gall et al 2016;Schumacher et al 2017;Uhía et al 2018). However, our effort so far focused on a highly simplified picture, it is unclear 1) whether and how this Brownian ratchet mechanism can adapt the plasmid segregation distance to the length of an elongating nucleoid, and 2) how the PC partition ensures its fidelity against cellular noises.…”