fluorescence-live-cell-imaging-complete-vegetative-cell-cycle-slow

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium <em>Myxococcus xanthus</em>

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

Max Planck Institute for Terrestrial Microbiology

Accurate positioning of the division site is essential to generate appropriately sized daughter cells with the correct chromosome number. In bacteria, division generally depends on assembly of the tubulin homologue FtsZ into the Z-ring at the division site. Here, we show that lack of the ParA-like protein PomZ in Myxococcus xanthus resulted in division defects with the formation of chromosome-free minicells and filamentous cells. Lack of PomZ also caused reduced formation of Z-rings and incorrect positioning of the few Z-rings formed. PomZ localization is cell cycle regulated, and PomZ accumulates at the division site at midcell after chromosome segregation but prior to FtsZ as well as in the absence of FtsZ. FtsZ displayed cooperative GTP hydrolysis in vitro but did not form detectable filaments in vitro. PomZ interacted with FtsZ in M. xanthus cell extracts. These data show that PomZ is important for Z-ring formation and is a spatial regulator of Z-ring formation and cell division. The cell cycle-dependent localization of PomZ at midcell provides a mechanism for coupling cell cycle progression and Z-ring formation. Moreover, the data suggest that PomZ is part of a system that recruits FtsZ to midcell, thereby, restricting Z-ring formation to this position.

PomZ, a ParA-like protein, regulates Z-ring formation and cell division in Myxococcus xanthus.

Cells closely coordinate cell division with chromosome replication and segregation; however, the mechanisms responsible for this coordination still remain largely unknown. Here, we analyzed the spatial arrangement and temporal dynamics of the 9.1 Mb circular chromosome in the rod-shaped cells of Myxococcus xanthus. For chromosome segregation, M. xanthus uses a parABS system, which is essential, and lack of ParB results in chromosome segregation defects as well as cell divisions over nucleoids and the formation of anucleate cells. From the determination of the dynamic subcellular location of six genetic loci, we conclude that in newborn cells ori, as monitored following the ParB/parS complex, and ter regions are localized in the subpolar regions of the old and new cell pole, respectively and each separated from the nearest pole by approximately 1 µm. The bulk of the chromosome is arranged between the two subpolar regions, thus leaving the two large subpolar regions devoid of DNA. Upon replication, one ori region remains in the original subpolar region while the second copy segregates unidirectionally to the opposite subpolar region followed by the rest of the chromosome. In parallel, the ter region of the mother chromosome relocates, most likely passively, to midcell, where it is replicated. Consequently, after completion of replication and segregation, the two chromosomes show an ori-ter-ter-ori arrangement with mirror symmetry about a transverse axis at midcell. Upon completion of segregation of the ParB/parS complex, ParA localizes in large patches in the DNA-free subpolar regions. Using an Ssb-YFP fusion as a proxy for replisome localization, we observed that the two replisomes track independently of each other from a subpolar region towards ter. We conclude that M. xanthus chromosome arrangement and dynamics combine features from previously described systems with new features leading to a novel spatiotemporal arrangement pattern. 

Tracking of chromosome and replisome dynamics in Myxococcus xanthus reveals a novel chromosome arrangement.

Cell division site positioning is precisely regulated to generate correctly sized and shaped daughters. We uncover the strategy used by the social bacterium Myxococcus xanthus to position the FtsZ cytokinetic ring at midcell. PomX, PomY, and the nucleoid-binding ParA/MinD ATPase PomZ self-assemble forming a large nucleoid-associated complex that localizes at the division site before FtsZ to directly guide and stimulate division. PomXYZ localization is generated through self-organized biased random motion on the nucleoid toward midcell and constrained motion at midcell. Experiments and theory show that PomXYZ motion is produced by diffusive PomZ fluxes on the nucleoid into the complex. Flux differences scale with the intracellular asymmetry of the complex and are converted into a local PomZ concentration gradient across the complex with translocation toward the higher PomZ concentration. At midcell, fluxes equalize resulting in constrained motion. Flux-based mechanisms may represent a general paradigm for positioning of macromolecular structures in bacteria.

The PomXYZ Proteins Self-Organize on the Bacterial Nucleoid to Stimulate Cell Division.

Rod-shaped Myxococcus xanthus cells are polarized with proteins asymmetrically localizing to specific positions. This spatial organization is important for regulation of motility and cell division and changes over time. Dedicated protein modules regulate motility independent of the cell cycle, and cell division dependent on the cell cycle. For motility, a leading-lagging cell polarity is established that is inverted during cellular reversals. Establishment and inversion of this polarity are regulated hierarchically by interfacing protein modules that sort polarized motility proteins to the correct cell poles or cause their relocation between cell poles during reversals akin to a spatial toggle switch. For division, a novel self-organizing protein module that incorporates a ParA ATPase positions the FtsZ-ring at midcell. This review covers recent findings concerning the spatiotemporal regulation of motility and cell division in M. xanthus and illustrates how the study of diverse bacteria may uncover novel mechanisms involved in regulating bacterial cell polarity.

Article	Year
PomZ, a ParA-like protein, regulates Z-ring formation and cell division in Myxococcus xanthus. Molecular microbiology\| PubMed ID: 23145985	2013
Tracking of chromosome and replisome dynamics in Myxococcus xanthus reveals a novel chromosome arrangement. PLoS genetics\| PubMed ID: 24068967	2013
The PomXYZ Proteins Self-Organize on the Bacterial Nucleoid to Stimulate Cell Division. Developmental cell\| PubMed ID: 28486132	2017
Regulation of Cell Polarity in Motility and Cell Division in Myxococcus xanthus. Annual review of microbiology\| PubMed ID: 28525300	2017

Dominik Schumacher

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