Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RCSB (Regulation of cell size and shape in bacteria)

Teaser

The RCSB project addresses a major question of bacterial cell biology: What determines cell shape and cell volume? This problem is interesting from a fundamental point of view, as cell size and shape are important for all cells, bacteria, eukaryotes and archaea. To that end we...

Summary

The RCSB project addresses a major question of bacterial cell biology: What determines cell shape and cell volume? This problem is interesting from a fundamental point of view, as cell size and shape are important for all cells, bacteria, eukaryotes and archaea. To that end we are using the bacterium Escherichia coli as a model system. In bacteria and other microorganisms cell shape is physically determined by the peptidoglycan cell wall. During growth the bacterial cell wall is enzymatically remodeled. New material is inserted and existing material is cut. This project therefore investigates how cells know where and when to remodel their cell walls to achieve a specific cell shape, a desired cell volume, and mechanical integrity. To that end we are using high-accuracy live-cell microscopy, physical modeling, molecular biology and genetics.

Work performed

We have made progress along the two major lines of research – a) understanding the determinants of cell-wall insertion and cell shape, and b) understanding the determinants of cell volume for a given physiological state of the cell.
a) The bacterial actin cytoskeleton MreB is thought to be responsible for important traits of bacterial cell shape: cylindrical shape, cell diameter, and cell straightness. By carefully reassessing previous claims we found that the bacterial actin cytoskeleton is not responsible for the straightness of rod-shaped cells [Wong et al. Nat Microbiology 2017]. Instead, mechanical strain is likely the physical cue responsible for cell-shape deformations. Along similar lines, we found strong evidence that the localization of cell-wall insertion is not predominantly determined by the MreB cytoskeleton, but that instead a cell-wall cross-linking enzyme initiates new sites of cell-wall insertion based on its binding to the cell wall itself.

MreB filaments have previously been suggested to dictate the orientation of newly inserted glycan strands – through filament curvature and twist. While MreB is important to stabilize circumferential glycan-strand orientation we found that the orientation of MreB filaments and rod-complex motion is dominated by the local orientation of the existing cell wall. This is seen both when reorienting the cell wall on average through transient chemical perturbation or when following correlations between the orientations of neighboring tracks. The common cell wall substrate tends to align different rod complexes in their motion, even if these complexes visit proximal locations in the cell wall at different time points. This work supports the importance of the cell wall for the determination of when and how new peptidoglycan is inserted. This work will be submitted as a manuscript in early 2019.
Cells use different machineries for building its cell wall, the processive rod complex and the bifunctional class-A penicillin-binding proteins. It remains unclear whether these machineries are truly independent or whether they work in concert. To assess this question we developed a tool to repress each of the systems by precise relative amounts from their native levels using an enzymatically-dead variant of CRISPR-Cas9 [see our publication Vigouroux et al. MSB 2018]. We then found functional evidence for their independence: While the rod complex is important for rod shape, the Class-A-PBPs are important for cell-wall integrity but not for shape.
We have also implemented tools to measure the amount of cell-wall insertion in bulk populations. To that end we use either the rate of MreB rotation as an indirect readout or radiolabeled precursors of cell-wall synthesis, which we then detect by scintillation counting.
b) To study the determinants of cell volume we established novel techniques to measure cell volume and cellular dry mass independently and in live cells. These techniques will be used to establish how cells expand their cell envelope in response to growth and other physiological states – by combining measurements with genetic, physical, and chemical perturbations. Preliminary results already suggest unexpected modes of bacterial cell-envelope remodeling as major players for volume regulation.

Final results

We have made great progress at narrowing down the determinants of bacterial cell shape and we will continue to do so by further investigation of different cell-envelope-modifying enzymes and structural proteins. We have also already made great progress at developing novel tools to control and measure bacterial traits: specifically, controlling gene expression through CRISPR-dCas9, measuring the localization and movement of individual proteins, and measuring cellular volume and dry mass with high precision in live cells.

Website & more info

More info: https://research.pasteur.fr/en/team/microbial-morphogenesis-and-growth/.