Periodic Reporting for period 4 - RCSB (Regulation of cell size and shape in bacteria)
Período documentado: 2020-10-01 hasta 2021-01-31
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.
This project dealt with the question of how rod-shaped bacteria control their own cell shape and size during growth. The project was separated into two work packages, the first dealing with the question of the spatial and temporal control of cell shape, and the second dealing with the question of how cell-envelope growth is coupled to cell physiology.
As central part of the first work package we studied how cells control the location of peptidoglycan insertion into the cell wall, which physically determines cell shape. We investigated two major machineries involved in cell-wall insertion, the so-called Rod complex and the class-A Penicillin Binding Proteins (aPBPs). We investigated the workings of both machineries using single-molecule tracking microscopy (Vigouroux et al. eLife 2020; Özbaykal et al. eLife 2020). We found that the two systems have fundamentally different roles: The Rod complex maintains rod-like cell shape, while aPBPs are responsible for the repair of cell-wall defects. Traditionally, the Rod complex is thought to be governed by the MreB actin cytoskeleton. Here, we found that a different cell-envelope component, which is likely the cell wall, has an at least equally important role to guide the Rod complex. As a secondary part of this work package we also tackled the question of how cells decide to divide into two. We found we found that cell-division is temporally coupled to two processes, chromosome replication and a different process that relates cell division to cell-size at birth (Colin et al. bioRxiv 2021). This work resolves a long-standing debate about the potential involvement of chromosome replication in cell-cycle control.
The main question of the second workpackage was to understand how cells control the growth of cell volume with the growth in biomass. We found that E. coli controls cell volume growth indirectly (Oldewurtel et al. bioRxiv 2019): Cells increase their cell-envelope in direct proportion to biomass while controlling cell width independently on the generation time scale. Therefore, the intracellular density of biopolymers changes as cells change cell width. However, on long time scales, dry-mass density can adapt and density is almost constant across different growth conditions. This work profited from the development of quantitative phase microscopy in our lab, which allowed us to measure single-cell mass in absolute terms and with unprecedented accuracy and precision.
Our results have changed multiple paradigms in cell-envelope biology, bacterial morphogenesis, and more generally in cell biology. Notably, we identified the cell wall as an important template for cell-wall insertion; we found that cell-wall insertion and cell-envelope growth are not strictly coupled but somewhat independent of each other; we found that biomass density is not a constant but changes depending on cell dimensions. At the technological level, we have developed tools and image-analysis methods to interpret single-molecule trajectories and to measure single-cell biomass. These techniques will be helpful not only for the study of bacterial morphogenesis but for many other problems in single-cell physiology.
As central part of the first work package we studied how cells control the location of peptidoglycan insertion into the cell wall, which physically determines cell shape. We investigated two major machineries involved in cell-wall insertion, the so-called Rod complex and the class-A Penicillin Binding Proteins (aPBPs). We investigated the workings of both machineries using single-molecule tracking microscopy (Vigouroux et al. eLife 2020; Özbaykal et al. eLife 2020). We found that the two systems have fundamentally different roles: The Rod complex maintains rod-like cell shape, while aPBPs are responsible for the repair of cell-wall defects. Traditionally, the Rod complex is thought to be governed by the MreB actin cytoskeleton. Here, we found that a different cell-envelope component, which is likely the cell wall, has an at least equally important role to guide the Rod complex. As a secondary part of this work package we also tackled the question of how cells decide to divide into two. We found we found that cell-division is temporally coupled to two processes, chromosome replication and a different process that relates cell division to cell-size at birth (Colin et al. bioRxiv 2021). This work resolves a long-standing debate about the potential involvement of chromosome replication in cell-cycle control.
The main question of the second workpackage was to understand how cells control the growth of cell volume with the growth in biomass. We found that E. coli controls cell volume growth indirectly (Oldewurtel et al. bioRxiv 2019): Cells increase their cell-envelope in direct proportion to biomass while controlling cell width independently on the generation time scale. Therefore, the intracellular density of biopolymers changes as cells change cell width. However, on long time scales, dry-mass density can adapt and density is almost constant across different growth conditions. This work profited from the development of quantitative phase microscopy in our lab, which allowed us to measure single-cell mass in absolute terms and with unprecedented accuracy and precision.
Our results have changed multiple paradigms in cell-envelope biology, bacterial morphogenesis, and more generally in cell biology. Notably, we identified the cell wall as an important template for cell-wall insertion; we found that cell-wall insertion and cell-envelope growth are not strictly coupled but somewhat independent of each other; we found that biomass density is not a constant but changes depending on cell dimensions. At the technological level, we have developed tools and image-analysis methods to interpret single-molecule trajectories and to measure single-cell biomass. These techniques will be helpful not only for the study of bacterial morphogenesis but for many other problems in single-cell physiology.
Our results have changed multiple paradigms in cell-envelope biology, bacterial morphogenesis, and more generally in cell biology. Notably, we identified the cell wall as an important template for cell-wall insertion; we found that cell-wall insertion and cell-envelope growth are not strictly coupled but somewhat independent of each other; we found that biomass density is not a constant but changes depending on cell dimensions. At the technological level, we have developed tools and image-analysis methods to interpret single-molecule trajectories and to measure single-cell biomass. These techniques will be helpful not only for the study of bacterial morphogenesis but for many other problems in single-cell physiology.