Research

Bacterial cell shape

Bacterial cell shape is determined by the cell envelope, and specifically by the peptidoglycan cell wall. We therefore study the spatial and temporal dynamics of enzymes and proteins involved in cell-envelope organization and growth, for example the MreB actin cytoskeleton or the enzymes responsible for cell-wall remodeling. Single-molecule observations are complemented by physical, genetic, and chemical perturbations, e.g. through CRISPRi-based control of protein levels (Vigouroux et al. MSB 2018) or by physically bending cells in microchambers (Wang et al. Nat Microbiology 2017).

Özbaykal G., E. Wollrab, F. Simon, A. Vigouroux, B. Cordier, A. Aristov, T. Chaze, M. Matondo, and S. van Teeffelen (2020). The transpeptidase PBP2 governs initial localization and activity of the major cell-wall synthesis machinery in E. coli. eLife 9:e50629

Vigouroux A., B. Cordier, A. Aristov, L. Alvarez, G. Özbaykal, T. Chaze, E.R. Oldewurtel, M. Matondo, F. Cava, D. Bikard, S. van Teeffelen (2020). Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects. eLife 9:e51998

Wong F., L.D. Renner, G. Özbaykal, J. Paulose, D.B. Weibel, S. van Teeffelen, A. Amir (2017) Mechanical strain-sensing implicated in cell shape recovery in Escherichia coli. Nat. Microbio. 2017; 2: 17115

Volume regulation and regulation of dry-mass density

During growth, cells need to increase their volume in coordination with biomass. How this happens remained fundamentally not understood in any organism until our recent work. We implemented and improved quantitative phase microscopy to study bacterial cell shape and cellular dry mass in single cells in absolute terms during time-lapse movies. We showed that bacteria growth their surface areas (rather than volume) in proportion to biomass growth. Cellular dry-mass density then changes in proportion to changes of cell width, observed both in the Gram-negative E. coli and in the Gram-positive B. subtilis.

Kitahara Y., E.R. Oldewurtel, S. Wilson, E. Garner, S. van Teeffelen (2021) Cell-envelope synthesis is required for surface-to-mass coupling, which determines dry-mass density in Bacillus subtilis. BioRxiv 2021.05.05.442853

Oldewurtel, E.R., Y. Kitahara, and S. van Teeffelen (2021) Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density Proc. Natl. Acad. Sci. U.S.A. 118(32) e2021416118.

Cell-wall integrity

How do protect their cell envelope against lysis? Using single-molecule tracking, precise gene regulation through CRISPRi, and time-dependent perturbations of the cell envelope, we recently demonstrated that so-called class-A Penicillin Binding Proteins likely constitute repair enzymes. In the future, we will study how exactly these enzymes and other proteins get recruited to different kinds of cell-envelope defects and how they repair the cell wall.

Vigouroux A., B. Cordier, A. Aristov, L. Alvarez, G. Özbaykal, T. Chaze, E.R. Oldewurtel, M. Matondo, F. Cava, D. Bikard, S. van Teeffelen (2020). Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects. eLife 9:e51998

Cell-cycle control

How bacterial cells decide to divide into two is a long-standing question. We study this question using single-cell investigations in microfluidic channels. Using single-cell correlations and mathematical modeling we recently demonstrated together with the lab of Marco Cosentino Lagomarsino that at least two processes are responsible for cell division: DNA replication, and a second process that relates cell size at division to cell size at birth. However, many questions remain open: What is the second process that relates division to previous birth? What determines the size at DNA replication?

Colin A., G. Micali, L. Faure, M. Cosentino Lagomarsino, S. van Teeffelen (2021) Two different cell-cycle processes determine the timing of cell division in Escherichia coli. BioRxiv 2021.03.08.434443v1 (under review, eLife)

Tools for precise regulation of gene expression

We are also interested in a range of other problems. In the past, we worked on the question how the catalytically-dead variant of CRISPR-Cas9 (dCas9) blocks transcription. Together with the lab of David Bikard, we established that CRISPRi can be used to control gene expression with unprecedented accuracy at the single-cell level, if expression is tuned by the homology between guideRNA and target DNA. In the course of our study, we could attribute precise regulation to an important feature of the system: RNA replicase kicks dCas9 off the target in a guideRNA-dependent manner. The system is now used to control gene repression with unprecedented precision.

Vigouroux A., E. Oldewurtel, C. Lun, D. Bikard, S. van Teeffelen. (2018). Tuning dCas9’s ability to block transcription enables robust, noiseless knockdown of bacterial genes. Molecular Systems Biology14 (3), e7899