Biology subjects

van Teeffelen, S.

Publications and source records attributed to van Teeffelen, S..

11 recordsLinked to original sources

bioRxiv2025

Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

The bacterial cell wall is a critical load-bearing structure, but is not thought to be an important permeability barrier since proteins freely diffuse through isolated cell wall sacculi and bacteria secrete proteins without the aid of any known channels or transporters in the wall. Using new genetically encoded probes to measure the permeability of the cell in situ at the single-cell level, we discovered that the size threshold determining whether proteins can pass through the Bacillus subtilis sacculus is smaller than was previously thought. We found that transport of small proteins (<10 kDa) through the sacculus was consistent with passive diffusion through discrete pores, while larger proteins (>15 kDa) required the generation of larger pores by inducing peptidoglycan hydrolysis unbalanced by synthesis. These data are consistent with physics-based models of diffusion through a random percolation network of finite thickness. Conversely, the ability of the innate immune factor phospholipase (15.2 kDa) to kill B. subtilis was inhibited by membrane de-polarization. The protective effect of de-polarization was dependent on latent peptidoglycan synthesis (decoupled from cell growth) by PBP1 - highlighting a new role for this enzyme - and on reduced peptidoglycan hydrolysis. These results demonstrate that the rapid peptidoglycan turnover that drives cell growth also promotes movement of phospholipase across the cell wall, identifying a quintessentially bacterial mechanism of active transport. Significance StatementGram-positive bacteria, which include many serious pathogens like Staphylococcus aureus, Listeria monocytogenes, and Clostridium difficile, are defined by their thick peptidoglycan cell wall. Here, we demonstrate that this structure is a critical permeability barrier that blocks antibacterial proteins like those used by the human innate immune system. Furthermore, this barrier function depends on the physiological state of the cell: the wall of non-growing cells is less permeable because peptidoglycan turnover during growth actively promotes transport of specific proteins through the cell wall. This prokaryotic paradigm for molecular transport has important implications for host-pathogen interactions since pathogenic bacteria often assume both non-growing and growing states during infection.

microbiology↗

bioRxiv2025

Condition-dependent, amorphous protein agglomerates control cytoplasmic rheology

Molecular crowding in the bacterial cytoplasm restricts the diffusion of large molecules, impacting cellular processes. However, how nutrient availability influences cytoplasmic rheology is not well understood. With single-particle tracking in Escherichia coli, we observed a threefold variation in the diffusion of a 40-nm particle across exponential growth conditions. Previously suggested determinants of rheology did not account for this variation; instead, we found a strong anticorrelation between the diffusion coefficient and the abundance of amino acid metabolism proteins, persisting upon genetic perturbations and showing that lower diffusion is associated with increased viscoelasticity. Photoactivated light microscopy revealed that some amino acid metabolism proteins form clusters. Electron microscopy showed that these proteins could form amorphous agglomerates at physiological concentrations in vitro, likely driven by their low intrinsic disorder, high compactness and hydropathy score. These findings show that protein agglomerates regulate cytoplasmic rheology in a condition-dependent manner, suggesting an underappreciated level of cytoplasmic organization. HighlightsO_LIDiffusion of 40-nm particles varies threefold across growth conditions in E. coli C_LIO_LICytoplasmic diffusion inversely correlates with COG-E protein abundance COG-E proteins form agglomerates that increase cytoplasmic viscoelasticity C_LIO_LIProtein compactness and hydrophobicity predict condition-dependent crowding effects C_LI

biophysics↗

bioRxiv2022

ExTrack characterizes transition kinetics and diffusion in noisy single-particle tracks

Single-particle tracking microscopy is a powerful technique to investigate how proteins dynamically interact with their environment in live cells. However, the analysis of tracks is confounded by noisy molecule localization, short tracks, and rapid transitions between different motion states, notably between immobile and diffusive states. Here, we propose a probabilistic method termed ExTrack that uses the full spatio-temporal information of tracks to extract global model parameters, to calculate state probabilities at every time point, to reveal distributions of state durations, and to refine the positions of bound molecules. ExTrack works for a wide range of diffusion coefficients and transition rates, even if experimental data deviate from model assumptions. We demonstrate its capacity by applying it to slowly diffusing and rapidly transitioning bacterial envelope proteins. ExTrack greatly increases the regime of computationally analyzable noisy single-particle tracks. The ExTrack package is available in ImageJ and Python.

biophysics↗

bioRxiv2021

Cell-envelope synthesis is required for surface-to-mass coupling, which determines dry-mass density in Bacillus subtilis

Cells must increase their volumes in response to biomass growth to maintain intracellular mass density, the ratio of dry mass to cell volume, within physiologically permissive bounds. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. Recently, we demonstrated that the Gram-negative bacterium Escherichia coli expands cell-surface area rather than volume in proportion to mass. Here, we investigate the regulation of cell-volume growth in the evolutionarily distant Bacillus subtilis. First, we demonstrate that the coupling of surface growth to mass growth is conserved in B. subtilis. Therefore, mass density changes with cell shape at the single-cell level. Interestingly, mass density varies by more than 30% when we systematically change cell width by modulation of cell-wall insertion, without any effect on mass-growth rate. Second, we demonstrate that the coupling of surface- and mass growth is broken if peptidoglycan or membrane synthesis are inhibited. Once transient perturbations are relieved, the surface-to-mass ratio is rapidly restored. In conclusion, we demonstrate that surface-to-mass coupling is a conserved principle for volume regulation in bacteria, and that envelope synthesis provides an important link between surface growth and biomass growth in B. subtilis.

microbiology↗

bioRxiv2021

Two different cell-cycle processes determine the timing of cell division in Escherichia coli

Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

systems biology↗

bioRxiv2019

Bacteria control cell volume by coupling cell-surface expansion to dry-mass growth

Cells exhibit a high degree of intracellular crowding. To control the level of crowding during growth cells must increase their volumes in response to the accumulation of biomass. Using Escherichia coli as a model organism, we found that cells control cell volume indirectly, by increasing cell-surface area in proportion to biomass growth. Thus, dry-mass density, a readout of intracellular crowding, varies in proportion to the surface-to-volume ratio, both during the cell cycle and during perturbations such as nutrient shifts. On long time scales after shifts, initial dry-mass density is nearly restored by slow variations of the surface-to-mass ratio. Contrary to a long-standing paradigm, cell-envelope expansion is controlled independently of cell-wall synthesis but responds to the activity of cell-wall cleaving hydrolases. Finally, we observed rapid changes of Turgor pressure after nutrient shifts, which were likely responsible for initial changes of cell diameter and dry-mass-density. Together, our experiments reveal important regulatory relationships for cell volume and shape.

microbiology↗

bioRxiv2019

Cell-wall synthases contribute to bacterial cell-envelope integrity by actively repairing defects

AbstracCell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell, the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that class-A PBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level we then demonstrate that PBP1b, the major class-A PBP, contributes to cell-wall integrity by localizing and inserting peptidoglycan in direct response to local cell-wall defects.

microbiology↗

bioRxiv2019

Transpeptidase PBP2 governs initial localization and activity of major cell-wall synthesis machinery in Escherichia coli

Bacterial shape is physically determined by the peptidoglycan cell wall. The cell-wall-synthesis machinery responsible for rod shape in Escherichia coli is the processive Rod complex. Previously, cytoplasmic MreB filaments were thought to govern formation and localization of Rod complexes based on local cell-envelope curvature. However, using single-particle tracking of the transpeptidase PBP2, we found strong evidence that PBP2 initiates new Rod complexes by binding to a substrate different from MreB or any known Rod-complex component. This substrate is likely the cell wall. Consistently, we found only weak correlations between MreB and envelope curvature in the cylindrical part of cells. Residual correlations do not require any curvature-based Rod-complex initiation but can be attributed to persistent rotational motion. Therefore, local cell-wall architecture likely provides the cue for PBP2 binding and subsequent Rod-complex initiation. We also found that PBP2 has a limiting role for Rod-complex activity, thus supporting its central role.

microbiology↗

bioRxiv2018

Cell Diameter in Bacillus subtilis is Determined by the Opposing Actions of Two Distinct Cell Wall Synthetic Systems

Rod shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod system moves around the cell circumference, while the class A penicillin-binding proteins (aPBPs) are unorganized. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each system affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. We find that rod diameter is not determined by MreB, rather it depends on the balance between the systems: The Rod system reduces diameter, while aPBPs increase it. RodA/PBP2A can both thin or widen cells, depending on its levels relative to MreBCD. Increased Rod system activity correlates with an increased density of directional MreB filaments, and a greater fraction of directionally moving PBP2A molecules. This increased circumferential synthesis increases the amount of oriented material within the sacculi, increasing their mechanical anisotropy and reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems build rods of different widths, a model that appears generalizable: Escherichia coli containing Rod system mutants show the same relationship between the density of directionally moving MreB filaments and cell width.

microbiology↗

bioRxiv2018

A matrix protein acts as a cue to preserve collective motility of B. subtilis biofilm cells

Bacteria in nature are usually found in complex multicellular communities, termed biofilms. Biofilms are generally seen as sessile structures, resulting from downregulation of motility. However, during interspecies competition and predation, biofilm cells were shown to migrate towards competitor colonies. Here, we show that a specific extracellular matrix (ECM) protein, TasA, is essential for collective migration toward potential competitors and serves as a developmental cue that increases the formation of motile offspring from sessile chains. We reveal an effective strategy to maintain migration capacities in bacterial biofilms: besides providing a three-dimensional adhesive scaffold for the cells, TasA acts as a signal within the bacterial community.

microbiology↗

bioRxiv2017

Engineered CRISPR-Cas9 system enables noiseless, fine-tuned and multiplexed repression of bacterial genes

Over the past few years, tools that make use of the Cas9 nuclease have led to many breakthroughs, including in the control of gene expression. The catalytically dead variant of Cas9 known as dCas9 can be guided by small RNAs to block transcription of target genes, in a strategy also known as CRISPRi. Here, we reveal that the level of complementarity between the guide RNA and the target controls the rate at which dCas9 successfully blocks the RNA polymerase. We use this mechanism to precisely and robustly reduce gene expression by defined relative amounts. We demonstrate broad applicability of this method to the study of genetic regulation and cellular physiology. First, we characterize feedback strength of a model auto-repressor. Second, we study the impact of copy-number variations of cell-wall synthesizing enzymes on cell morphology. Finally, we demonstrate that this system can be multiplexed to obtain any combination of fractional repression of two genes.

synthetic biology↗