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

Kitahara, Yuki; Er, Oldewurtel; Wilson, Sean; Ec, Garner; Teeffelen, Sven van

doi:10.1101/2021.05.05.442853

Cited by 3 publications

(5 citation statements)

References 76 publications

(119 reference statements)

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“…3) while causing a strong reduction of peptidoglycan insertion (45,46). In future work, we will therefore test peptidoglycan and other envelope components for their potentially limiting role for envelope expansion in E. coli and other bacteria, similar to our recent work in B. subtilis (47).…”

Section: Discussionmentioning

confidence: 81%

“…3E), even though this perturbation is known to cause a strong reduction of the rate of peptidoglycan insertion (45,46). Cell-wall synthesis is therefore likely not rate limiting for surface growth, in contrast to our recent reports on the gram-positive bacterium Bacillus subtilis (47), and a surface-limiting envelope component potentially responsible for the surface-growth law (Eq. 3) remains to be identified.…”

Section: Surface-to-mass Coupling Is Robust With Respect To Drug-inducedmentioning

confidence: 71%

“…Independently of the molecular mechanism, the robust coupling between surface growth and mass growth might be a general design feature for walled cells. Indeed, our recent work in B. subtilis (47) suggests that the surface-to-mass coupling is conserved in gram-positive bacteria, despite their fundamentally different envelope architectures. Recent cell-cycle-dependent variations of dry-mass density in fission yeast are also compatible with this picture (50).…”

Section: Discussionmentioning

confidence: 99%

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Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

Oldewurtel

Kitahara

Teeffelen

2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

102

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During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth—according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density.

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Section: Discussionmentioning

confidence: 81%

Section: Surface-to-mass Coupling Is Robust With Respect To Drug-inducedmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

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Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

Oldewurtel

Kitahara

Teeffelen

2021

Proc. Natl. Acad. Sci. U.S.A.

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102

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“…Note that tunicamycin primarily blocks WTAs synthesis but at the high concentrations used here (> 10 μg/ml) it additionally blocks PG synthesis (Brandish et al 1996; Campbell et al 2011). Polymerisation and crosslinking of the PG meshwork was targeted by vancomycin and penicillin G. We also analysed the effect of the hydrolytic enzyme lysozyme, and used the antibiotic chloramphenicol, which blocks protein synthesis by targeting ribosomes but does not impact cell surface expansion (Kitahara et al 2021), as negative control. We aimed to investigate the direct responses as well as the adaptation of B. subtilis to CW targeting antibiotics.…”

Section: Resultsmentioning

confidence: 99%

Peptidoglycan remodeling in response to cell wall acting antibiotics inBacillus subtilis

Cornilleau

Álvarez

Wegler

et al. 2023

Preprint

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Most bacteria are encased into a load-bearing rigid framework, the cell wall (CW). The peptidoglycan (PG) layer, a network composed of glycan strands cross-linked by stem peptides, is the main component of the CW. During PG synthesis, precursors are first synthetized intracellularly, before being incorporated into the existing PG meshwork by transglycosylation (TG) and transpeptidation (TP) reactions. Covalent modificatinos of the PG meshwork such as amidation and acetylation participate in PG homeostasis by regulating PG-associated enzymes like PG hydrolases. Because of its essential role, PG synthesis represents a primary target for antibiotic action. Here, we investigated the effect on PG composition of antibiotics targeting intracellular and extracellular steps of PG synthesis: inhibitors of PG precursors synthesis (fosfomycin, D-cycloserine, bacitracin and tunicamycin) and TG/TP inhibitors (vancomycin and penicillin G), respectively. Our study revealed interesting correlations between crosslinking and both de-N-acetylation and amidation of the sacculus. A thorough analysis of muropeptides composition put into light an unexpected anti-correlation between the degree of PG crosslinking and accumulation of de-amidated disaccharide-tripeptide monomer subunit (M3) in the presence of TP inhibitors. We confirmed these observations by analyzing mutants of the PG synthesis pathway.

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“…Figures 2g and 3c show that the relative width estimation bias from the membrane image decreases as cell width increases, while the estimates from the cytoplasmic marker exhibit a non-monotonic dependence. This size-dependent nature of biases complicates the use of calibration for computational correction 27 . In the following section, we explore methods for incorporating these effects into training deep learning image segmentation models, enabling these models to accurately estimate cell sizes from 2D images.…”

Section: Projection and Diffraction Effects On Cell Size Estimation F...mentioning

confidence: 99%

Quantitative microbiology with widefield microscopy: navigating optical artefacts for accurate interpretations

Hardo

Bakshi

2023

Preprint

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Live cell imaging of microbial cells with microscopy has revolutionised quantitative microbiology. Micrographs are one of the most information-rich data types captured about a microbe, allowing quantification of the size and morphology of individual cells and their gene-expression over time. However, an optical microscope is a diffraction-limited system, and the comparable size of the point spread function of the microscope to the size of a microbial cell can lead to imaging artefacts which corrupt and bias the data. Additionally, the comparable thickness of a microbe to the depth of field of the microscope means that the 2D image contains compressed, projected 3D information. This makes it difficult to extract the underlying 3D distribution of photon emitters. For unknown distributions, the problem can be as ill-posed as a deconvolution problem, usually not having a unique solution. Together, the diffraction and projection effects affect our ability to accurately quantify the size and shape of microbial cells from their images and their contents from intensity measurements. In this paper, we use a mixture of simulations and experiments of microscopic image formation of microbial cells to illustrate the effects of diffraction and projection on cell segmentation and signal quantification. We use targeted experiments to validate the predictions where possible. Finally, we use the knowledge of these effects to design experiments which can help to reduce the errors and biases in our analysis. Awareness of these effects and the approaches towards alleviating them will help to accurately quantify microbiology from microscopy data.

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

Cited by 3 publications

References 76 publications

Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

Robust surface-to-mass coupling and turgor-dependent cell width determine bacterial dry-mass density

Peptidoglycan remodeling in response to cell wall acting antibiotics inBacillus subtilis

Quantitative microbiology with widefield microscopy: navigating optical artefacts for accurate interpretations

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