Carolina Tropini

Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity

Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user‐defined stiffness, measured the growth rate of individual cells and fit data to a thin‐shell mechanical model to extract the effective longitudinal Youngs modulus of the cell envelope of Escherichia coli (50–150 MPa), Bacillus subtilis (100–200 MPa) and Pseudomonas aeruginosa (100–200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour‐intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22‐induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Youngs modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium

Spatial asymmetry is crucial to development. One mechanism for generating asymmetry involves the localized synthesis of a key regulatory protein that diffuses away from its source, forming a spatial gradient. Although gradients are prevalent in eukaryotes, at both the tissue and intracellular levels, it is unclear whether gradients of freely diffusible proteins can form within bacterial cells given their small size and the speed of diffusion. Here, we show that the bacterium Caulobacter crescentus generates a gradient of the active, phosphorylated form of the master regulator CtrA, which directly regulates DNA replication. Using a combination of mathematical modeling, single-cell microscopy, and genetic manipulation, we demonstrate that this gradient is produced by the polarly localized phosphorylation and dephosphorylation of CtrA. Our data indicate that cells robustly establish the asymmetric fates of daughter cells before cell division causes physical compartmentalization. More generally, our results demonstrate that uniform protein abundance may belie gradients and other sophisticated spatial patterns of protein activity in bacterial cells.

A dynamically assembled cell wall synthesis machinery buffers cell growth

Significance For complex biological processes, the formation of protein complexes is a strategy for coordinating the activities of many enzymes in space and time. It has been hypothesized that growth of the bacterial cell wall involves stable synthetic complexes, but neither the existence of such complexes nor the consequences of such a mechanism for growth efficiency have been demonstrated. Here, we use single-molecule tracking to demonstrate that the association between an essential cell wall synthesis enzyme and the cytoskeleton is highly dynamic, which allows the cell to buffer growth rate against large fluctuations in enzyme abundance. This indicates that dynamic association can be an efficient strategy for coordination of multiple enzymes, especially those for which excess abundance can be harmful to cells. Assembly of protein complexes is a key mechanism for achieving spatial and temporal coordination in processes involving many enzymes. Growth of rod-shaped bacteria is a well-studied example requiring such coordination; expansion of the cell wall is thought to involve coordination of the activity of synthetic enzymes with the cytoskeleton via a stable complex. Here, we use single-molecule tracking to demonstrate that the bacterial actin homolog MreB and the essential cell wall enzyme PBP2 move on timescales orders of magnitude apart, with drastically different characteristic motions. Our observations suggest that PBP2 interacts with the rest of the synthesis machinery through a dynamic cycle of transient association. Consistent with this model, growth is robust to large fluctuations in PBP2 abundance. In contrast to stable complex formation, dynamic association of PBP2 is less dependent on the function of other components of the synthesis machinery, and buffers spatially distributed growth against fluctuations in pathway component concentrations and the presence of defective components. Dynamic association could generally represent an efficient strategy for spatiotemporal coordination of protein activities, especially when excess concentrations of system components are inhibitory to the overall process or deleterious to the cell.

Nonexponential Kinetics of DNA Escape from α-Hemolysin Nanopores ☆

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

The Gut Microbiome: Connecting Spatial Organization to Function

The first rudimentary evidence that the human body harbors a microbiota hinted at the complexity of host-associated microbial ecosystems. Now, almost 400 years later, a renaissance in the study of microbiota spatial organization, driven by coincident revolutions in imaging and sequencing technologies, is revealing functional relationships between biogeography and health, particularly in the vertebrate gut. In this Review, we present our current understanding of principles governing the localization of intestinal bacteria, and spatial relationships between bacteria and their hosts. We further discuss important emerging directions that will enable progressing from the inherently descriptive nature of localization and -omics technologies to provide functional, quantitative, and mechanistic insight into this complex ecosystem.

Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.

BackgroundThe determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features.ResultsHere we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics.ConclusionsMorphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.

Background: HPLC enables quantification of bacterial cell-wall composition, yet systematic studies across strains, species, and chemical perturbations are lacking. Results: UPLC coupled to computational modeling enables submicroliter injection volumes, and was applied to systematic analysis of several Gram-negative species. Conclusion: Composition is largely decoupled from morphology, although large interspecies differences were evident. Significance: UPLC and automated analysis accelerate discovery regarding peptidoglycan and physiology. The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

Islands Containing Slowly Hydrolyzable GTP Analogs Promote Microtubule Rescues

Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5′-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro.

Deep Phenotypic Mapping of Bacterial Cytoskeletal Mutants Reveals Physiological Robustness to Cell Size

Size is a universally defining characteristic of all living cells and tissues and is intrinsically linked with cell genotype, growth, and physiology. Many mutations have been identified to alter cell size, but pleiotropic effects have largely hampered our ability to probe how cell size specifically affects fundamental cellular properties, such as DNA content and intracellular localization. To systematically interrogate the impact of cell morphology on bacterial physiology, we used fluorescence-activated cell sorting to enrich a library of hundreds of Escherichia coli mutants in the essential cytoskeletal protein MreB for subtle changes in cell shape, cumulatively spanning ∼5-fold variation in average cell volume. Critically, pleiotropic effects in the mutated library are most likely minimized because only one gene was mutated and because growth rate was unaffected, thereby allowing us to query the general effects of morphology on cellular physiology over a large range of cell sizes with high resolution. We discovered linear scaling of the abundance of DNA and the key division protein FtsZ with cell volume, a strong dependency of sensitivity to specific antibiotics on cell width, and a simple correlation between MreB localization pattern and cell width. Our systematic, quantitative approach reveals complex and dynamic links between bacterial morphology and physiology and should be generally applicable for probing size-related genotype-phenotype relationships.