Justin Sonnenburg, Postdoctoral Faculty Sponsor
Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.
View details for DOI 10.1128/mBio.01378-20
View details for PubMedID 33082255
The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and beta-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ∆yhdP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.
View details for DOI 10.1073/pnas.2015556117
View details for PubMedID 33046656
Despite their small sizes, bacterial cells within a host-associated microbial community often form highly structured complexes determined by environmental factors and interspecies interactions. Wilbert et al. combined species-specific fluorescent labels and high-resolution microscopy to visualize human tongue dorsum microbiomes and to highlight their structure and dynamics.
View details for DOI 10.1016/j.tem.2020.05.003
View details for PubMedID 32475653
In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its organization in vivo. Here, we perform molecular dynamics simulations for Caulobacter crescentus MreB to extract mechanical parameters for inputs into a coarse-grained biophysical polymer model that successfully predicts MreB filament properties in vivo. Our analyses indicate that MreB double protofilaments can exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting correlates with the length and orientation of MreB filaments observed in vitro and in vivo. Our molecular dynamics simulations also suggest that membrane binding of MreB double protofilaments induces a stable membrane curvature of similar magnitude to that observed in vivo. Thus, our multiscale modeling correlates cytoskeletal filament size with conformational changes inferred from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular organization and function.
View details for DOI 10.1038/s41467-020-14752-9
View details for PubMedID 32179732
Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.
View details for DOI 10.1038/s41564-019-0658-4
View details for PubMedID 31959968
The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeleton systems, few MreB-interacting proteins have been well characterized. Here, we identify a small protein from Caulobacter crescentus, an assembly inhibitor of MreB (AimB). AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Furthermore, aimB appears to be essential, and its depletion results in decreased cell width and increased resistance to A22, a small-molecule inhibitor of MreB assembly. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft and that this interaction is favored for C. crescentus MreB over Escherichia coli MreB because of a closer match in the degree of opening with AimB size, suggesting coevolution of AimB with MreB conformational dynamics in C. crescentus. We support this model through functional analysis of point mutants in both AimB and MreB, photo-cross-linking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings are consistent with AimB promoting MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor. Because AimB has only 104 amino acids and small proteins are often poorly characterized, our work suggests the possibility of more bacterial cytoskeletal regulators to be found in this class. Thus, like FtsZ and eukaryotic actin, MreB may have a rich repertoire of regulators to tune its precise assembly and dynamics.
View details for DOI 10.1016/j.bpj.2020.04.029
View details for PubMedID 32416080
Despite their small size and lack of membrane-based DNA encapsulation, prokaryotic cells still organize and scale their nucleoid in specific subcellular regions. Two studies show that the DNA-free regions in prokaryotes are full of large biomolecules, which exclude DNA via entropic forces.
View details for DOI 10.1016/j.cub.2019.06.002
View details for PubMedID 31287980
The actin family of cytoskeletal proteins is essential to the physiology of virtually all archaea, bacteria, and eukaryotes. While X-ray crystallography and electron microscopy have revealed structural homologies among actin-family proteins, these techniques cannot probe molecular-scale conformational dynamics. Here, we use all-atom molecular dynamic simulations to reveal conserved dynamical behaviors in four prokaryotic actin homologs: MreB, FtsA, ParM, and crenactin. We demonstrate that the majority of the conformational dynamics of prokaryotic actins can be explained by treating the four subdomains as rigid bodies. MreB, ParM, and FtsA monomers exhibited nucleotide-dependent dihedral and opening angles, while crenactin monomer dynamics were nucleotide-independent. We further show that the opening angle of ParM is sensitive to a specific interaction between subdomains. Steered molecular dynamics simulations of MreB, FtsA, and crenactin dimers revealed that changes in subunit dihedral angle lead to intersubunit bending or twist, suggesting a conserved mechanism for regulating filament structure. Taken together, our results provide molecular-scale insights into the nucleotide and polymerization dependencies of the structure of prokaryotic actins, suggesting mechanisms for how these structural features are linked to their diverse functions.
View details for PubMedID 30951524
While cell division is a critical process in cellular proliferation, very few antibiotics have been identified that target the bacterial cell-division machinery. Recent studies have shown that the small molecule PC190723 inhibits cell division in several Gram-positive bacteria, with a hypothesized mechanism of action involving direct targeting of the tubulin homolog FtsZ, which is essential for division in virtually all bacterial species. Here, it is shown that PC190723 also inhibits cell division in the Gram-negative bacterium Escherichia coli if the outer membrane permeability barrier is compromised genetically or chemically. The results show that the equivalent FtsZ mutations conferring PC190723 resistance in Staphylococcus aureus do not protect E. coli against PC190723, and that suppressors of PC190723 sensitivity in E. coli, which do not generically decrease outer membrane permeability, do not map to FtsZ or other division proteins. These suppressors display a wide range of morphological and growth phenotypes, and one exhibits a death phenotype in the stationary phase similar to that of a mutant with disrupted lipid homeostasis. Finally, a complementing FtsZ-msfGFP fusion is used to show that PC190723 does not affect the Z-ring structure. Taken together, the findings suggest that PC190723 inhibits growth and division in E. coli without targeting FtsZ. This study highlights the importance of utilizing a combination of genetic, chemical, and single-cell approaches to dissect the mechanisms of action of new antibiotics, which are not necessarily conserved across bacterial species.
View details for DOI 10.1002/adbi.201900021
View details for PubMedID 32648693
In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape, although the molecular mechanism by which cell width is regulated remains unknown. Here we demonstrate that the membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization of E. coli cell-wall growth. The relative expression levels of MreB and RodZ change in a manner commensurate with variations in growth rate and cell width, and RodZ systematically alters the curvature-based localization of MreB and cell width in a concentration-dependent manner. We identify MreB mutants that alter the bending properties of MreB filaments in molecular dynamics simulations similar to RodZ binding, and show that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Thus, E. coli can control its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.
View details for PubMedID 29599448
Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.
View details for PubMedID 29522748
View details for PubMedCentralID PMC5846203
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.
View details for PubMedID 29103935
The 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.Here 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.Morphometrics 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.
View details for DOI 10.1186/s12915-017-0348-8
View details for PubMedID 28222723
View details for PubMedCentralID PMC5320674
Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.
View details for DOI 10.1038/nprot.2016.181
View details for PubMedID 28125106
The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.
View details for DOI 10.1038/ncomms13170
View details for Web of Science ID 000385916600001
View details for PubMedID 27774981
View details for PubMedCentralID PMC5078992
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
View details for DOI 10.1016/j.cell.2016.05.003
View details for Web of Science ID 000377045400021
View details for PubMedID 27238023
View details for PubMedCentralID PMC4894308
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