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Temperature is a key determinant of microbial behaviour and survival in the environment and within hosts. At intermediate temperatures, growth rate varies according to the Arrhenius law of thermodynamics, which describes the effect of temperature on the rate of a chemical reaction. However, the mechanistic basis for this behaviour remains unclear. Here we use single-cell microscopy to show that Escherichia coli exhibits a gradual response to temperature upshifts with a timescale of ~1.5 doublings at the higher temperature. The response was largely independent of initial or final temperature and nutrient source. Proteomic and genomic approaches demonstrated that adaptation to temperature is independent of transcriptional, translational or membrane fluidity changes. Instead, an autocatalytic enzyme network model incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics through metabolome rearrangement, resulting in a transient temperature memory. The model successfully predicts alterations in the temperature response across nutrient conditions, diverse E. coli strains from hosts with different body temperatures, soil-dwelling Bacillus subtilis and fission yeast. In sum, our model provides a mechanistic framework for Arrhenius-dependent growth., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The key bacterial cell division protein FtsZ can adopt multiple conformations, and prevailing models suggest that transitions of FtsZ subunits from the closed to open state are necessary for filament formation and stability. Using all-atom molecular dynamics simulations, we analyzed state transitions of Staphylococcus aureus FtsZ as a monomer, dimer, and hexamer. We found that monomers can adopt intermediate states but preferentially adopt a closed state that is robust to forced reopening. Dimer subunits transitioned between open and closed states, and dimers with both subunits in the closed state remained highly stable, suggesting that open-state conformations are not necessary for filament formation. Mg 2+ strongly stabilized the conformation of GTP-bound subunits and the dimer filament interface. Our hexamer simulations indicate that the plus end subunit preferentially closes and that other subunits can transition between states without affecting inter-subunit stability. We found that rather than being correlated with subunit opening, inter-subunit stability was strongly correlated with catalytic site interactions. By leveraging deep-learning models, we identified key intrasubunit interactions governing state transitions. Our findings suggest a greater range of possible monomer and filament states than previously considered and offer new insights into the nuanced interplay between subunit states and the critical role of nucleotide hydrolysis and Mg 2+ in FtsZ filament dynamics.

Temperature is one of the key determinants of microbial behavior and survival, whose impact is typically studied under heat- or cold-shock conditions that elicit specific regulation to combat lethal stress. At intermediate temperatures, cellular growth rate varies according to the Arrhenius law of thermodynamics without stress responses, a behavior whose origins have not yet been elucidated. Using single-cell microscopy during temperature perturbations, we show that bacteria exhibit a highly conserved, gradual response to temperature upshifts with a time scale of ~1.5 doublings at the higher temperature, regardless of initial/final temperature or nutrient source. We find that this behavior is coupled to a temperature memory, which we rule out as being neither transcriptional, translational, nor membrane dependent. Instead, we demonstrate that an autocatalytic enzyme network incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics through metabolome rearrangement, which encodes a temperature memory and successfully predicts alterations in the upshift response observed under simple-sugar, low-nutrient conditions, and in fungi. This model also provides a mechanistic framework for both Arrhenius-dependent growth and the classical Monod Equation through temperature-dependent metabolite flux.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve ( Bb ). We generated a high density, randomly barcoded transposon insertion pool in Bb , and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

Filament formation by cytoskeletal proteins is critical to their involvement in myriad cellular processes. The bacterial actin homolog MreB, which is essential for cell-shape determination in many rod-shaped bacteria, has served as a model system for studying the mechanics of cytoskeletal filaments. Previous molecular dynamics (MD) simulations revealed that the twist of MreB double protofilaments is dependent on the bound nucleotide, as well as binding to the membrane or the accessory protein RodZ, and MreB mutations that modulate twist also affect MreB spatial organization and cell shape. Here, we show that MreB double protofilaments can adopt multiple twist states during microsecond-scale MD simulations. A deep learning algorithm trained only on high- and low-twist states robustly identified all twist conformations across most perturbations of ATP-bound MreB, suggesting the existence of a conserved set of states whose occupancy is affected by each perturbation to MreB. Simulations replacing ATP with ADP indicated that twist states were generally stable after hydrolysis. These findings suggest a rich twist landscape that could provide the capacity to tune MreB activity and therefore its effects on cell shape., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kerwyn Casey Huang reports financial support was provided by National Science Foundation. Kerwyn Casey Huang reports financial support was provided by Chan Zuckerberg Biohub. Handuo Shi reports financial support was provided by James S McDonnell Foundation., (© 2022 The Authors.)

Temperature impacts biological systems across all length and timescales. Cells and the enzymes that comprise them respond to temperature fluctuations on short timescales, and temperature can affect protein folding, the molecular composition of cells, and volume expansion. Entire ecosystems exhibit temperature-dependent behaviors, and global warming threatens to disrupt thermal homeostasis in microbes that are important for human and planetary health. Intriguingly, the growth rate of most species follows the Arrhenius law of equilibrium thermodynamics, with an activation energy similar to that of individual enzymes but with maximal growth rates and over temperature ranges that are species specific. In this review, we discuss how the temperature dependence of critical cellular processes, such as the central dogma and membrane fluidity, contributes to the temperature dependence of growth. We conclude with a discussion of adaptation to temperature shifts and the effects of temperature on evolution and on the properties of microbial ecosystems.

There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes. 3-Hydroxychromone (3HC) dyes, known for having high fluorescence quantum yields, exhibit shifts in fluorescence intensity in response to changes in environmental polarity. We synthesized two analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3-Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial cells displayed a 10-fold increase in fluorescence intensity compared to our previous reports on the dye 4,4- N,N -dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb cells within 10 min of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-resource settings., Competing Interests: The authors declare the following competing financial interest(s): M.K. and C.R.B. are cofounders of OliLux Biosciences which has licensed patents related to the work in this paper. B.K. is a scientific advisor to OliLux Biosciences. B.K is cofounder of SmartSpot Quality CC and scientific advisor to SmartSpot Quality CC and Pulmosat. C.R.B. is a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the board of directors of Eli Lilly & Company., (© 2021 The Authors. Published by American Chemical Society.)

Single-cell imaging, combined with recent advances in image analysis and microfluidic technologies, have enabled fundamental discoveries of cellular responses to chemical perturbations that are often obscured by traditional liquid-culture experiments. Temperature is an environmental variable well known to impact growth and to elicit specific stress responses at extreme values; it is often used as a genetic tool to interrogate essential genes. However, the dynamic effects of temperature shifts have remained mostly unstudied at the single-cell level, due largely to engineering challenges related to sample stability, heatsink considerations, and temperature measurement and feedback. Additionally, the few commercially available temperature-control platforms are costly. Here, we report an inexpensive (<$110) and modular Single-Cell Temperature Controller (SiCTeC) device for microbial imaging-based on straightforward modifications of the typical slide-sample-coverslip approach to microbial imaging-that controls temperature using a ring-shaped Peltier module and microcontroller feedback. Through stable and precise (±0.15°C) temperature control, SiCTeC achieves reproducible and fast (1-2 min) temperature transitions with programmable waveforms between room temperature and 45°C with an air objective. At the device's maximum temperature of 89°C, SiCTeC revealed that Escherichia coli cells progressively shrink and lose cellular contents. During oscillations between 30°C and 37°C, cells rapidly adapted their response to temperature upshifts. Furthermore, SiCTeC enabled the discovery of rapid morphological changes and enhanced sensitivity to substrate stiffness during upshifts to nonpermissive temperatures in temperature-sensitive mutants of cell-wall synthesis enzymes. Overall, the simplicity and affordability of SiCTeC empowers future studies of the temperature dependence of single-cell physiology., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2020 Atolia et al.)

Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis or the consequences when this homeostasis is perturbed. Here, we identify conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to general accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of "supergrowth" wherein rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a mechanism for global proteome homeostasis based on modulation of volume growth and dilution., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The cytoplasm is a highly crowded and complex environment, and the regulation of its physical properties has only recently begun to be revealed. In this issue of Cell, Delarue et al. demonstrate that the control of ribosome concentration through mTORC1 sets limits on the diffusion of large particles and controls phase separation in eukaryotic cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)