Knapp, B. D. | Search bioRxiv

bioRxiv2023

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions

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.

microbiology↗

bioRxiv2020

SiCTeC: an inexpensive, easily assembled Peltier device for rapid temperature shifting during single-cell imaging

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 {degrees}C) temperature control, SiCTeC achieves reproducible and fast (1-2 min) temperature transitions with programmable waveforms between room temperature and 45 {degrees}C with an air objective. At the devices maximum temperature of 89 {degrees}C, SiCTeC revealed that Escherichia coli cells progressively shrink and lose cellular contents. During oscillations between 30 {degrees}C and 37 {degrees}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.

microbiology↗

bioRxiv2020

SiCTeC: an inexpensive, easily assembled Peltier device for rapid temperature shifting during single-cell imaging

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 {degrees}C) temperature control, SiCTeC achieves reproducible and fast (1-2 min) temperature transitions with programmable waveforms between room temperature and 45 {degrees}C with an air objective. At the devices maximum temperature of 89 {degrees}C, SiCTeC revealed that Escherichia coli cells progressively shrink and lose cellular contents. During oscillations between 30 {degrees}C and 37 {degrees}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.

microbiology↗

bioRxiv2018

Decoupling of rates of protein synthesis from cell expansion leads to supergrowth

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 identified conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to global 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\" in which rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a novel mechanism for global proteome homeostasis based on modulation of volume growth and dilution.

cell biology↗