Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier that defines cell shape and allows the cell to sustain large mechanical loads such as turgor pressure. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

Effects of fixation on cellular dimensions and fluorescence patterns were quantified Length and cytoplasmic GFP exhibited rapid dynamics during formaldehyde fixation Methanol preserved fluorescence but did not fully inhibit growth and caused lysis Extended storage faces tradeoffs of fluorescence maintenance and membrane integrity

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.

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17 18 Keywords: open-source experimentation, Arduino, PBP2, PBP3, temperature-sensitive 19 mutants 20 Abstract 21 Single-cell imaging, combined with recent advances in image analysis and microfluidic 22 technologies, have enabled fundamental discoveries of cellular responses to chemical 23 perturbations that are often obscured by traditional liquid-culture experiments.24 Temperature is an environmental variable well known to impact growth and to elicit 25 specific stress responses at extreme values; it is often used as a genetic tool to 26 interrogate essential genes. However, the dynamic effects of temperature shifts have 27 remained mostly unstudied at the single-cell level, due largely to engineering 28 challenges related to sample stability, heatsink considerations, and temperature 29 measurement and feedback. Additionally, the few commercially available temperature-30 control platforms are costly. Here, we report an inexpensive (<$110) and modular 31 Single-Cell Temperature Controller (SiCTeC) device for microbial imaging, based on 32 straightforward modifications of the typical slide-sample-coverslip approach to 33 microbial imaging, that controls temperature using a ring-shaped Peltier module and 34 microcontroller feedback. Through stable and precise (±0.15 °C) temperature control, 35 SiCTeC achieves reproducible and fast (1-2 min) temperature transitions with 36 programmable waveforms between room temperature and 45 °C with an air objective. 37 At the device's maximum temperature of 89 °C, SiCTeC revealed that Escherichia coli 38 cells progressively shrink and lose cellular contents. During oscillations between 30 °C 39 and 37 °C, cells rapidly adapted their response to temperature upshifts. Furthermore, 40 SiCTeC enabled the discovery of rapid morphological changes and enhanced sensitivity 41 to substrate stiffness during upshifts to nonpermissive temperatures in temperature-42 sensitive mutants of cell-wall synthesis enzymes. Overall, the simplicity and 43 affordability of SiCTeC empowers future studies of the temperature dependence of 44 single-cell physiology. 45 Introduction 46 47 While chemical perturbations during single-cell imaging experiments have been made 48 possible by microfluidic technologies [1, 2], other environmental variables such as 49 temperature have been more difficult to precisely and rapidly manipulate during an 50 experiment. Temperature has dramatic effects on virtually all cellular processes, 51 including polymer behavior [3, 4], RNA and DNA polymerases [5, 6], ribosomal 52 elongation [7], and overall enzyme kinetics and function [8]. These diverse, 53 temperature-dependent processes have global impacts on cell growth, which cells must 54 integrate and collectively optimize at each temperature [9]. 55 56 Two predominant elements of experimental design limit our understanding of how 57 cells respond to changes in temperature. First, bulk experiments are the standard for 58 investigating the effects of temperature on steady-state cellular growth [10, 11]. By 59 contrast, single-...