Single-Cell Analysis of the Physiology of Mechanosensation in Bacteria

Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels

The Rate of Osmotic Downshock Determines the Survival Probability of Bacterial Mechanosensitive Channel Mutants

Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been characterized primarily by a standardized assay, where aliquots of batch-cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in Escherichia coli) demonstrate limited effectiveness against shock, typically rescuing less than 10% of the cells when expressed at native levels. We introduce a single-cell-based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of a second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a reevaluation of our basic understanding of the physiology of MS channels

Methylation at the C-2 position of hopanoids increases rigidity in native bacterial membranes

Sedimentary rocks host a vast reservoir of organic carbon, such as 2-methylhopane biomarkers, whose evolutionary significance we poorly understand. Our ability to interpret this molecular fossil record is constrained by ignorance of the function of their molecular antecedents. To gain insight into the meaning of 2-methylhopanes, we quantified the dominant (des)methylated hopanoid species in the membranes of the model hopanoid-producing bacterium Rhodopseudomonas palustris TIE-1. Fluorescence polarization studies of small unilamellar vesicles revealed that hopanoid 2-methylation specifically renders native bacterial membranes more rigid at concentrations that are relevant in vivo. That hopanoids differentially modify native membrane rigidity as a function of their methylation state indicates that methylation itself promotes fitness under stress. Moreover, knowing the in vivo (2Me)-hopanoid concentration range in different cell membranes, and appreciating that (2Me)-hopanoids' biophysical effects are tuned by the lipid environment, permits the design of more relevant in vitro experiments to study their physiological functions

Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories

Hopanoids are steroid-like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2-methylhopanoid production in Rhodopseudomonas palustris TIE-1 and purified 2Me-diplopterol, 2Me-bacteriohopanetetrol (2Me-BHT), and their unmethylated species (diplopterol and BHT). We found that 2-methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me-diplopterol produces 10× higher ion counts than equivalent quantities of 2Me-BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC-MS, however, 2Me-BHT produces 11× higher ion counts than 2Me-diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D_4) diplopterol, a precursor for a variety of hopanoids. LC-MS analysis on a mixture of (D4)-diplopterol and phospholipids showed that under the influence of co-eluted phospholipids, the D_4-diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples

Bacterial mechanosensitive channels : progress towards an understanding of their roles in cell physiology

Open Access funded by Wellcome Trust Under a Creative Commons license Thanks to all members of the Aberdeen group, collaborators and friends whose discussions have spurred the development of the MS channel field. Special thanks to Doug Rees, Diane Newman and Rob Phillips for their support and hospitality at Caltech. Unique insights have been provided by members of the Newman and Phillips research groups, particularly, Caj Neubauer, Gargi Kulkarni and Megan Bergkessel, Heun Jin Lee and Maja Bialecka-Fornal. The author's research on MS channels is supported by a grant from The Wellcome Trust (WT092552MA) and the BBSRC (BB/H017917/1). The author is a Leverhulme Emeritus Fellow and this work was supported in part by a CEMI Visiting Faculty Fellowship from Caltech.Peer reviewedPublisher PD

Stability for Receding-horizon Stochastic Model Predictive Control

A stochastic model predictive control (SMPC) approach is presented for discrete-time linear systems with arbitrary time-invariant probabilistic uncertainties and additive Gaussian process noise. Closed-loop stability of the SMPC approach is established by appropriate selection of the cost function. Polynomial chaos is used for uncertainty propagation through system dynamics. The performance of the SMPC approach is demonstrated using the Van de Vusse reactions.Comment: American Control Conference (ACC) 201

Membranes by the Numbers

Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.Comment: 27 pages, 12 figure

Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering

The Rate of Osmotic Shock Determines Bacterial Survival

Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been primarily characterized by a standardized assay, where aliquots of batch cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in E. coli) demonstrate questionable effectiveness against shock. We introduce a single-cell based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, even "ineffective" MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a re-evaluation of our basic understanding of the physiology of MS channels

Mean channel counts per cell determined by fluorescence microscopy (FM) for various media versus OD₆₀₀.

<p>(A) M9+glycerol. Fusion strains with (MLG910, light blue squares) and without RpoS (MLG-Δ<i>rpoS</i>, yellow squares). (B) M9+glucose. Fusion strains with (MLG910, green squares) and without RpoS (MLG-Δ<i>rpoS</i>, red squares). (C) LB-Miller. Fusion strains with RpoS (MLG910, black squares). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone-0033077-g003" target="_blank">Figures 3A, 3B, and 3C</a>, the corresponding mean number of channels determined by Western blots (WB) for the MLG910 strain (open squares) and the MG1655 strain (open triangles) are shown for reference. (D) Comparison of fluorescence microscopy results from MLG910 grown in three different media. (E) Comparison of fluorescence microscopy results from MLG910 grown in M9+glucose supplemented with four different NaCl concentrations: 0 mM (green squares), 100 mM (dark blue squares), 250 mM (gray-blue squares), and 500 mM (dark gray squares). The error bar of each fluorescence microscopy results measurement is dominated by systematic uncertainties in the absolute calibration related to single-molecule fluorescence calibration. The standard error of the mean of the uncalibrated fluorescence counts per cell is typically less than 5% of the total error bar.</p