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

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

Summary of reports on the number of MscL channels per <i>E. coli</i> cell.

<p>Summary of reports on the number of MscL channels per <i>E. coli</i> cell.</p

The dependence of critical tension needed to open one channel on the total number of channels.

<p>Estimates of E_open-E_closed show considerable variation due to experimental uncertainties and the effect of lipid composition <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Perozo1" target="_blank">[15]</a>. For illustration, we chose representative values found in the literature that reflect the range of values: 10.0 k_BT (red solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Ursell3" target="_blank">[62]</a>, 18.6 k_BT (green solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Sukharev2" target="_blank">[63]</a>, and 51.0 k_BT (blue solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Chiang1" target="_blank">[64]</a>. Dashed lines show τ_1/2 the tension, where P_open = 0.5. The area change ΔA was taken to be 10 nm² .</p

Representative Western blots showing expression of MscL, MscL-sfGFP, and RpoS.

<p>Arrows indicate the protein of interest. Other bands are the result of non-specific binding. The strains of interest were cultured to exponential (exp) and stationary (stat) phase in LB-Miller (LB), M9+glucose (U), and M9+glycerol (Y). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone-0033077-g001" target="_blank">Figures 1A, 1B, and 1C</a> lanes 1 through 5 are a concentration series of a known number of purified channels diluted into lysate from the MJF612 strain (References). The numbers under lanes 6 through 12 represent the average number of channels from three independent Western blots for the respective conditions. The total error of each measurement includes contributions from the standard deviation of 3 repetitions and the systematic uncertainties in the absolute calibration related to chemiluminescence linearity, initial cell culture density, and lysis efficiency. (A) Western blot performed with MscL antibodies. Lysate from the MJF612 strain (612) was used as a negative control (lane 6). Lanes 7 through 12 show the MscL levels in the MG1655 strain (WT). (B) Western blot performed with GFP antibodies. Lysate from the MJF612 strain (612) was used as a negative control (lane 6). Lanes 7 through 12 show the MscL-sfGFP levels in the MLG910 strain (MLG). (C) Western blot performed with GFP antibodies. Lysate from MJF612 strain (612) was used as a negative control (lane 12). Lanes 6 to 11 show the MscL-sfGFP levels in the MLG910-Δ<i>rpoS</i> strain (ΔR). (D) Western blot performed with RpoS antibodies. Lysate from the MLG910-Δ<i>rpoS</i> strain (ΔR) was used as a negative control (lane 13). Lanes 1 to 12 show the RpoS levels in the MLG910 (MLG) and MG1655 (WT) strains. The numbers under the lanes are the relative amount of RpoS, as compared to the lysate from M9+glucose (lane 11), determined by the average of three independent repetitions. The errors are the standard deviation.</p

Comparison of channel counts per cell from quantitative Western blots and fluorescent microscopy.

a<p>Western blots performed with MscL antibody.</p>b<p>Western blots performed with GFP antibody.</p><p>MG1655 and MLG910 were cultured in LB-Miller media (LB), M9 minimal media supplemented with glucose (U), and M9 minimal media supplemented with glycerol (Y) to the indicated optical density (OD600).</p