The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.
The Escherichia coli cell envelope is responsible for chemically isolating the cell interior from the surrounding environment and consists of the outer membrane, the periplasmic space that contains the cell wall, and the inner membrane. Each of these elements contributes to the overall permeability of the cell to different solutes. A host of inner and outer membrane-proteins facilitate the passage of ions and molecules across the two lipid bilayers, greatly increasing their permeability. Note that here we use the term membrane permeability to represent the passage of solutes through the combined lipid-protein sheet. The cell wall in the periplasm consists of porous peptidoglycan chains, although the periplasm itself is thought to be a packed physical environment.
Escherichia coli cells actively regulate their internal osmolality to maintain a favorable turgor pressure. Ordinarily, the concentration of solutes within the cytoplasm is higher than the environment resulting in a positive pressure on the cell wall. An increase in external solute concentration, termed hyperosmotic shock, causes fast water efflux and a pressure drop across the semipermeable cell envelope. This results in altered cell size, cell shape, and membrane stress levels. Proteins responsible for the detection of the internal osmolality changes as well as subsequent recovery, such as ProP, TrkA, KdpA, and BetT, presumably respond to one or more of these morphological parameters. Some of these sensors localize at specific places along the cell surface, such as the cell poles, whereas others form a more diffuse pattern (1). It is therefore of considerable interest for understanding their specific roles in osmorecovery to compare these localization patterns with the localized morphological changes, which occur upon osmotic shock. Here, we use quantitative analysis of the E. coli periplasmic and cytoplasmic spaces to investigate cell shape and size changes during shocks of different magnitudes and compositions and find that the kinetics of membrane permeability plays a significant role in defining the shocked state.
The response of bacteria to osmotic shock has been studied for decades (2–16). However, the methods used in these experiments vary considerably, particularly in time resolution (typically 30 s up to 15 min) and the number of conditions investigated (12,14,16,17). In addition, morphological changes have largely been discussed in qualitative and binary terms, e.g., when investigating plasmolysis, a process in which the inner membrane detaches from the cell wall. This work has therefore led to an incomplete, and at points contradictory, set of conclusions. In particular, the role that solute membrane permeability plays in defining the dynamics of cell shape change has been difficult to elucidate.
What is clear from previous work is that shape change does not solely depend on the magnitude of a shock. Hyperosmotic shock produced using solutes such as pentose or sodium polyglutamate that cannot penetrate the outer and inner membranes cause both the cytoplasmic and periplasmic volumes to shrink extensively, up to 60%, without noticeable plasmolysis (16,18). On the other hand, plasmolysis does occur when using solutes that can easily penetrate the outer membrane as the periplasm and external environment quickly equilibrate tonicity (2,3,5,7,8,11,19–21). A large number of solutes fall somewhere between these two extremes and penetrate the inner and outer membranes on slow timescales. Two solutes that are commonly used to increase the external concentration in osmotic shock experiments, sodium chloride and sucrose, have been reported to exhibit very different membrane permeation kinetics. Sodium chloride is relatively fast and crosses the outer membrane on the order of seconds, whereas sucrose equilibration proceeds on the order of minutes (5). Thus, it is not a surprise that the studies of the initial response with limited time resolution using these two solutes have led to seemingly contradictory observations.
Escherichia coli strain YD133 (ΔFimA, ΔFliC, ΔFlgE derived from the K12 Keio collection strain) with plasmid pWR20 (carrying enhanced green fluorescent protein, EGFP, and kanamycin resistance) was grown from frozen stocks as described previously (22) to an optical density (OD) of 0.5. Upon reaching the required OD, cells were kept at room temperature and used for sample preparation for up to 3.5 h (up to a maximum OD of 0.85).
For cytoplasmic and total cell volume measurements, cells were prepared as previously described (22). Microscope coverslips were assembled into tunnel slides and both cells and microspheres were immobilized on the glass coverslip surface (22,23).
Cells were observed in epifluorescence and differential interference contrast using a modified Nikon TE2000 microscope as described previously (22,24). To stabilize the sample during the measurements, the position of a microsphere attached to the coverslip surface was kept fixed in the x-, y-, and z-directions using proportional-integral-derivative feedback of the stage position (22,23) and back focal plane interferometry (25,26). s were acquired 1 Hz for sucrose shocks and 0.5 Hz for dextran and sodium chloride shocks. Trans- and epiillumination light was shuttered in between recordings to reduce photobleaching of the GFP.
To change the osmolality of the external environment in the tunnel slide, LB is exchanged with 10 mM Tris-HCl (pH = 7.5) with defined concentrations of sodium chloride, sucrose, or dextran (Mw = 1000). Osmolalities of solutions were calibrated with an osmometer (Osmomat30, Genotec, Germany).
A small rectangle around each analyzed cell is chosen and the cell long axis is aligned horizontally with the axis. A record of volume, length, and width (derived from the red, outer membrane dye signal) and cytoplasmic cell volume, length, and width (derived from the green GFP signal) were obtained by a process of background subtraction and thresholding we previously described (22). Briefly, after initial alignment and background subtraction the pixels above a certain threshold were identified and a mask of cell area obtained (22). The total cell length and width, and cytoplasmic length and width correspond to the length and width of the cell along the cell midline. The length of the cell along three pixel columns aligned with the cell axis was averaged to obtain the total cell and cytoplasmic lengths. Similarly, the length of three rows in the middle of the cell was averaged to obtain the total cell and cytoplasmic width (Fig. S1 A in the Supporting Material). To obtain the total cell volume and cytoplasmic volume the cell was assumed to be rotationally symmetric along the long axis. We recorded the width as a function of distance along the cell axis. The volume for each pixel along the length of the cell was calculated based on this width profile (see also Fig. S1) and summed to obtain the record of the total cell and cytoplasmic volume.
The resultant records were aligned at the time of osmotic shock and normalized by dividing the entire trace with the average value of volume, length, or width calculated using the first 5 s of recording that occur before osmotic shock. The average and standard deviation trace was then computed from these normalized and aligned data sets. For osmolality changes induced with sodium chloride, the FM4-64 dye bleaches significantly within ∼90 s after shock, depending on the cell. Thus, the data sets showing cytoplasmic cell volume, length, and width for high sodium chloride shocks contains more cells then the data sets showing total cell volume, length, and width (where the smaller data set is part of the larger).
Polar kymographs were calculated from cytoplasmic and total cell fluorescent s. Cytoplasmic area changes were normalized by subtracting the initial from each subsequent frame. For periplasmic area, both the cytoplasmic and total cell areas were normalized as previously mentioned, and then cytoplasmic subtracted from the total cell area. Cytoplasmic and periplasmic area changes where then converted into polar coordinates, where we define the polar extent to be the distance from the center of the cell (defined as half a distance between minimum and maximum value of the cell boundary in x and y) to the edge of the cell. Initial alignment of cells along the long axis did not take into account possible postshock morphological asymmetries, thus the polar extent in [π, 2π] interval was added to that of the [0, π] interval.
caused by high osmotic pressure outside the cell. the shrinkage of microbial cells due to water loss.
Initial response to an outer-membrane impermeable solute
To gain detailed information about morphological changes in E. coli cells upon a hyperosmotic shock caused by an outer and inner membrane impermeable solute, we looked at the effect of shocks of different magnitudes caused by dextran (5). shows an averaged response of a population of E. coli cells upon transition from LB into 10 mM Tris-HCl buffer with different dextran concentrations: 0.11, 0.42, 0.95, and 1.5 Osmol/kg. The smallest dextran concentration used, 0.11 Osmol/kg, is hyposmotic. As a result, the total and cytoplasmic volumes expand by a few percent when shocked with this concentration. When the osmolyte cannot penetrate the outer membrane, the total cell volume and cytoplasmic cell volume both decrease in unison upon hyperosmotic shock. The extent of the total and cytoplasmic cell volume, length, and width decrease is plotted against the magnitude of the shock in . The cell volume increases with magnitude of the dextran hyperosmotic shock, up to ∼40% total volume reduction for the largest shocks ( A). For small hyperosmotic shocks cell’s cytoplasm shrinks in length (up to 10%) with hardly any reduction in width ( C). As the magnitude of the shock increases a further reduction is achieved by predominantly decreasing the width of the cell ( , B and C).
, D and E, show polar kymographs of average changes in the polar extent of the cytoplasmic and periplasmic spaces before, during, and after osmotic shock, as the shock magnitude increases from left to right. No increase in periplasmic polar extent for an inner and outer-membrane impermeable solute is visible. Plasmolysis does not occur regardless of the shock magnitude.
Quantifying changes in volume
To obtain a record of total and cytoplasmic cell volume changes during the initial response of E. coli to hyperosmotic shock, we simultaneously monitored a cytoplasmicly-expressed GFP and fluorescent outer membrane dye, as described previously (22). We kept the observed field of view fixed in a three-dimensional position (22) and used a sampling rate of either 0.5 or 1 Hz. To isolate the initial response from the recovery, we induced the hyperosmotic shock by transferring the cells from the growth medium (Material and Methods section) to 10 mM Tris-HCl buffer supplemented with a given amount of dextran, sucrose, or sodium chloride. shows the example traces from individual cells shocked with three different solutes. The fluorescent s of the outer membrane and cytoplasm prior and upon hyperosmotic shock as well as the corresponding time traces of the total and cytoplasmic length, width, and volume in response to a dextran ( A), sucrose ( , B and C), and sodium chloride ( D) hyperosmotic shock are given. To obtain a record of normalized cell length, width, and volume trace from a time sequence of fluorescent s, we used a previously reported process of background subtraction and thresholding ((22), see also Materials and Methods).
, E–H, shows polar kymographs of changes in the polar extent of the cytoplasmic and periplasmic spaces before, during, and after osmotic shock for each of the example cells in , A–D. We used polar kymographs as a way of displaying morphological changes throughout the cell. The periplasmic polar extent kymographs are useful to show the occurrence of plasmolysis, the periplasmic space increases where detachment of inner and outer membrane (with the cell wall) occurs. To obtain the kymographs each cell was aligned with the long axis horizontally and converted to polar coordinates (inset of E and Materials and Methods), such that changes in length translate into the changes in polar extent at angle π, and changes in the middle of the cell to changes in polar extent at angle π/2.
Plasmolysis and deplasmolysis | Biology | Osmosis
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How is plasmolysis induced?
Plasmolysis only occurs in extreme conditions and rarely occurs in nature. It is induced in the laboratory by immersing cells in strong saline or sugar (sucrose) solutions to cause exosmosis, often using Elodea plants or onion epidermal cells, which have colored cell sap so that the process is clearly visible.
What causes plasmolysis during osmotic shock?
Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock.
Why does plasmolysis occur along the poles of a cell?
The poles of the cell are DNA free ( 33 ), potentially explaining the occurrence of plasmolysis along the poles. A recent report shows that the nucleoid has a large-scale coiled organization ( 34 ), which could explain constriction and subsequent occurrence of plasmolysis along the cell width.
Does plasmolysis have an ER-resident protein?
Although the presence of the ER in components of the cell ‘left behind’ at the cell wall by the process of plasmolysis has been indicated by electron microscopy ( Oparka, 1994) and vital staining ( Lang-Pauluzzi and Gunning, 2000 ), the use of an ER-resident protein, GFP-HDEL ( Sparkes et al., 2009) definitively demonstrates this ( Fig. 2 ).