Because bacterial cells lack a cytoskeleton, they rely on an internal pressure to push the cell membrane outward. This pressure serves both to help maintain the cell shape and to allow the cytoplasm to increase in volume as the cell grows. The internal pressure is created by the presence of concentrated solutes within the cytoplasm, though it is unclear which solutes play the dominant role. Solutes by definition have an affinity for water, so that there is an free-energy decrease associated with transferring a small volume ∆Vwater of water into the cell,
∆Fosm=−Π·∆Vwater, (2.47)
whereΠis the cytoplasmicosmotic pressure, a number that depends on the concentrations of various solutes in the cytoplasm relative to the extracellular space. This osmotic pressure can be measured in various ways: Stocket al.(1977) measured the cellular contents ofE. coliunder various osmotic conditions and computed a theoretical pressure of 0.33 pN/nm2forE. coligrowing rapidly but approaching saturation; Knaysi (1951) appliedplasmolysis, a contraction of the cytoplasm away from the outer membrane, at various times during growth of an E. coliculture, finding an osmotic pressure up to 1.5 pN/nm2 during initial rapid growth, which stabilized at 0.2 pN/nm2. More references and discussion about the osmotic pressure withinE. coli
can be found in Neidhardt (1996), p. 1211. We will takeΠ = 0.3pN/nm2, or 3 atm as a standard value. In the absence of any constraints, water will flow into the cytoplasm, diluting the internal solutes, until the Π = 0; the cytoplasm will then be in equilibrium with the extracellular space. However, if the cell volume is constrained by a rigid cell membrane, the transfer of water results in an increase of internal hydrostatic
pressure, defined by a free energy cost
∆Fhyd=P ·∆Vwater. (2.48)
In this case, water will continue to flow into the cell until an equilibrium is reached where the net free energy benefit of transferring water is zero; where
P = Π. (2.49)
In many situations, this equilibrium is reached quickly, so there is no need to make a distinction between osmotic pressure and the resulting hydrostatic pressure.
However, during rapid cell growth, the cytoplasm is clearly out of equilibrium to some extent, since water is continuously flowing into the cell. In addition, there are various experimental techniques that involve subjecting a cell to osmotic shocks, causing the cytoplasm to shrink or swell; in these experiments, the cell is therefore temporarily out of equilibrium. The remainder of this section is concerned with the problem of quantifying the extent to which the cell is out of equilibrium under these different conditions. In particular, we will try to quantify the pressure imbalanceQ ≡Π−P, which will be positive whenever water is flowing into the cell, and negative when water is flowing out.
For the following estimates we will assume thatE. colihas a cell volume of0.4µm3 = 4×108nm3. During rapid growth,E. colidivides once every 1200 s. This means that water must flow into the cell at a rate of
Φ = 4×108nm3/1200s= 3.3×105nm3/s. (2.50)
The pressure imbalance depends on the permeability of the cytoplasmic membrane to water: a more permeable membrane will allow the cell to stay closer to equilibrium. To estimate this permeability, we turn to experiments in which measured osmotic shocks cause measurable changes in cytoplasmic volume. Delamarche et al. (1999) report a water channel, AqpZ, that is present in the cytoplasmic membrane of
E. coli. In their experiments, cells are subjected to hyperosmotic shocks (Q <0), causing a shrinkage of the
cytoplasm, observable under cryo-electron microscopy, known as plasmolysis. From their graphs we see that a 1 osM (2.6 pN/nm2) shock causes AqpZ+ cells to begin shrinking at a rate of∼0.2cell volumes/15 s= 5.3×106nm3/s, while AqpZ- cells shrink much more slowly, at about6.7×105nm3/s. From this we can infer that AqpZ is the primary water device for water transport across the cytoplasmic membrane, and we can estimate the rate of water flow as a function of pressure. We will assume a linear relationship between
the influxΦof water andQ:
Φ = Q
2.6pN/nm2 ·5.3×10
6nm3/s=Q·2.1×106nm3/s·nm2/pN. (2.51)
The coefficient multiplyingQon the right side of Equation 2.51 is the permeability of the cell membrane to water; the assumption of linearity allows us to make estimates for other conditions. For example, during rapid growth, the rate of water influx implies a pressure imbalance of
Q= 3.3×105nm3/s· 2.1×106nm3/s·nm2/pN−1 = 0.16pN/nm2. (2.52)
Is the cell permeability given in Equation 2.51 reasonable? A simple hydrodynamics calculation shows that it is. Laminar fluid flow through a cylindrical pipe is traditionally calculated using a formula owed to Poiseuille: Φ = Q l π 8ηr 4 =Q·1.4×104nm3/s, (2.53)
usingl = 2nm andr = 0.15nm as the length and radius of the AqpZ pore. This would imply that about 150 copies of AqpZ are required to give the cytoplasmic membrane the permeability given in Equation 2.51, which is a very reasonable number for a membrane channel.