PLANTACIONES AGROFORESTALES EN ZONAS ÁRIDAS

It is known that E. coli changes the fatty-acid composition of membrane lipids as a function of the temperature of growth [216], which impacts the lipid bilayer’s fluidity. This phenomenon is known as “homeoviscous adaptation”. Los and Murata [217] re-

ported that hyperosmotic stress might have an impact on membrane fluidity similar in its consequences to low-temperature stress. The dependence of membrane fluidity on the degree of unsaturation of fatty acids is a well-known phenomenon, so one may expect that the rigidity of the membrane in hyperosmotically shocked cells is a result of desaturation of fatty acids, as was shown for Bacillus subtilis [217]. When the lipid membranes of the cultures grown in LB medium with and without a 1.5 M NaCl were compared, the difference in viscosity and cardiolipin (CL) content was apparent [218]. Interestingly, membrane fluidizers cause very similar effects to hyperosmotic stimulation [219]. Morein et al. [220] reported that the addition of a 0.1 M NaCl to a lipid extract from an inner and outer membrane ofE. coli grown at 37◦C caused a 15 – 20◦C decrease in the temperature at which the phase transition occurred.

The facts listed above indicate that the salt content in the growth medium may have an impact on membrane fluidity and, as a consequence, on mechanosensitive channels. The degree of impact of salt on functioning of MS channels may be tested by a series of volume change measurements performed on cells grown in media of different salt concentrations and exposed to an identical osmotic shock (e.g., osmotic shock from 0.5 M to 0.2 M and from 0.3 M to 0 M). The hypothesis of salt indirect impact on MS channels could also be proven by showing that the “temperature effect” may be reversed by the “salt effect”.

The most common solute used to change the osmolarity of the medium is NaCl. However, it was reported [221] that salt may have an impact on peptidoglycan con- traction. This appeared to be the effect of the electrostatic interaction with the peptidoglycan rather than cell volume change due to the osmotic effect. Isolated pep- tidoglycan showed a similar behavior: upon addition of water it released protons and, as a result, the structure shrank (however, intact cells and extracted peptidoglycan do not show sensitivity to salt in well-buffered media). Interestingly, this effect was not observed when sucrose was used instead of salt. The salt content may also have an impact on the peptidoglycan geometry: in high-salt media cells are smaller and shorter compared to the ones grown in a low-salt medium.

The presence of PEG (polyethylene glycol) in the growth medium (used to change the osmolarity of the medium) caused very severe damage in the cells during the osmotic shock. One could notice the presence of a few inclusion bodies in the cyto- plasm, which might have been aggregates of denaturated protein (Figure 7.7). The experiments on cells grown in media of the same osmolarity but in the presence of various solutes would show the role and importance of the peptidoglycan mesh in cell protection from the osmotic shock and any other potential changes in cell physiology due to high concentration of a given solute.

Figure 7.7: Inclusion bodies localization in E. coli cells. As reported in [222], such aggre- gates may appear in the cell growing in the non-stressing environment as natural transcrip- tion and translation errors, or in protein overproducing mutants, and as a result of stress conditions. The induced inclusion bodies were found to be located at the cellular poles, in mid- or quarter-cell positions. The results suggest that the presence of the aggregate caused a reduced growth rate. Hence, inclusion bodies appear to act as an intracellular sink for abnormal proteins.

The majority of experiments are done on cells in the exponential phase of growth. Such a solution is believed to be optimal since all cells should be in the same physi- ological state and their reaction is expected to be identical. However, Makinoshima

et al. [122] reported that E. coli culture in an exponential phase of growth may be separated into at least five discrete subpopulations after the Percoll gradient centrifu- gation (in stationary phase even ten separate subpopulations may be distinguished). Bacterial populations, even those in an exponential phase of growth, show some het- erogeneity.

The physiology of a bacterial cell changes upon transition into a stationary phase. The two most meaningful parameters (from the osmotic shock survival point of view) are structural modification of the peptidoglycan and changes in gene expression. It was reported that murein from cells in a stationary phase of growth is more cross- linked compared to that from an exponential phase. However, the length of the glycan chains was longer in the peptidoglycan from the cells in the exponential phase of growth, compared to that from cells in a stationary phase [223].

All the changes described above may modify the cell response to a given stress. The results of experiments performed on cells in an exponential phase of growth separated in the Percoll gradient centrifugation may indicate the importance of the overall cell density on survival of osmotic shock. If the variation in cell density (or size) is the main source of heterogeneity, separation of cell population in the Percoll gradient and then exposure of each subpopulation to an identical osmotic shock is expected to result in a much lower cell-to-cell variation among the cells from a given subpopulation.