Hyperosmotic Shock in Non-Halophilic Bacteria

Hyperosmotic shock was used in this thesis as an example for environmental stress. The process of osmoadaption in bacteria will be explained in the following.

In many non-halophilic bacteria an osmotic upshift leads to loss of water and turgor pressure. To prevent the loss of water bacteria engage in a two-phase adaption reaction (Figure 13). The reaction to plasmolysis is marked by inhibition of a variety of cellular processes including nutrient uptake and replication (206). Cell growth stops immediately upon a hyperosmotic shock and is first resumed after adaption (207). First the osmotic pressure across the membrane is reestablished by a temporary, massive uptake of K⁺ ions from the environment. E. coli constitutively expresses the low affinity K⁺ transport systems, Trk and Kup (208,209).

Additionally the two component system KdpDE is induced at high osmolarity. The kdpFABC high affinity K⁺ transporter has been shown to be upregulated under hyperosmolal conditions (210,211) and its transcription is under positive control of the KdpE and KdpD proteins.

Figure 13: Biphasic Osmoadaption in Bacteria

After an osmotic increase in the surrounding medium bacteria engage in a biphasic osmoadpation process. A: The osmotic pressure across the membrane is re-established by massive uptake of K⁺ ions. Glutamate, glutamine or glutathione are taken up or synthesized de novo and serve as counter ions. B: High intracellular concentrations of K⁺ are in the long run detrimental to cellular functions and replaced by compatible solutes such as polyols, amino acid derivatives, urea and methylamines.

As counter ions glutamate, glutamine or glutathione are taken up or synthesized de novo (212,213). In gram-negative bacteria hyperosmotic stress can lead to greater than 10-fold increase in the intracellular levels of glutamate, likewise glutamine accumulates, the precursor of glutamate (206). An increase of the osmolarity of the medium from 0.1 to 1.2 osm lead to an increase in the intracellular K⁺ concentration from 0.15 to 0.55 M in exponentially growing E. coli (206). Ohwada and Sagisaka report an immediate increase of intracellular ATP after induction of osmotic stress with sodium chloride, possibly as a result of inhibition of macromolecular biosynthesis (214). An increase in the ATP to ADP ratio was also reported by Meury and Kohiyama (215). The accumulation of K⁺ ions has an overall negative effect on the cellular metabolism by disturbing protein function, DNA-protein interactions and protein synthesis (207). Therefore intracellular K⁺ is subsequently replaced by compatible solutes. More effective osmoprotectants include polyols (e.g. glycerol, trehalose), amino acids and their derivatives, urea and methylamines (e.g. glycine betaine, proline betaine) (206,216). ProU and ProP are transport systems for proline and needed for the accumulation of proline under hyperosmotic stress.

A hyperosmotic shock induces transcription of the proU operon (206,217). OmpF and OmpC are outer membrane porins with opposite behavior to osmostress, high osmolarity of the medium causes a reduction of OmpF and an increase in OmpC and vice versa (218-220). Weber and Jung carried out a profiling of the early osmostress dependent gene expression in E. coli after treating the bacteria with 0.4 M NaCl for 9 min using a DNA macroarray (221). In total 152 genes were detected that showed changed expression after shock compared to before. Genes were considered differentially regulated when the change in expression level was greater than 1.4 fold. Retesting a number of genes by Northern Blotting showing upregulation of kdpFABC, osmY, rpoS and dps and downregulation of ompF and ompT. In general 11% of all decreased genes were found to encode amino acid biosynthetic enzymes and Weber and Jung argue that this could be an additional effect triggering rpoS expression and probably underlines the slow growth rates of E. coli under osmotic stress.

Balaji et al. examined the timing of induction of osmotically controlled genes in Salmonella enterica serovar typhimorium by quantitative real-time reverse transcription-PCR (qPCR). 0.3 M sodium chloride and 0.6 M sucrose were used as stressors (222). The following time course was detected after shock with 0.3 M NaCl: The proU operon encoding the ProVXW transport system for the osmoprotectants glycine betaine and proline was induced early on after 4 min. Next, genes encoding the proline glycine betaine permease proP and the stationary phase sigma factor rpoS were induced at 4 to 6 min. Increasing levels of the kdpABC operon were detected after 8 to 9 min.

After 10 to 12 min the trehalose-6-phosphate phosphatase otsB and ompC were induced. Shock with 0.6 M sucrose followed similar kinetics, but kdp levels were found to be reduced in comparison to shock with sodium chloride. The response to osmostress occurs rapidly, changes in gene expression peak after 15-20 min and have returned to normal levels after 60 min (222).

While cell division stops during the early phase of adaption, osmoadaption growth resumes after completed osmoadatption to high osmolality in the medium (223).

Higgins et al. studied the effects of growth under high osmolarity on DNA supercoiling and detected an increase of negative supercoiling in a plasmid system upon hyperosmotic shock (224).

In this case DNA supercoiling is proposed to be important for the osmotic induction of proU transcription. An increase in negative supercoiling upon osmotic stress was also published by Meury and Kohiyama (215). Cheung et al. report that an increase in negative supercoiling is crucial for the induction of many genes upregulated during hyperosmotic conditions in E. coli (225). In addition Cameron et al. describe vast changes in the level of supercoiling in E. coli during different growth phases, upon osmotic challenge or antibiotic treatment. However, the changes in supercoiling were less prominent in Salmonella under the same conditions (226). Generally, movement of the RNA polymerase along the DNA during transcription generates negative supercoiling behind the enzyme and positive supercoiling in front of the enzyme (227-229). Zhang et al. reported G-quadruplex formation in response to transcriptional activity at remote (~kbp) downstream locations due to the mechanical torsion propagated through the double helix. They propose that G-quadruplex formation at distal sites may work as a relay of the transcriptional activity for instance by G-quadruplex binding proteins (230).