Cell membranes are permeable to water but not to most solutes. When the external solute concentration is greater (or smaller) than the internal water tends to move out of (or into) the cell. This may change the size of the cell. However, if there is a cell wall, as in most bacteria, this prevents cell expansion in medium of lower OP, and the pressure exerted against the wall (the turgor pressure) may change instead. In medium of higher OP the cytoplasm will lose water and shrink away from the cell wall. Cells try to adapt to changes in the external solute concentration by adjusting their internal solute levels to balance the outside. However cells need 100-150mM K+ and about the same concentration of metabolic intermediates so cannot reduce their internal solute levels below a minimum of approx 250milli-osmolar. Conversely, increasing the internal salt concentration too far results in the inhibition of many enzymes. The secret to adapting to high OP is the use of "compatible solutes". These are certain solutes which, even in high concentrations do not inhibit enzyme function. The most common are: glutamate, proline, trehalose and glycine betaine.

For E. coli growing in a typical mineral salts medium plus glucose.the internal OP is approximately 300 milli-osmolar. The pressure outwards on the cell wall is approximately 3.5 atmospheres. E.coli can adapt to grow in media containing upto about 4% NaCl if provided with osmoprotectants. Marine bacteria live at substantially greater OPs than this

DEFINITIONS AND NUMBERS

It is important to distinguish between Osmotic Pressure and Ionic strength:

Osmotic Pressure P = cRT

Where c = concentration in moles/liter, R = gas constant, T = absolute temperature. For ionic compounds, c = total concentration of all the ions e.g. for 1.0 molar sucrose c = 1.0 but for 1.0 molar NaCl, c = 2.0 and for 1.0 molar MgCl2, c = 3.0 and so on. Concentrations calculated this way are in "osmolar". Osmotic pressure is proportional to the total number of "molecular particles" (molecules and/or separate ions).

Ionic strength I = 1/2 ·cizi2

Where c = concentration of each ion and z = ionic charge.

For neutral molecules like sucrose I = 0 (no charged ions).

For 1.0 molar Na+Cl-, I = 1/2 [12 + 12] = 1.0.

For 1.0 molar Mg2+SO42-, I = 1/2 [22 + 22] = 4.0.

Osmotic Pressure versus Ionic Strength. An osmotic pressure differential between inside and outside puts pressure on the cell membrane. Furthermore, at high external OP the cell would be sucked dry if it did not increase its internal OP. Thus OP must be regulated. However, enzymes need an environment where the ionic strength does not vary too drastically. Enzymes don't care much about OP per se, it is the ionic environment that affects their structure. Hence increasing the concentration of trehalose, proline etc does not hurt enzymes much whereas increasing K+ does. Glycine betaine (and to a lesser extent proline and trehalose) are not merely just "extra" osmotic pressure they are actual osmoprotectants and counteract the effect of increased ionic strength.

ADAPTING TO HIGH OSMOTIC PRESSURE

Potassium. This is the major inorganic ion in most bacteria. E. coli contains 100 to 150 mM K+ under normal circumstances and the K+ concentration increases with increasing osmotic pressure. Like most cells E. coli accumulates K+ and expels Na+.

The high affinity K+ transport system of E. coli is regulated by osmotic pressure. Mutants in the kdp (K+ dependent) operon cannot grow in low K+ medium (less than 0.1 mM K+). The kdpABC operon is induced by high osmolarity and encodes a transport system which takes up K+ to increase the internal concentration. The three transport proteins, A, B & C form a complex in the inner membrane. ATP is the energy source and the high energy phosphate is transferred to protein KdpB. The sensor protein KdpD, also in the inner membrane responds to a change in turgor pressure and activates the kdpABC genes via the DNA binding protein, KdpE.

Amino acids: Glutamate and Proline. These, and some derivatives, are the major osmoregulatory solutes for many bacteria which synthesize more Glt and Pro as the OP of the culture medium rises. E. coli makes more Glt (and glutamate containing peptides and glutamine) in response to high OP but unlike many bacteria does not increase Pro production much. However, at high OP a special transport system for proline (proU) is induced and E. coli will transport and accumulate high levels of proline. Addition of proline to the culture medium protects against growth inhibition by high salt. The ProU system consists of three genes, proVWX. ProX is a periplasmic binding protein for proline and betaine. ProV &W comprise the inner membrane transporter. Glutamate and proline are only of moderate effect as compatible osmoregulatory solutes.

Sugars: Trehalose. Many eukaryotes accumulate mannitol, glyceryl-glucose or glyceryl-galactose at high OP. Enterobacteria synthesize trehalose, a disaccharide of a1-2 linked glucose units. Trehalose is made from UDP-glucose and glucose-1-P which give trehalose-1-P.

UDP-Glc + Glc-1-P ÆÆÆ Glc-Glc-1-P (= Tre-1-P)

The trehalose-1-P is converted to trehalose by removal of the phosphate. The genes coding for the two enzymes trehalose-P synthase and trehalose-P phosphatase are induced by high OP. When E.coli is shifted to high OP it first makes glutamate and takes up more K+. Then trehalose is synthesized and as its concentration rises the cells excrete some of the K+ and glutamate so replacing them with trehalose which is a more efficient osmoprotectant.

Glycine Betaine. E. coli cannot make this but the proU transport system takes it up, even better than proline. Glycine betaine protects E. coli from inhibition by high salt better than other osmoprotective solutes. Glycine betaine is made by certain eukaryotes, Pseudomonads and Cyanobacteria which are adapted to live at very high osmotic pressure (halophiles). E. coli will convert choline into glycine betaine (via the aldehyde) and will transport choline at high OP. The two enzymes for this interconversion are only expressed at high OP.

Many enzymes of normal bacteria are inhibited by high salt concentrations. Glycine betaine is the best compatible solute known and is not merely harmless but actually counteracts the effect on enzymes of high concentrations of salt. Thus the presence of glycine betaine also allows E. coli to greatly increase its internal K+ concentration without inhibiting most of its enzymes. Certain other betaine derivatives may also be used. If glycine betaine is available synthesis of trehalose is switched off and levels of other solutes are reduced. Glycine betaine is made by cyanobacteria and algae from which some leaks out and is thus found in fresh or salt water environments. Choline is a major component of eukaryotic phospholipids.

GENUINE HALOPHILES

The extreme halophiles eg archebacteria such as Halobacterium, accumulate very high salt concentrations inside the cell. Their enzymes have evolved to work in high salt and can no longer work at normal salt concentrations, hence they do not use compatible solutes. The metabolism and lifestyle of Halobacteria will be discussed later.

Marine halophiles (eubacteria) eg Ectothiorhodospira make trehalose and also synthesize other compatible solutes eg ectoine, (1,4,5,6-tetrahydro-2-methylpyrimidine-4-carboxylate), a uracil derivative. Nonetheless, if glycine betaine is available they prefer to take this up rather than make either trehalose or ectoine.

PORINS & OSMOTIC PRESSURE

The porins of the OM vary in response to changes in OP in E.coli K12:

At low OP: OmpF increases and OmpC decreases

At high OP: OmpC increases and OmpF decreases

No one really knows why. The pore size of OmpF is 1.2 nm diameter - slightly larger than OmpC which is 1.1 nm. It has been suggested that the larger OmpF pores aid solute uptake at low OP when solutes are scarcer. However, the closely related E.coli B has only one porin, OmpF and it is expressed constitutively without regard to OP.

Nevertheless, a complicated scheme of regulation has been found which involves two regulatory genes: envZ a sensor of osmotic pressure and ompR a response regulator which regulates gene expression. Many responses of bacteria to a changing environment involve such two component regulatory systems. The sensor protein is typically found in the cytoplasmic membrane where it can monitor the environment. Upon detecting a signal the sensor protein phosphorylates itself on its active site histidine using ATP:

Sensor-His + ATP ÆÆ Sensor-His~P + ADP

Very quickly the phosphate is transferred to an aspartate group on the regulator protein.

Regulator + Sensor-His~P ÆÆ Regulator-Asp~P + Sensor-His

The phosphorylated version of the regulator binds to DNA in the promotor of the genes which

need to be switched on or off. The same phosphorylated regulator protein may act as a repressor for some genes and an activator for others. For example NarL~P switches genes for nitrate reductase on but represses genes for fermentation or TMAO reductase (see section on anaerobic respiration). Some sensor proteins share the same regulator (e.g. ArcB & CpxA share ArcB).

Examples of Two Component Regulatory Systems

Stimulus Sensor Regulator

osmolarity EnvZ OmpR
KdpD KdpE

phosphate PhoR PhoB

lack of oxygen ArcB ArcA

conjugation CpxA ArcA

nitrate NarX NarL
NarQ NarP

High osmotic pressure changes the conformmation of the outer segment of EnvZ sensor protein. The change is transmitted inwards and EnvZ phosphorylates itself using ATP. It then transfers the phosphate group to OmpR. The OmpR-P form binds DNA. There are two types of binding sites for OmpR-P - low and high affinity. When OP is low, there is only a trace of OmpR-P, but this is sufficient to bind to the high affinity site in front of the ompF gene and activate transcription. At high OP, the concentration of OmpR-P rises and it can now occupy the low affinity sites. This stops transcription of the ompF gene and activates transcription of the ompC gene.

In addition the micF gene is transcribed to give MicF RNA. This binds to the frontof the ompF message and prevents translation. Thus whenever expression of ompC is increased, expression of ompF is decreased. (Actually micF is more probably important for temperature control than for osmoregulation.)

ADAPTING TO LOW OSMOTIC PRESSURE

The cell may also adapt to conditions of external OP lower than the normal cytoplasmic concentration. The cell would tend to take up water and burst so it must lower its internal K+ - but this would also disturb enzyme function. At low OP, K+ is excreted and replaced by putrescine H2NCH2CH2CH2CH2NH2. Putrescine has two amino groups and will ionize at physiological pH to carry two positive charges. If we replace 4 moles of K+ with 1 mole of putrescine2+, the osmotic pressure is greatly reduced but the ionic strength stays the same. If the external OP suddenly increases again the putrescine is rapidly excreted and replaced by K+. Conversely, at low OP, putrescine and related polyamines will be taken up if present in the culture medium. Putrescine is made by decarboxylation of omithine, an intermediate in the synthesis of arginine. The enzyme ornithine decarboxylase is directly inhibited by high ionic strength.

Membrane Derived Oligosaccharides. Unlike all the other osmotically controlled solutes the MDO are found in the periplasmic space. They are made at low OP only. At low OP the periplasmic space would empty out since small ions such as K+ etc would diffuse out through the porins. To maintain some ionic strength/OP in the periplasmic space the cell makes oligosaccharides of 8 to 10 glucose units (hence cannot escape through porins) and which carry several negative charges each; due to glycerol phosphate side chains. The negative charges attract positive ions which would otherwise drift away. The glycerol-P groups come from the phospholipids of the cytoplasmic membrane - hence the name Membrane Derived Oligosaccharides.