Chapter 8: Temperature, Pressure & pH

Textbook describes temperature on the basis of Arrhenius plots (log growth rate vs. 1/T). While this may be chemically more correct, it is probably conceptually simpler to plot growth rate as a function as temperature. For E. coli:

All organisms exhibit similarly shaped plots, although the range of temperatures vary considerably. Plots define three important temperatures: CARDINAL TEMPERATURES

T_min = minimum T at which growth occurs

T_opt = T at which fastest growth occurs

T_max = maximum T at which growth occurs

For E. coli, the cardinal temperatures are 8°C, 39°C, and 48°C. NOTE: T_opt is always closer to T_max than to T_min.

Note that in the "normal range of growth, that growth rate increases approximately 2-fold for a change of 10°C in temperature. This is typical of enzymatic reactions. (So-called "Q₁₀ = 2" rule).

Medium composition has little effect on the general shape and T_opt; however, medium can alter the T_max and T_min somewhat. Blockage of biosynthetic paths may cause limitation for a critical component. Example: E. coli cannot synthesize methionine above 45°C, but will grow until 48°C if methionine is provided.

T_min and T_max typically differ by approximately 40°C; the reason for this relatively narrow range of growth temperature is not known. Mutations can cause cells to be more HEAT-SENSITIVE or or more COLD-SENSITIVE, but these do not alter in general the other maximum or minimum (do not affect the same component).

Bacteria are quite versatile in their ability to adapt to different temperatures.

Psychrophiles -10°C to 20°C

Mesophiles 10°C to 50°C

Thermophiles 40°C to 70°C

Hyperthermophiles 60°C to 110°C

PSYCHROPHILES: common in the oceans (~5°C), the Arctic and the Antarctic. T_opt of 15°C or lower. Psychrotolerant organisms are mesophiles that grow slowly at lower T. Typically exhibit enzymes that are adapted to function at lower temperatures ("looser" structures) and that are denatured at moderate temperatures (less thermostable). Also have polyunsaturated fatty acids in lipids.

MESOPHILES: includes majority of bacteria, including all pathogens of mammals. Have T_opt between 20°C and 45°C. Some exhibt slow growth at lower temperatures, especially soil microbes that must survive extremes of T.

THERMOPHILES: T_opt >45°C.

HYPERTHERMOPHILES: T_opt > 80°C

Mostly found associated with volcanic phenomena, although some soil temperatures can approach thermophilic temperatures.

Thermophily adaptations: more heat stable proteins. Proteins have more salt bridges, are more densely packed to exclude internal water, have higher degree of hydrophobicity, have more saturated and longer chain fatty acids (archaea have ether-linked, branched chain fatty acids that are more hydrophobic still).

Eukaryotes do not usually grow above ~ 60°C, possibly because of the sensitivity of organellar membranes (somewhat porous) to heat. Some plants will tolerate 45-50°C; protozoa, algae, and fungi ~55-60°C.

Hyperthermophilic eubacteria tolerate T's from 70-90°C. Some cyanobacteria grow well at 70-75°C, as do some anoxygenic photosynthetic bactertia. Only a few eubacteria, including Thermotoga maritima (T_opt = 80°C) and Aquifex pyrophilus (T_opt = 85°C)are capable of growth at temperatures characteristic of hyperthermophilic Archaea. Thermotoga sp. are anaerobic chemoorganotrophs. Aquifex sp. are obligate chemolithotrophs that use H₂, S⁰, and S₂O₃^-2 as electron donors and O2 or NO₃^-1 as acceptors. Aquifex sp. are the deepest branch of the eubacterial group (see week 1 notes).

The high-temperature kings are the anaerobic Archaea. Some methanogens will grow at 110°C, and other Archaea (Pyrolobus sp., Pyrodictium, Pyrococcus sp. grow at up to 115°C). Growth at these temperatures only occurs at increased pressures that keep water in the liquid phase by raising its boiling point. The latter grow fermentatively, or grow chemolitho-trophically (with O₂, NO₃^-1 or S⁰ as electron acceptors).

When growth temperature is shifted in the normal range, cells do not exhibit significant compositional changes. However, shifts from or to the extremes of temperature range are typically accompanied by changes in composition. Shifts to high temperature (HEAT-SHOCK) induce the production of ~24 protein, including many proteases, folding chaperones, etc. Under the control of a special heat-shock sigma factor, RpoH (HtpR in book).

Shifts to low temperature are also accompanied by changes. Cold-shock also induces a special set of proteins, that differs from heat-shock proteins. Low temperature also causes a shift to fatty acids with more double bonds (polyunsaturated); some species also decrease the length of the fatty acid chains. These changes help to maintain lipid fluidity at low temperature, which in turn helps to maintain membrane protein function.

Upper limit for growth is set by protein stability.

The T_max is the lowest temperature at which a required protein is destroyed faster than it can be synthesized.

Protein stability is determined by sequence, and most mutations cause destabilization (more so than loss of catalytic activity). Other adaptations are important to thermophily, but this is the major adaptation. Note that hydrophobic interactions are strengthened at higher temperature. However, few proteins in mesophiles are thermostable, although selection can cause them to evolve in this direction rather rapidly.

Insight into low-T minima are provided by cold-sensitive mutants. These typically grow nearly as well as wild-type at higher temperature, but grow much slower at low T. Allosteric proteins are one problem. Can become more (or in some cases less) sensitive to effectors, and hence cells literally starve to death at low T. Other effects are assembly defects, especially in ribosomes; also protein synthesis is sensitive to low T. As for high-temperature limits: the types of changes that are limiting can be specified, but the target in general is not easy to identify. Low-T limits are more complex to explain in general than high-T limits.

NOTE: the book says that inability to change lipid composition is not a cause of temperature minima, but this is not exactly so. This may be the case for E. coli, but cyanobacterial mutants unable to synthesize polyunsaturated fatty acids lose transport (permease) functions at higher temperatures than wild-type organisms that can increase their polyunsaturated fatty acid contents. Starvation for nitrogen or other nutrients then occurs.

Solute concentration differences across the CM establish an OSMOTIC PRESSURE due to movement of water into the cytoplasm. At external solute concentrations greater than about 0.15 M, water will move OUT of the cell and cell will shrink, or PLASMOLYZE, away from cell wall. At external concentrations lower than 0.15 M, water will move into the cell causing it to swell and press against the peptidoglycan layer.

Plants and animals maintain constant environments, by and large, to combat this problem. Bacteria are contained within a rigid wall and maintain high turgor pressure (G+ higher than G-). Eliminates the need for rapid and careful balancing of osmolarity inside cell to external conditions, and may even be required for expansion of cell wall during growth.

There are a few exceptions: halobacteria and mycoplasmas have low turgor pressure. Mycoplasmas maintain approximately iso-osmotic conditions by means of a Na⁺-ATPase; they pump Na⁺ out of the cell in an ATP dependent fashion. Additionally, mycoplasmas incorporate sterols from host into their CM to strengthen the membrane.

Bacteria typically respond to hyperosmotic stress by increasing their internal concentration of "COMPATIBLE SOLUTES."

Examples include: K⁺, amino acids such as glutamate, glutamine, proline, g-aminobutyrate, alanine, glycinebetaine, and sugars such as sucrose, trehalose, and glucosylglycerol.

Quantitatively, K⁺ is most important, and its concentration increases (along with counterion, usually Cl^-) when external osmolarity increases.

K⁺ uptake occurs by 3 transporters in Salmonella sp. and is driven by H⁺ extrusion.

Turgor pressure, not external osmolarity, signals K⁺ pump to increase or decrease K⁺ concentration. How? Mechano-sensitive?

Glutamate is also important in G- bacteria, less so in G+, which sometimes use glutamine or alanine instead.

High osmolarity in cytoplasm will eventually inhibit enzyme activity leading to cessation of growth. OSMOPROTECTANTS, e.g. proline or glycinebetaine, can protect against harmful effects. Salmonella sp. transport proline, choline, and betaine into cytoplasm in response to hyperosmotic conditions. G+ bacteria tend to synthesize the osmoprotectant (such as proline).

The details of how osmoprotectants/compatible solutes function is not known. Are they harmless solutes to balancing osmotic strength, or do they interact proteins to prevent inactivation?

An additional adaptation, not fully understood: OmpF synthesis declines and OmpC synthesis rises in response to medium osmotic strengthen. Both proteins are porins found in the outer membrane of E. coli/Salmonella sp. The benefit of this adaptation is not understood. OmpF has a slightly larger channel width; OmpC is typically used inside the human body (for pathogenic strains).

High pressure environments are found in the deep ocean, where organisms must survive: low temperature (~2°C), dilute nutrients, and very high pressures (up to ~1100 atm at 10,500 m). This is a high-stress environment, yet bacteria survive (thrive) there.

Do deep-sea bacteria TOLERATE (BAROTOLERANT) high pressure, or are they DEPENDENT (BAROPHILIC) upon high pressure? Depends on where the organism comes from. Organisms from depths to 4000 m (about 400 atm) are barotolerant. They grow better at 1 atm than at high pressure, but can still grow up to a limiting pressure after which growth no longer occurs.

Organisms from 5000 to 6000 m are barophilic and grow better at high pressure (500-600 atm) than at 1 atm or 1000 atm (i.e., they have an optimum pressure for growth which is very high). Extreme barophiles also exist, however, that only grow at very high pressures (700-800 atm) and do not grow at all at 1 atm.

Pressure affects volume-change-dependent reactions in cells (conformational changes affect protein volume); allosteric proteins and catalysis would be affected, and pressure changes the affinity of enzymes for substrates and the ability of proteins to change conformation. Changes that increase volume would be slowed at high pressure, while changes that decrease volume would be speeded up at high pressure. Also, protein synthesis appears to be very sensitive to high pressures. One change that is known to occur when barophiles are grown under different pressures is that they alter the porins of their outer membranes. Apparently porins may be pressure-sensitive. Pressure-regulated gene expression is an interesting phenomenon, but the control mechanism is not understood at present. However, it is known that transcriptional regulation occurs, so pressure signal is somehow transduced into a DNA binding protein.

Side note: yeasts are only capable of growth up to about 8 atm. The build-up of pressure in a champagne bottle inhibits their further growth.

Bacteria have remarkable adaptive capabilities, and have adapted biochemically and physiologically to growth conditions ranging from pH 1 to pH 11. Bacteria are roughly divided into 3 groups:

Acidophiles grow at pH 1-5

Neutrophiles grow at pH 6-8

Alkaliphiles grow at pH 9-11

Bacteria maintain a constant internal pH no matter what the external pH, and most neutrophiles have internal pH values that are close to neutrality (E. coli has pH_i = 7.6). However, internal pH is variable, with acidophiles having somewhat lower pH_i = 6.5 and alkaliphiles having pH_i = 8.0

Remember that protonmotive force (Dp) from respiration is the result of two components:
Dp = DY - 60DpH

DY = electrical potential component

DpH = chemical energy component due to H⁺ conc.

In neutrophiles, such as E. coli and most other pathogens, etc. 70-80% of the stored energy of Dp comes from the electrical potential component (DY) and 20-30% comes from DpH. Since the internal pH is 7.6, only a small DpH can exist across the CM.

Primary proton pumps of respiration are electrogenic and create a large charge difference. pH differences are electroneutral (You can't walk to a shelf and find a container of protons, rather you find HCl or H₂SO₄ which dissociate to provide protons and anions in solution).

Also note: A given proton cannot simultaneously be used to create a charge difference and a pH difference! However, exchanging H⁺ and K⁺ across the membrane can establish a DpH. K⁺ influx reduces the membrane charge potential, and exchanging H⁺ across the membrane for K⁺ allows pH control of cytoplasm.

Acidophiles must maintain a very large DpH across the CM, since internal pH is about 6.5 and optimal external pH is about pH 2. The force in Dp is mostly DpH, and the DY component must be kept very low and generally positive rather than negative (as is true in E. coli). K⁺ influx at the expensive of ATP (K⁺ ATPase) is probably used to maintain this inverted chemical potential.

Alkaliphiles live in environments rich in sodium carbonate and have the opposite situation, since they must constantly pump H⁺ into the cell to maintain their low pH relative to the high external pH. ATP synthesis in alkaliphiles is a particular problem, since the Dp seems to be too low to allow ATP synthesis to occur. The answer to this dilemma is not completely understood, but it may be the local H⁺ flow is more important than bulk H⁺ effects; respiration could directly couple to the ATP synthase to deliver protons for ATP synthesis.

Most of the Dp must come from DY. Alkaliphiles probably use a Na⁺/H⁺ antiporter system to maintain low internal pH. Protons generated by respiratory activities are used to pump Na⁺ out of the cell. In turn, Na⁺ gradient is used to pump solutes, etc. into the cell via Na⁺ symports, completing the circuitry.

In addition to pH homeostasis, many bacteria can induce an ACID TOLERANCE RESPONSE (ATR) upon exposure to acid. The response is triggered at moderate pH 5-6, but can protect against much stronger acidic conditions (pH 3 or so). More than 40 proteins are induced by this response, which helps pathogens to survive passage through stomach and phagocytic vesicles of host defense cells.