Acidophiles and alkaliphiles

Question 1

Definition of acidophiles and alkaliphiles

Answer 1

Most microbes grow best around pH 7 (10-7 M H+) and tolerate pH range from 4 to 9.

Acidophiles - optimum growth at pH 4 or less.

Alkaliphiles - optimum growth at pH 9 or above.

Question 2

What are the habitats of acidophiles

Answer 2

Rare in nature tend to be created in part by the organisms e.g. Fe(II) and sulphide rich environments. core reaction (iron creates sulfuric acid):

4 FeS + 7.5 O2 + H2O 2 H+ + 4 SO42- + 4 Fe(III)

There are two habitats rich in FeS:

1) mining areas - H2SO4 leaches heavy metals such as copper into solution - organisms must tolerate these metals- lot of secretion systems that transport toxic metals out of the cell.

2) geothermal areas - hot springs, organisms must also be thermophiles.

Question 3

What are some examples of acidophiles

Answer 3

Majority are bacteria that oxidise FeS e.g. Acidithiobacillus ferrooxidans (mesophilic Bacterium) and Sulfolobus acidocaldarius (thermophilic Archaeon).

Also, heterotrophic bacteria e.g. Bacillus acidocaldarius.

Few eukaryotes - fungi such as Aspergillus (pH 2 -10) and alga Cyanidium caldarium, which is found near hot springs - optimum temperature 45 oC.

Question 4

What are some habitats of alkaliphiles

Answer 4

Desert soils e.g. Atacama desert, best studied are soda lakes e.g. Lake Natron, Africa (also high salt) - Rift Valley, Kenya.

Few are due to industrial waste.

Alkalinity is due to excess sodium carbonate - geological

Question 5

What are some examples of alkaliphiles

Answer 5

Wide range of taxonomic categories e.g. photosynthetic Bacteria - Spirulina (cyanobacterium) and Ectothiorhodospira (purple bacterium).

Range of fungi, algae and heterotrophic Bacteria, latter best studied e.g. Bacillus alcalophilus.

Question 6

Case study: Arthrospira Harvest Lake Chad

Answer 6

Harvest of Arthrospira platensis from Lake Chad as part of the project “Harvest of Hope”, a project that is sponsored and funded by the Global Climate Change Alliance of the European Union. This project provides employment, cash income and a source of high value food in Chad which is part of the Sahel, an arid region that has seen a rapidly growing population and will be particularly affected by climate change .

Question 7

Whats the internal pH of acidophiles and alkaliphiles

Answer 7

1) If it is similar to external pH, cell constituents must tolerate acid/alkali conditions.

2) If it is about pH 7, processes involved in generating proton motive force (especially in bacteria) must be modified.

Question 8

How is internal pH measured

Answer 8

Difficult to do for microbial cells due to small size – cannot use micro pH probes.

Different internal pH creates a problem for the proton motor force

Question 9

How was internal pH measured in older methods

Answer 9

31P nuclear magnetic resonance (31P NMR) of titratable phosphate and methylphosphonate (Drawback: highly concentrated cell suspensions necessary, OD600= 20-200).

Transmembrane equilibration of radiolabeled permanent acids (Drawback: low sensitivity, measure only the transmembrane pH difference; they do not measure the cytoplasmic pH independent of the external pH.

Question 10

How is internal pH measured in newer methods

Answer 10

The arrival of highly pH-sensitive fluorescent proteins such as GFP (naturally responsive) or YFP offers new and improved ways of measuring the internal pH of bacterial cells. Moreover, use of signal peptides allows pH measurement in the periplasm.

Advantages: high sensitivity, no indicator loading necessary, lack of phototoxicity (issue when you use inorganic fluroscent probes), allow rapid detection (1ms, simple protonation reaction), ideally suited for kinetic studies.

Detection: Direct observation using a fluorescent light microscope (cumbersome as signal needs to be quantified); better fluorimetry (allows observation of live cell culture).

Question 11

Measurement of internal pH of E. coli using fluorescent Protein Fluorimetry

Answer 11

Effect of external acid shift on cytoplasmic pH of E. coli.

Cell cultures were suspended at pH 7.5 in a buffered M63 minimal media (5 mM HOMOPIPES).

Fluorescence was converted to pH units using standard curves of the pH dependence of the fluorescence signal.

At time point zero, 8.5 mM HCl was added to shift the external pH from pH 7.5 to 5.5.

The rapid decrease in fluorescence (1 ms) is the consequence of the simple protonation reaction of the two proteins.

For each strain, three independent cultures were tested.

Question 12

Whats the internal pH of alkaliphiles and acidophiles

Answer 12

Maintain an internal pH within Ph 4-9 range, cell constituents are normal with respect to pH

Question 13

What’s the proton motor force and its implications

Answer 13

Used for all of the transport processes and to drive the multiprotein machinery such as flagellum in order to be active

Large pH gradients between cells and medium have implications for bioenergetics.

Photosynthesis or respiration in bacterial cells generate a proton-motive force (PMF) across the cell membrane due to vectorial electron transport.

1. Cells remove protons and high-energy electrons from energy sources like glucose
2. shuttle molecules like NAD move the electrons to a series of membrane proteins called cytochromes
3. Cytochromes extract energy from the electrons and use that energy to pump H+ out of the cell

Question 14

What two components does the proton motor force consist of

Answer 14

• Difference in pH (internal pH minus the outside pH)
• Difference between electrical activity (transmembrane electrical potential due to positive charges- established through the activity of ion transporters and pumps)

Question 15

The Proton Motive Force in Neutralophiles

Answer 15

PMF = -∆Ψ – (+∆pH) = negative value- combination so the proton motor force will be larger

The transmembrane potential ∆Ψ is normally negative inside the cells

Question 16

What are some bioenergetic problems of acidophiles

Answer 16

The evidence for K+ involvement is:

1.) K+ is required for proton extrusion in acidophilic Sulfolobus species (potassium is important for the reverse of membrane potential)

2. ) a lack of K+ in the medium decreased the ability of Sulfolobus to adapt to acidic pHs.
3. K+ is required to generate a ΔΨ in Acidithiobacillus.

May be a role for other cations (including Na+, but K+ is the cation most compatible with cellular structures.

To confirm the role of K+ in reversing the ΔΨ, gene knockouts of K+ transporter genes need to be carried out.

Question 17

What are the bioenergetic problems of alkaliphiles

Answer 17

More challenged because the pH is reversed- external pH is higher than the internal pH- the pH is reversed and opposite of the productive gradient

All transport processes that rely on protons are driven by sodium ions- flagellum is driven by a proton gradient

Alkaliphiles have a pHi that is significantly lower than pHo. This protects their cellular components from the extreme pHo. However, as a consequence the pH gradient is in the reverse of the productive orientation for bioenergetic work. The reversed pH gradient reduces the available PMF to i.e. generate ATP. They require Na+ in the medium, to increase ∆Ψ using a Na+/H+ antiport system.