Halophiles

Question 1

Whats the habitat of halophiles

Answer 1

They are salt loving and can be found:

• Inland lakes in tropical areas, high evaporation exceeds input, temperature is high and pH can be variable
• Man-made evaporation ponds for salt production- salterns
• Hypersaline microbial mats
• Underground saline deposits
• Salted food products

Question 1

Whats happening in the Aral sea

Answer 1

Destruction of halophilic habitats.

Agriculture related redirection of rivers feeding into the Aral seas has resulted in the disappearance of large parts of the lake destroying in the process a unique environment containing an unknown number of unique organisms.

It remains to be seen whether recent efforts to restore some parts of this unique ecosystem will be successful

Question 2

Classification of halophiles

Answer 2

Halotolerant: low optimum (mM or less), but can range up to 1M NaCl (not permenantly) and occasionally to higher levels. They can tolerate salt but do not prefer to grow on these conditions

Moderate halophiles - optimum growth between 0.5 and 2.5 M NaCl, range up to 4.5 M NaCl

Extreme halophiles - optimum growth above 3M NaCl, range from 1.5 - 5.2 M (saturated salt). They thrive under these conditions.

Question 3

What are the two main mechanisms of salt tolerance

Answer 3

Osmotic balance must be maintained

• “High salt in” - KCL is accumulated in place of NaCL. Hydrophilic organisms use this.
• “Low salt in” - small molecular weight organic compounds (compatible solutes) are synthesized to replace NaCl.

Question 4

What is the high salt-in option utilised by

Answer 4

Archaeal order- Halobacteriales (aerobic extreme halophiles)
Bacterial order- Haloanaerobiales (anaerobic low G + C gram positive bacteria)
Bacterium Salinibacter ruber - identified in salterns

Holoquadratum - swimming bacteria.

Question 5

What is low-salt in utilised by

Answer 5

• Methanogenic Archaea
  -Alkaliphilic members of Halobacteriales
  -Heterotrophic and phototrophic Bacteria
  -Cyanobacteria
  -Eukarya

Question 6

What are compatible solutes

Answer 6

They are highly soluble organic compounds which are either uncharged or zwitterionic at physiological pH.

Question 7

What are the key compatible solutes

Answer 7

Glycerol, glycine betaine and ectoine as well as hydroxyectoine

Question 8

Properties of compatible solutes

Answer 8

• At low concentrations protect enzymes from inhibition by high salt. They protect enzymes by directly competing with sodium chloride
• At high concentrations they exclude NaCl from inside cells
• Even at high concentrations they do not affect cell metabolism, because they are very poor inhibitors of enzymes (don’t infringe on the activity of the cell)

The low salt in mechanism gives great flexibility to grow over a range of salinities which is a big advantage if you don’t occupy extreme environments.

Question 9

What the physiological costs of compatible solutes

Answer 9

The three cheapest compatible solutes are:
1. Glycerol- 20-30 ATP equivalents per molecule.

-Glycine betaine - 40-50 ATP equivalents per molecule

• Ectoine - 40-54 ATP equivalents per molecule

Question 10

Where can organisms acquire compatible solutes when in low energy

Answer 10

To reduce the bioenergetic cost of de novo synthesis, halophiles will take up compatible solutes from the medium when available

Question 11

What are some examples of Aerobic heterotrophic bacteria

Answer 11

Mainly moderate halophiles e.g. Salinvibrio costicola but some are halotolerant e.g. Halomonas elongata

Question 12

Case study for compatible solutes

Answer 12

Initial experiments suggested that glycine betaine was the first compatible solute.

A component of yeast extract, choline was taken up by the cells and converted by a two enzyme pathway into glycine betaine.

When grown on minimal medium, Salinivibrio and Halomonas both grew well at high salinity, but no compatible solute was detected.

Solution: used 13-C-NMR to discover a novel compatible solute in haloalkaliphilic anoxygenic photosynthetic bacterium Ectothiorhodospira.

It was found to be a heteroclylic amino acid known as ectoine

In minimal medium, only ectoine is accumulated by H. elongata at high salinity, 3 enzyme steps are switched on to synthesise ectoine from central amino acid pathways: 2,4 - diaminobutyrate trans-aminase, 2,4- diaminobuyrate acetylase and extoine synthase

Question 13

What is ectoine

Answer 13

It has no amine group (NH2) so was initially missed so had nothing to reacct with ninhydrin

Question 14

What is Dunaliella salina

Answer 14

A halophilic eukaryotic green algae, they are the main primary producer in many aquatic saline environments.

They grow over a wide range of salinities by synthesis/ removal of glycerol using the glycerol cycle

They’re unicellular, large cup shape chloroplasts that undergo interesting morphological change while the cell undergoes cell division, the chloroplast disintergrates. They have keratanoids controlled by sensors that are light receptors related to UV light.

They have an interesting life cycle, usually haploid but can undergo a sexual cycle. High percentage of sexual development increases diversity, high salt concentration leads to little sexual development

Question 15

Whats the glycerol cycle of Dunaliella salina

Answer 15

1) Glycerol phosphate dehydrogenase (chloroplast only, reversible reaction);

2) glycerol phosphate phosphatase (chloroplast and cytoplasm, irreversible reaction);

3) dihydroxy-actone reductase (cytoplasm only, reversible reaction);

4) dihydroxyacetone kinase (cytoplasm only, irreversible reaction).

Glycerol concentration high enough to allow for the production but is currently not done commercially. Cells can release glycerol into medium

Question 16

Whats the relationship between external NaCl and intracellular glycerol in Dunaliella salina

Answer 16

There is a linear relationship between NaCl in the medium and intracellular glycerol, with virtually constant levels of Na+ and K+. Increasing salt- 4 molar is high. Sodium is constant until high concentrations. Potassium is rare- easier to control their levels in the environment

Question 17

Why is Glycerol only rarely used?

Answer 17

Glycerol is the cheapest compatible solute to produce and it is entirely non-toxic to cells but because membranes are highly permeable to glycerol, it leaks out of the cells into the medium.

Dunaliella and some fungi have adapted their membrane structure to retain glycerol within cells, how this is done is not known

Question 18

What is the mode of action of compatible solutes

Answer 18

The key characteristic of compatible solutes is their lack of interaction with proteins.

Compatible solutes are strong water structure formers i.e. bind a number of H2O molecules. Thus they are preferentially excluded from the hydration shells of proteins.

All share a large number of hydroxyl groups- bind water. Inside the cell.

Question 19

What can ectoine and hydroxyectoine do

Answer 19

They have both been shown to protect enzyme activity against high temperature and freeze-thaw cycles

Ecotoine may replace NH4SO4 as the protective compound for commercially available enzymes

Question 20

How are high salt-in halobacteriales adapted and example

Answer 20

They are irreversibly adapted to high salinity.

The glycoprotein of their cell wall, their cytoplasmic proteins and ribosomes are all highly acidic (COO-) as are their membrane lipids

Halobacterium is stable and able to produce gas vesicles and in most cases used by regulation.

Question 21

Halobacterium halobium: gas vesicle and purple membrane.

What are gas vesicles, what could they be used for?

Answer 21

They are large cylindrical structures, have large ribbons and have super hydrophobic proteins that make up vast part of shell, stabilising the structure.

Gas vesicles could be used biotechnologically for vaccination (advantage is they don’t contain DNA)

Question 22

The high salt-in strategy used by Halobacteriales

Answer 22

A high level of external Na+ ions is required to stabilise the negative charges on the cell wall and stop the molecule falling apart. Therefore inside the cell K+ ions are accumulated instead of Na+

When grown in 4.5 M NaCl, Halobacterium cells contain 4 M KCl and 0.7 M NaCl.

Halobacterial cytoplasmic proteins also contain low levels of hydrophobic amino acids making them highly polar. In the presence of high levels of K+ polar proteins tend to remain in solution.

High salt in mechanism is less energetically costly than low salt in mechanisms but there is no flexibility to colonise lower salinity habitats