Halobacterium
Above: a 3D computer model of Halobacterium. (Click image to enlarge). As we shall see, Halobacterium is a
very unusual organism which thrives in very harsh conditions and is able to endure tremendous hardship.

Halobacterium is a member of the Archaea or
Archaebacteria. Officially, although prokaryotes, archaeans
are no longer classified as bacteria on the basis that they are genetically very distinct and so appear distantly
related. However, some dispute this, hypothesising that many of the unusual features of archeans are due to
their evolution in harsh environments. I prefer to keep 'bacteria' as a descriptive term (in the same manner as
the word 'worm') and personally prefer the designation Archaebacteria, with Eubacteria refering to the
remaining 'true bacteria' in the prokaryote kingdom. As an example of an archaebacterium, we shall consider
Halobacterium.

Halobacterium requires a solution of at least 2 M (2 Molar) NaCl (sodium chloride, salt) in order to grow, and
grows best in 4 to 5 M NaCl (compared to 0.6 M for typical sea water). Such extremely salty conditions occur
in the Dead Sea and evaporating salt lakes, which are environments with high light-intensity. In bright
sunlight, Halobacteria are brick=red due to the presence of red
carotenoid pigments in their cell
membrane, which protects the cells from damage (especially against UV light). they often bloom, turning the
water red.

Archaebacteria are often (though not exclusively) found in harsh environments - many are
extremophiles.
They appear to be a very ancient group and may have evolved on Earth at a time when conditions were quite
different and harsh to life as we know it and continue to dominate harsh environments. That said, it could be
that much of their unusual chemistry has been acquired through secondary adaptation to harsh extremes and
they are also being increasingly discovered in more widespread habitats. Many species of bacteria and
archaebacteria no doubt remain to be discovered. Many do not grow in standard culture conditions and
plating out environmental samples onto agar typically fails to detect many strains and species.

Archaebacteria include a number of extremophiles, such as archaebacteria that live in hot springs
(
thermophiles), in highly acidic solutions (acidophiles). Halobacterium thrives in high salt concentrations,
and so is a
halophile, and grows best at a very warm 42C. The study of extremophiles is a very rewarding
one. It is remarkable to see the adaptations of living organisms to extremes and is of great importance to the
study of evolution and astrobiology.

Photophosphorylation - making ATP from light, Solar Power

Plants, algae and some bacteria undergo photosynthesis, the process of harvesting light-energy to drive
growth.
Halobacterium is not photosynthetic, however, it has a novel mechanism for making ATP by using
sunlight to generate a proton gradient (an electrical potential difference or voltage) directly. Recall, that in
respiration mitochondria and bacteria use protons as a form of positive electricity to drive the ATPase, an
enzyme which spans the membrane and spins like an electric motor when the protons flow through it. This
rotary energy is then used to generate ATP, with the ATP storing some of the rotational energy as chemical
energy (from ADP and inorganic phosphate). Thus, we have the conversion or transduction of energy from
electrical, to rotary mechanical, to chemical; and this process drives living cells. ATP is the
universal energy
currency
of the living cell. Most active processes in most cells require ATP as a source of energy.

Some strains of
Halobacterium have a novel mechanism for generating ATP. In aerobic conditions they will
undergo normal respiration using oxygen to synthesise ATP. However, if starved of oxygen, they will
synthesise a new membrane component, the purple pigment
bacteriorhodopsin, which is deposited in
dense patches in the cell membrane, forming patches of
purple membrane. Purple membrane may account
for half of the total cell membrane surface area. Although the organism appears not to grow under these
conditions, it is able to keep itself ticking-over. Purple membrane is 25% lipid and 75% of a single type of
protein, bacteriorhodopsin (consisting of a form of the carotenoid chromophore retinal bound to an opsin
protein). Vertebrate retinas contain rhodopsin as a visual light-sensitive pigment. Bacteriorhodopsin is the
bacterial form of rhodopsin and associates into trimers (groups of three bacterirhodopsin molecules) that
span the membrane. A Schiff base links the retinal to the opsin protein (via a lysine amino acid residue). A
Schiff base is an organic molecule containing a nitrogen atom bound to a C atom on one side by a C=N
double bond and to a carbon on the other side (in this case to the lysine residue) by a C-N single bond (the N
is not bound to hydrogen). A proton can reversibly attach to the N, which thus acts as a proton store.

When the retinal absorbs a photon of light of the right wavelength band, it becomes energised and
undergoes a shape-change (conformational change to a higher energy state) which causes the stored proton
to be released. Thus, when light strikes the purple membrane protons are released into the space outside the
cell membrane. These protons do not leak far, but are conducted back into the cell - protons are positively
charged and the inside of the cell membrane is negatively charged with a voltage drop of about -200 mV
across the membrane (the cell membrane is acting as an electrical capacitor or a store of electric charge
difference).

In most bacteria and in
mitochondria, ATPase has a proton-permeable channel running through its centre,
and the protons flow into the cell (completing the circuit by flowing towards the negatively charged cell
interior) through the ATPase. Many thousands of ATPase molecules span the non-purple patches of
membrane (the 'brown membrane'). It has been shown that ATPase in general (not specifically that of
Halobacterium) spins around like an electric motor as the proton current flows through it and this rotational
mechanical energy is used to make ATP.

Another light-driven transport system, independent of bacteriorhodopsin is found in
Halobacterium. This
system is driven by the retinal-containing pigment
halorhopsin (peak absorption at 590 nm, yellow light).
However, the contribution of halorhodopsin to ATP synthesis appears to be small. Mutants lacking
bacteriorhodopsin, but not halorhodopsin do not release protons to the outside upon stimulation by light, but
do take-up protons on illumination. This appears to be a passive transport process (see transport across
membranes) and is accompanied by sodium ion export (we have a proton-sodium antiporter). This generates
a sodium ion gradient and it is thought that in
Halobacterium, sodium ions, rather than protons (as is usual)
carry the charge that drives the ATPase. Thus, light stimulates bacteriorhopsin to release protons and light
also stimulates halorhodopsin to open a proton-sodium importer which allows the protons to flow back into the
cell whilst sodium ions flow out and then the sodium ions flow back in to the cell through the ATPase,
synthesising ATP. Bacterirhodopsin in
Halobacterium also drives passive potassium import accompanied by
sodium export (a potassium-sodium antiporter) which also contributes to the sodium gradient and ATP
synthesis. Protons are still needed by
Halobacterium, however, for pH regulation.

The solubility of oxygen in brine is very low, so oxygen starvation must be a common occurrence in the very
salty waters in which
Halobacterium lives. Oxygen is required for the synthesis of the retinal in
bacteriorhodopsin, so the cells can not grow without oxygen, however they may grow by using light to
generate ATP if small amounts of oxygen are available, and then they may grow photoheterotrophically as
photoheterotrophs (that is by using light as a source of energy but not for carbon assimilation from
inorganic sources, as the cells would require pre-assimilated organic carbon as a food-source). Amino acids
(the building blocks of proteins) are the preferred carbon and energy source.

Flagella

Halobacterium is typically bipolarly flagellated (though sometime monopolarly) with a bundle of 5-10 flagella at
each end of the cell. The filaments form right handed helices. The flagella are inserted into a distinct polar
cap structure in the cytoplasm, which presumably anchors the flagella (and may contain the molecular
switches to switch from clockwise to counterclockwise rotation). Halobacterial cells are pushed forward by
clockwise rotation and pulled backward by counterclockwise rotation of the right handed flagellar bundles.
Thus, one bundle rotates clockwise (the trailing bundle) and the other rotates counterclockwise (the leading
bundle) with periodic synchronised reversals in rotation direction of both bundles. However, in bipolarly
flagellated forms, the leading bundle is depicted as curved backwards, so it is not clear what role it plays.

The eubacterial flagellum filament is thicker than that of archaebacteria like
Halobacterium, as it contains a
central channel through which flagellin monomers are transported to the growing tip. It seems as though the
archaebacterial flagellum is more like a (type IV)
pilus and so likely grows from the base. Bacterial flagella are
typically powered (like ATPase) by proton currents, which cause the flagella to rotate by driving an electric
motor at the base of each flagellum. In
Halobacterium, it is possible that sodium currents drive the flagellar
motor instead (as they do in some alkalophilic bacteria). ATP is required for flagellar rotation in
Halobacterium, though this might be an indirect requirement on ATP to establish/maintain ion gradients.

Photokinesis

Click here for an explanation of teh usage of the terms taxis and kinesis on Cronodon.

The optimum wavelength of light absorbed as an energy source by bacterirhodopsin in
Halobacterium is
about 560 nm (yellow-green, almost yellow light). Halobacteria are motile, they can swim equally well in either
direction by means of a tuft of flagella on each cell pole. Every 10-50 seconds or so a
Halobacterium cell
spontaneously reverses direction. The trailing flagella come together to form a propulsive bundle.

A sudden increase of yellow-green light leads to a suppression of spontaneous reversals for about 10
seconds. A sudden decrease in yellow-green light elicits an extra reversal response (after a few seconds).
The end result of these behaviours is that the bacteria tend to congregate in areas of bright (yellow-green)
light, which their purple membranes can utilise.

Conversely, a sudden increase in blue or UV light elicits an extra reversal and a decrease in blue/UV
suppresses spontaneous reversals (again for about 10 seconds). This leads to avoidance of harmful UV light.

When a
Halobacterium cell reverses, it does not set-of in exactly the opposite direction (if they could only
move along a straight line they would have problems!) but rather the new direction of locomotion is something
like 160 to 200 degrees offset from the original direction of motion - the cell turns slightly during reversal. Our
stochastic computer simulations of kinesis suggest two optimal strategies for finding or avoiding a source of
light (or chemicals) - random tumbling (as in chemokinesis in
Escherichia coli and Salmonella) or a set
change in direction a few degrees either side of 180, as in
Halobacterium. (Persistently making small turns of
less than 90 degrees appears to be highly inefficient) so kinesis in
Halobacterium is still optimised (though
very different from the tumbles of
Escherichia coli).

Two photosensory pigments have been found in
Halobacterium. PS 565 (photosystem 565) has peak
absorption at 565 nm and is used to respond to yellow-green light to enable the cells to find optimum
conditions for photophosphorylation and ATP synthesis. The second system, PS 370 has peak absorption at
370 nm and so appears responsible for the protective avoidance of blue/UV light. these photosensors also
contain retinal and are more variants of bacteriorhodopsin.

Cell Wall and Cell Membrane

Outside the cell membrane is a cell wall that is quite different to the usual peptidoglycan cell wall of
eubacteria. The
Halobacterium cell wall is made of glycoprotein. This glycoprotein can be removed by placing
the cells in dilute solution (salt appears to be essential for the glycoprotein's stability in the wall) and such
cells lose their shape, rounding up and become susceptible to osmotic stress (they will lyse (burst) in very
dilute solution).

The
cell membrane is overall about 20% lipid (the remainder being protein). This is low, but similar values
may be found in mitochondria and other bacteria - bacteria (and their mitochondrial descendants) have
membranes with high
protein contents. The lipids of archaebacteria, including Halobacterium, are very
different to those in other cells. Most organisms have cell membranes composed of glycerol esters, with two
fatty acid carbon-chains or tails attached to a glycerol backbone by ester linkages (-C-(C=O)-O-C-) forming a
glycerol diester, and a charged head that usually contains phosphorous. In archaebacteria, the carbon tails
are instead bound to glycerol by ether linkages (-C-O-C-), the so-called glycerol diethers. In both types of
membrane the lipids arrange into two opposing leaflets. However, in some archaebacteria, the lipids are
diglycerol tetraethers, in which two carbon chains join two glycerols together, so that a single layer can form a
membrane, with one glycerol at each surface. In all cases, the glycerols contain charged (polar) heads,
usually containing phosphorous, which are 'water-liking' (hydrophilic) and so sit on the surfaces of the
membranes, which are in contact with water, with water largely excluded from the 'water-hating' (hydrophobic)
membrane cores formed from the fatty-acid carbon tails/chains.

The membrane of
Halobacterium halobium (Halobacterium salinarum) the main lipid is a
phosphatidylglycerophosphate diether, with two carbon tails and a phosphate head joined to a glycerol
molecule by bonds (ether bonds for the tails). Detergent is required to solubulise most cell membranes, but
that of
Halobacterium halobium dissolves in water (though not in strong saline of course!)! It is thought by
some that the unusual ether lipids of archaebacterial cell membranes is evidence for their distant relationship
from other cellular life (Eubacteria and eukaryotes), however, it could be a secondary adaptation to extreme
conditions, with the ethers (and perhaps especially the tetraethers) being more stable in high temperatures.
Gas Vacuoles

Halobacterium frequently possesses gas vacuoles - arrays of gas-filled proteinaceous cylinders in the
cytoplasm which confer buoyancy, helping it float upwards and maintain its position in oxygenated waters,
away from the anoxic (oxygen-free) sediments.

Halophiles

Halobacterium is one of a number of halophilic (salt-loving) bacteria abundant in very salty (hypersaline)
waters. Halophilic archaebacteria, including
Halobacterium, are sometimes generically called halobacteria.
these include Many are extreme halophiles, meaning they thrive in salt concentrations greater than 3.4 to 5.1
Molar (20-30%). To prevent excessive movement of water into or out of the cell, these archaebacteria have a
high concentration of potassium chloride salt in their cytoplasm, achieving isotonicity with their environment
(see osmosis). In contrast non-halophiles thrive below 0.2 Molar. (Sea water is 3.5% salts).
Halobacterium
thrives in a variety of natural hypersaline waters and also in solar salterns - vast artificial pools of water which
are slowly evaporated by the Sun to precipitate salt which is harvested. In these salterns
Halobacterium may
reach a concentration of 10 million cells per ml and turns the water red!

Such hypersaline pools are often covered in a microbial mat, essentially a thick biofilm formed from several
species. Photosynthetic
Cyanobacteria, like Aphanothece halophytica, forms the top brown layer of these
microbial mats and thrives in 2-5 Molar salt and lyses (bursts) in distilled water due to osmosis (this is unusual
for bacteria because of their tough cell walls, but just goes to show how concentrated the cytoplasm is, which
in this case contains high concentrations of an
osmolyte called glycine betaine rather than potassium chloride
which is the major osmolyte in
Halobacterium). Filamentous cyanobacteria, like species of Oscillatoria, form
the second layer in the microbial mats, which is green and these thrive at 1-2.5 Molar salt concentration and
possess nitrogen-fixing heterocysts.

Beneath the cyanobacterial-layer are phototrophic bacteria, such as green sulphur-bacteria (e.g.
Chlorobium), green non-sulphur bacteria (e.g. Chloroflexus), purple sulphur-bacteria (e.g. Chromatium) and
purple non-sulphur bacteria (e.g.
Rhodospirlillum species).

Beneath the phototrophic layer are sulphur-oxidising bacteria, such as
Beggiatoa alba and Thiobacillus
halophilus
. Some of these fix carbon-dioxide, and oxidise sulphides and sulphur to sulphate.

Finally, anaerobes and archea inhabit the anoxic bottom sediments.

Bibliography

Eilo Hildebrand. Halobacteria: the role of retinal-protein complexes. (Ref details missing).

Wagner G, Hartmann R, Oesterhelt D. Potassium uniport and ATP synthesis in
Halobacterium halobium.
Eur J Biochem. 1978 Aug 15;89(1):169-79.

R.Y. stanier, J.L. Ingraham, M.L. wheelis and P.R. painter,  1989. General Microbiology (5th ed.). Pub:
Macmillan Education Ltd.

http://science.nasa.gov/science-news/science-at-nasa/2004/10sep_radmicrobe/

http://www.biochem.mpg.de/en/eg/oesterhelt/web_page_list/Topic_flagella_Hasal/index.html

Carolyn L. Marshall and A. D. Brown, 1968. The Membrane Lipids of Halobacterium halobium. Biochem. J.
(1968) 110: 441-448.

E.L. Chang, 1994. Unusual thermal stability of liposomes made from bipolar tetraether lipids.
Biochem
Biophys Res Commun
. 202:673-679 (http://www.ncbi.nlm.nih.gov/pubmed/8048936)
Genome

Halobacterium has a genome size of about 2.5 Gbp (Gbp = giga-base-pairs or 10^9 bp). There is a main
chromosome and a large
plasmid. The large plasmid encodes gas vacuoles and the pigment
bacteriorhodopsin.  There are many different transposable sequences (
transposons), up to some 5000 bp
in length, and each present as 20 or more copies. These transposons are highly mobile, being able to create
a copy that leaves the host
DNA molecule (the original stays where it is) and reinsert back into the host DNA
molecule in a different position (a process termed replicative recombination).

Remarkably it has been found that even if the DNA of
Halobacterium is completely fragmented by radiation,
the cell can repair and rebuild it within hours! Such radiation would destroy the vast majority of living
organisms. Salt and UV can damage DNA in a way similar to ionising radiation and so such versatile repair
mechanisms are probably a part of the survival kit of these halophiles. Often, when water evaporates, cells of
Halobacterium may become encased in salt (along with a small amount of water). This is a very harsh
environment (salt desiccates living cells which is why it helps preserve food), but the layer of salt may prevent
further dehydration and in this state
Halobacterium can remain dormant, repairing its DNA and
recommencing growth in more favourable conditions. There are controversial claims that Halobacteria have
been revived from salt deposits that are 250 million years old. Could the cells have remained dormant for this
length of time? With such efficient DNA repair mechanisms it seems likely to me.

We have seen that
Halobacterium is remarkable in a number of ways and is certainly an extremophile able to
thrive in conditions that would destroy most living organisms. It can tolerate extreme radiation, high light
intensities and very salty conditions whilst being able to endure desiccation in a dormant state. However, it is
worth remembering that dilute water kills it! In non-salty water its cell wall dissolves and then it will swell as
water enters the cell by osmosis and burst. Extremophiles are superbly adapted for their extreme
environments, but our own environment may well be extreme and intolerable to them. Organisms are each
adapted to their own environments and an unfamiliar environment may often seem extreme! extremophilism
is relative to some extent.