Halobacterium and the Archaebacteria

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. Archaebacteria were once thought to be exotic and ancient relics of the earliest cells, predating bacteria. The truth is more subtle. To understand what follows the reader should be aware of the key distinctions between prokaryotes, such as bacteria, and eukaryotes, such as animals, algae, plants, protozoa and fungi.

Modern classification systems, as given in textbooks, typically have three principal domains of life: bacteria, archaea and eukaryotes. Furthermore, phylogenetic trees (evolutionary trees) often have archaea more closely related to eukaryotes than bacteria. I have never been convinced by this and recently this triple scheme has been called into question. Although they are prokaryotes, archaeans are no longer classified as bacteria by most scientists on the basis that they are genetically very distinct and so appear distantly related, or so it was claimed. Hence the name change from 'archaebacteria' to 'archaea'. It was also thought, based on incomplete molecular data, that archaea were more closely related to eukaryotes than to bacteria and an evolutionary scheme was devised in which bacteria gave rise to a form that later split into archaea and eukaryotes. However, although archaebacteria do share certain core features with eukaryotes (including features of DNA and RNA packaging and processing) this is only the minority of their genetic makeup. Most of their genes, based on those that have been fully sequenced, are more closely related to those of bacteria. The same is also true for eukaryotes, which also share more genetic similarity to bacteria than to archaebacteria.

I would caution against accepting 'official' taxonomies as written in stone, however, since science is not determined by committee. Some scientists dispute the scenarios mentioned thus far. Some hypothesise that many of the unusual features of archeans are due to their evolution in harsh environments, as archaeobacteria were first discovered from harsh environments. In other words their exotic nature may be due more to selection pressure and less to evolutionary relatedness. More recently, it has been discovered that archaebacteria are found in many more usual habitats too, including the oceans, but were previously overlooked due to the difficulties growing them by traditional culture methods. We now know that not all of them are extremophiles and about 20% of oceanic prokaryotes are archaebacteria, perhaps the majority beneath the surface of the sediments. Interestingly, however, no known archaebacteria are human pathogens.

The chief problem with phylogenetic trees is the assertion that life evolves as an organic tree, with each branch representing a lineage and dividing when a species evolves into two or more new species. This linear and vertical view of evolution is a considerable approximation, especially near the base of the tree which grew in ancient times when prokaryotes dominated the world. Horizontal gene transfer, or the exchange of genes between prokaryotes is common place and suggests the tree of life is more of a reticulated web, at least where the prokaryotes are concerned (see: Mallet et al. 2015, https://onlinelibrary.wiley.com/doi/epdf/10.1002/bies.201500149).  Thus, the base of the tree is something of a mythical construct, since the relations between the various groups is better described as a web or net. Archaebacteria and bacteria appear to also exchange genetic information according to recent evidence.

One plausible scheme has early life consisting of several main groups of prokaryotes. Horizontal gene transfer by several means, including endosymbiosis (the incorporation of one cell inside another) produced the eukaryotes as a chimeric mixture of these types. Archaebacteria are similarly chimeric, but have retained the essential features of bacteria.

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 (or simply 'bacteria') referring to the remaining 'true bacteria' in the prokaryote kingdom. Archaebacteria have the morphological features of bacteria and just as the various groups of worms are not all closely related, they are nevertheless worms. Given the overall genetic similarities between archaebacteria and eubacteria, the term 'archaebacteria' certainly seems justified. 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 halorhodopsin (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 the usage of the terms taxis and kinesis.

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 solubulize 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.

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 ionizing 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.

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.


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