Halobacterium is a member of the Archebacteria, and has many unusual features. Halobacteria live in high salt, 4 to 5M NaCl typically and die in more dilute media (e.g., seawater). High Na+ concentrations are required for membrane stability, and the salt cannot be replaced by high concentrations of sucrose or other solutes.
Lipids of Halobacteria
The cytoplasmic membrane contains unusual lipids, which are made up from C5 isoprenoid units (as for the side chains of ubiquinone) rather than C2 units as in normal fatty acids. Moreover, the isoprenoid chains are attached to glycerol by ether linkages instead of esters. The major components are the diphytanyl ether analogs of phosphatidyl glycerol (4%) phosphatidyl glycerol phosphate (65%), phosphatidyl glycerol sulfate (4%), and a 3-sugar glycolipid sulfate (25%).
The phytane chain has 20 carbon atoms with four branches (3,7,11,15-tetramethyl hexadecyl):
The sulfate containing lipids are only found in the purple membrane (photosynthetic membrane).
Neutral lipids make up around 10% of the lipids and include squalene and its derivatives (C30), menaquinone-8, carotenoids (C40), bacterioruberins (C50 analogs of carotenoids) and diphytanyl-glycerol -- all isoprenoid derivatives. Note that isoprenoid lipid chains of 40-50 carbon atoms can stretch across the whole membrane. Bacterioruberin has hydroxyl groups on the inside and the outside of the membrane.
Some Halobacteria do contain a vestigial fatty acid synthetase - which is strongly inhibited by high NaCl concentrations! In contrast the mevalonate pathway for isoprenoid synthesis is dependent on high salt concentrations (4M).
Surface Glycoprotein
Halobacteria have no peptidoglycan. Instead their rod shape is maintained by an outer layer of structural protein. This is a glycoprotein i.e. a protein with attached sugar residues. If the salt concentration drops much below 4M Halobacteria become spherical and finally lyse. The first step is due to disintegration of the glycoprotein envelope.
The glycoprotein of H. salinarium is 200,000 MW and contains about 10% carbohydrate. The structural glycoprotein contains all of the envelope sugar derivatives not bound to the glycolipids. It comprises about 50% of the envelope proteins (there are about 15-20 proteins in the envelope). Glycoprotein is extremely acidic due to sugar acid derivatives. One N-linked oligosaccharide and 36-38 O-linked di/trisaccharides are found, all clustered in a region of the protein of 55,000 MW. It resembles eukaryotic surface glycoproteins. Bacitracin inhibits the attachment of the sugars to this protein due to the assembly of the oligosaccharide units on polyisoprenoid carrier lipids.
Ionic Relationships
Halobacteria live in approximately 4M NaCl containing much lower amounts of K+, Mg2+ etc. They accumulate K+ inside and keep the Na+ outside. The PMF of Halobacteria is mostly used for ion pumping.
Na+ is pumped out by proton antiport, which uses 2H+ per Na+ (energy is provided both by the charge gradient and the pH gradient components of the proton motive force). K+ is accumulated to replace the Na+ and its uptake is driven by the charge gradient. Ca2+ is expelled by antiport, in exchange for 2Na+. The resulting ion gradients (4M NaCl outside, 4M KCl inside) are extremely large and contain a lot of energy, which may be used in transport of nutrients.
Amino acid uptake by Halobacteria is driven by the Na+ gradient and the charge gradient. The mechanism is sodium symport. For serine, an uncharged amino acid one Na+/serine is sufficient whereas for aspartate which is negatively charged two Na+/aspartate are needed. In the absence of Dy and DpH the gradient of Na+ itself can drive amino acid uptake though less efficiently (twice as much Na+/amino acid is needed).
Sources of the Proton Motive Force
Two mechanisms exist to generate the proton motive force in Halobacteria: respiration and photosynthesis. Both occur in the cytoplasmic membrane.
The red membrane is the site of respiration. It is colored by carotenoids and other pigments. The respiratory chain contains b and c cytochromes, and cytochrome oxidase and is not particularly unusual (except in being salt dependent). Three pairs of protons are ejected per oxygen atom reduced i.e. there are three coupling sites. Light absorbed by the purple membrane can inhibit respiration.
The purple membrane is the site of photosynthesis. Halobacteria do not perform true photosynthesis. They obtain energy from light but they do not use light to generate reducing power and they cannot fix carbon dioxide. Halobacteria are heterotrophic and need a source of organic food. The extra energy they obtain from light is mostly used to maintain their ionic composition.
Patches of purple membrane form in the light especially if the oxygen level is low. Protons are pumped out by operation of the purple membrane just as in the case of the respiratory chain. The purple membrane contains 25% lipids and 75% protein all of which is bacteriorhodopsin. Bacteriorhodopsin is arranged in groups of 3 molecules surrounded by lipid and with a few lipid molecules in the hole in the center of the trimer.
Photochemical Cycle
Bacteriorhodopsin (MW = approximately 25,000) has 7 alpha-helical segments each spanning the membrane. It contains one molecule of retinal per protein molecule which is bound via a Schiff base to the side chain -NH2 group of a lysine residue. The Schiff base lysine is 40 amino acids from the N-terminus and is near the outside of the membrane in the second helical segment. Interaction of retinal with the protein causes a large red shift - and hence light absorption is at 560-570 nm and the pigment looks purple when bound to the protein.
The Schiff base between the retinal and the lysine of the rhodopsin protein may exist either protonated or unprotonated and either in the cis or trans conformation. The trans form is of lower energy and has a higher affinity for the proton. The cis form is the high-energy intermediate and has a lower affinity for the proton.
Details of the photochemical cycle were revealed by low-temperature flash spectrometry. The intermediates are very short lived and their code numbers refer to the absorption wavelengths. The resting state (bR 570) has all double bonds of the retinal/Schiff base in the trans conformation and is protonated on the Schiff base.
Absorption of light energy converts the trans isomer to the high-energy cis isomer - K590. This is spontaneously converted to L550, also cis. The energy is used to expel the proton to the outside of the membrane. This leaves behind the low energy intermediate M412 that decays back to the trans form - O640. A proton is taken up on the inside of the membrane and we are back to bR568, the ground state.
The protons are taken up from inside the cell and released on the outside. Therefore some sort of proton channel through the membrane is needed. In fact no aqueous channel is present. The structure of bacteriorhodopsin is known from neutron scattering. The protein cannot move due to the very rigid membrane structure. The proton travels down a "proton-wire" i.e., a chain of proton exchanging groups, which runs along the surface of the a-helical protein segments which span the membrane.
The proton wire consists of two segments with a gap in the middle. The inner half of the proton wire allows the proton to reach the Schiff base. In order to jump the gap, the lysine/Schiff base/retinal has to move sideways. It does this when light energy is absorbed. So, in addition to the change from cis to trans, the whole pigment structure swings across from one alpha-helix to another. This allows the proton to reach the outside half of the proton wire. This conformational change in the Schiff base of bacteriorhodopsin allows one way proton transfer only.
Halorhodopsin
Halorhodopsin is also found in the purple membrane of some Halobacteria. It contains the same retinal pigment as bacteriorhodopsin and absorbs light which it converts to an ion gradient. However instead of pumping protons out, like bacteriorhodopsin, it pumps chloride ions inwards! Since chloride ions have a negative charge, moving chloride inwards is equivalent in terms of energy to moving a proton outwards. Thus halorhodopsin generates a chloride ion gradient which also supplies energy.