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Comparative studies on the fine structure of purple membrane from Halobacterium Cutirubrum and Halobacterium Halobium.

Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane from Halobacterium cutirubrum and Halobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane from Halobacterium halobium, it must be similarly so for the membrane from Halobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.

Bacteriorhodopsins

Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement.

A halophilic bacterium was isolated from bottom sediment from the Dead Sea. The organism possessed the properties of the halobacteria, but differed from the known species in two important respects, 1) the cells were disc shaped and often cupped when grown under optimum conditions, 2) the optimum requirements for sodium chloride was in the range 1.7--2.5 molar which is about half of that generally reported for the halobacteria. The organism was assigned to the genus Halobacterium and described as Halobacterium volcanni spec. rov. The optimum sodium chloride concentration for growth was close to that found in the Dead Sea. The tolerance for magnesium chloride was very high; the organism grew well in media containing magnesium chloride in the concentrations found in the Dead Sea. Halobacterium volcanii is therefore remarkably well fitted for life in the Dead Sea.

Amino Acids

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.

Bacteriorhodopsins

Distribution of non-electrolytes in Halobacterium cells. I. Halobacterium marismortui.

Halobacterium marismortui is an obligatorily halophilic species isolated from the Dead Sea. When inulin, fructose or glycerol are added to suspensions of bacteria, the amounts of these substances recovered from centrifuges pellets are more than could have been present in the extracellular space. Thus a certain amount becomes associated with the bacteria, though not enough to equilibrate with all the cell water. The inulin or fructose concentration found after uptake of these substances was correlated with the cell sodium concentration. It is argued that inulin, fructose or glycerol is unlikely to be adsorbed on the outside of the bacteria and more probably crosses the plasma membrane. A possible scheme for explaining the data is presented.

Cell Membrane Permeability

Passive electrical properties of Halobacterium species. I. Low-frequency range.

The electrical conductivity of suspensions of two species of Halobacterium was measured at low A. C. frequency. The results obtained from Halobacterium halobium suspensions show that the bacteria act as non-conducting particles. In contrast, the cells of a Halobacterium obtained from the Dead Sea (Halobacterium marismortui) had an apparently high conductivity which can be explained partly in terms of the cell-membrane being pierced by pores through which ions can move freely and partly in terms of highly concentrated cell ions, all of which are mobile.

Cell Count

What does Halobacterium tell us about photoreception?

A photosensory mechanism is proposed for Halobacterium halobium based on the observation of light-induced motor responses. Possible mechanisms of signal transduction in Halobacterium are discussed. Bacteriorhodopsin and the visual pigment rhodopsin are compared with respect to their structural and functional properties. The conclusion is drawn that Halobacterium may help to understand primary photochemical events of rhodopsin rather than the transduction mechanism of visual photoreceptors.

Bacteriorhodopsins

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes.

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl, suspended in 3 M NaCl, and the loss of K+ was followed at various temperatures. The Arrhenius plot of the K+-efflux rates shows a break at 30 degrees C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K+ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K+-efflux data would be related to the passive permeability of the membranes to K+. The apparent K+ permeability at 30 degrees C is 1--2 - 10(-10) cm - s-1. This value corresponds to a 5-h half-life for retained K+ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K+ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.

Cell Membrane

Chemosensory responses of Halobacterium halobium.

Responses of Halobacterium halobium cells to chemical stimuli have been shown by a capillary technique. Cells were attacted by D-glucose and several amino acids and repelled by phenol. Certain chemicals, such as acetate, benzoate, indole, and NiSO4, that are known to act as repellents of Escherichia coli cells served as attractants for Halobacterium. In the presence of ethionine, sensitivity to attractants was reduced. Arsenate prevented the attraction by glucose without lowering the cellular adenosine 5'-triphosphate level. The ability for chemo-accumulation toward glucose and histidine was interfered with by the formation of photosensory systems. Light-induced motor responses and chemosensory behavior toward glucose and histidine became detectable in the late stationary growth phase only. The behavior toward acetate and indole was not connected to photobehavior in that way: both substances acted as attractants already in the late log phase. Inhibition of bacteriorhodopsin synthesis by L-nicotine allowed chemo-accumulation toward glucose and histidine already in the late logarithmic phase.

Adenosine Triphosphate

Halobacterium saccharovorum sp. nov., a carbohydrate-metabolizing, extremely halophilic bacterium.

The previously described extremely halophilic bacterium, strain M6, metabolizes a variety of carbohydrates with the production of acid. In addition, the organism produces nitrite (but no gas) from nitrate, is motile, and grows most rapidly at about 50 degrees C. These characteristics distinguish it from all previously described halophilic bacteria in the genus Halobacterium. It is suggested that it be designated as a new species, Halobacterium saccharovorum.

California

Halobacterium vallismortis sp. nov. An amylolytic and carbohydrate-metabolizing, extremely halophilic bacterium.

The extremely halophilic bacterium (formerly designated as strain J.F. 54) isolated from salt pools of the Death Valley, California, is a motile, Gram-negative, extremely pleomorphic organism, aerobe, and facultative anaerobe. A variety of carbohydrates are assimilated with or without acid production; soluble starch is hydrolyzed. The organism is not proteolytic; catalase, oxidase, and DNase reactions are positive; Tween 20 is slightly hydrolyzed, but Tweens 40, 60, and 80 are not. Nitrates are reduced to nitrites with gas production; nitrites are not reduced. Optimum growth temperature is 40 degrees C. Growth is inhibited by bacitracin and by novobiocin. The type strain J.F. 54 differs from described species of the genus Halobacterium and is assigned to a new species, Halobacterium vallismortis sp. nov.

Carbohydrate Metabolism

The purple membrane of Halobacterium halobium: a new system for light energy conversion.

Patches of a distinctly different structure from the rest of the cell membrane occur in Halobacterium halobium. The isolated patches are called the purple membrane. It derives its colour from a retinal-protein complex, bacteriorhodopsin, which is the only protein species in this membrane serving a phototransducing function. Light energy is converted by a photochemical cycle going on continuously under illumination and accompanied by a cyclic release and uptake of protons. In the intact cell, this cycle operates as a vectorial process and therefore builds up an electrochemical gradient across the cell membrane conserving part of the absorbed light energy. The cell apparently uses this electrochemical gradient for the synthesis of ATP. Photophosphorylation is shown to be insensitive to cyanide but sensitive to dicyclohexylcarbodiimide (DCCD) and uncouplers. The concentration of ATP and the pH are tightly coupled but can be uncoupled by DCCD. Bacteriorhodopsin, as a light-driven proton pump, can then be studied in the cell as an isolated process. Quantitation of light energy conversion is possible by the indirect method of inhibition of respiration by light and the comparison of the number of absorbed quanta which prevent consumption of one molecule of oxygen.

Adenosine Triphosphate

Temperature related alterations in the acidic alanine-rich "A" protein from the 50S ribosomal particle of the extreme halophile, Halobacterium cutirubrum.

50-S ribosomal subunits from the extreme halophilic bacterium, Halobacterium cutirubrum, contain an alanine-rich acidic "A" protein which resembles the L7--L12 multimer (Kaltschmidt and Wittmann, 1970) found in the 50-S ribosomal subunit of Escherichia coli cells. The protein contains 24 mole % alanine and is devoid of histidine, tryptophan and cysteine. Unlike E. coli which has two forms of the "A" protein distinguished solely by the acetylation state of the serine amino terminus. H. cutirubrum 50-S subunits contain only one unsubstituted form of the "A" protein in vivo. However, during purification of ribosomes from cells grown between 25 and 37 degrees C the latter "A" protein undergoes rapid, specific, in vitro enzymatic alteration at its carboxy-terminal end. When the halophile is grown in the temperature range of 40 to 42 degrees C the cleaving enzyme is not active and only one form of the "A" protein is found on the ribosomes.

Alanine

Non-aerated cultivation of Halobacterium cutirubrum and its effect on cellular squalenes.

Halobacterium cutirubrum was successfully cultivated under aerobic and microaerobic conditions. The early stationary phase of growth was obtained at 2.2 days and 45--55 days for aerated and non-aerated cultures, respectively. The dry cell yields were 0.7--1.2 gm/1 in all preparations grown to early stationary growth phase. The cellular ratio of squalene to dihydro- and tetra-hydrosqualene decreased proportionately with decreased aeration rates.

Anaerobiosis

Regulation of cell volume and ion concentrations in a Halobacterium.

Changes in cell volume and ion content of a Halobacterium species are described in terms of the NaCl concentration (0.5--3.5M) and pH(4-8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O2 consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.

Bacterial Proteins

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium.

The light-dependent uptake of triphenylmethylphosphonium (TPMP+) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (deltapH) across the membrane, and TPMP+ was used as an index of the electrical potential difference, deltapsi. Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, deltapH amounts to 0.6-0.8 pH unit. Its value can be increased to 1.5-2.0 by either incubating the cells with TPMP+ (10(-3) M) or at low external pH (5.5). --deltapH can be lowered by uncoupler or by nigericin. The TPMP+ uptake by the cells indicates a large deltapsi across the membrane, negative inside. It was estimated that in the light, at pH 6.6, deltapsi might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, deltamuH+/F, amounts under these conditions to about 140 mV. The effects of different ionophores on the light-drive proton extrusion by the cells were in agreement with the effects of these compounds on --deltapH.

Biological Transport, Active

Light-driven proton translocations in Halobacterium halobium.

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.

Bacteriorhodopsins