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The malic enzyme from Halobacterium cutirubrum requires monovalent cations for both activation and stabilization. NaCl, the best stabilizer, is ineffective as activator; NH(4)Cl, the best activator, is a poor stabilizer. These results support the idea that the roles of salts in both processes are different.
Properties of the aspartate transcarbamylase of the extremly halophilic bacterium Halobacterium cutirubrum, an enzyme that needs high salt concentrations for activity and regulation, were studied in cell-free extracts. The enzyme was stable on prolonged incubation at 4 C in concentrated extracts (50 mg of protein per ml) but not in diluted extracts. Mg(2+) ions and beta-mercaptoethanol stabilized enzyme activity. At salt concentrations below the maximum for activity (3.5 m), the enzyme was rapidly inactivated. Carbamyl phosphate stabilized the enzyme under these conditions; aspartate had a smaller effect. The enzyme was most stable at 0 C; raising or lowering the temperature from this point increased the rate of inactivation. On exposure to lowered salt concentrations, enzyme activity was more sensitive than feedback inhibition. Hyperbolic substrate saturation curves were found for carbamyl phosphate. The K(m) obtained varied with the salt concentration used. With aspartate, sigmoidal curves were found when extracts were assayed immediately after preparation, but hyperbolic curves were obtained with extracts allowed to stand 1 to 2 hr. The presence of cytidine triphosphate (CTP) decreased the V(max) but did not change the K(m); this is thus a V-type enzyme. Low concentrations of succinate activated the enzyme, in the presence and absence of CTP; higher concentrations did not affect its activity. CTP increased the activation energy of the enzyme in 3.5 m salt but decreased it in 2.0 m salt. At both salt concentrations, the sensitivity of the enzyme to feedback inhibition diminished with increasing temperatures. Gel chromatography suggested that the enzyme in crude extracts had a molecular weight of 160,000. Precipitating the enzyme with polyethylene glycol decreased the molecular weight to 34,000, and this activity was no longer sensitive to CTP. The presence of either substrate of the enzyme during polyethylene glycol treatment prevented dissociation of the enzyme and loss of feedback inhibition. Thus, as with other aspartate transcarbamylases, association of subunits seems to be required for regulation of activity by end product.
Gas vesicles, isolated from lysed Halobacterium halobium cells, gave an amino acid analysis which accounted for 78% of the weight, and the balance was mainly salt and water. One percent of tightly bound d-galactose was found, as well as 2% of phosphate that was not released by treatment which promotes beta-elimination, by hydrolytic release of the galactose, by carboxymethylation of lysine, or by alkaline phosphatase digestion. Only a trace of lipid was detected, and it appeared to have a polyisoprenoid structure. The vesicles were not solubilized by extremes of pH, by agents such as urea, guanidine hydrochloride, formic acid, and detergents, or by organic solvents. Succinylation and carboxymethylation gave partial dispersion, but the products were heterogeneous and of high molecular weight. The amino acid composition of vesicles was independent of fragment size. No band was obtained by polyacrylamide gel electrophoresis, with neutral, acidic, and alkaline systems, with or without sodium dodecyl sulfate and urea, before or after chemical modification. No amino terminus was detected. Electrofocusing of a vesicle dispersion showed a major component with a pI of 4.0 and an amino acid composition of the whole vesicles, and a minor band with pI 3.4 which had an amino acid composition different from whole vesicles. Vesicle protein was resistant to digestion by Pronase, trypsin, thermolysin, and papain. The precipitin reaction with rabbit antivesicle serum was not inhibited by galactose or inorganic phosphate. Succinylated and carboxymethylated vesicles cross-reacted with antivesicle serum. Cell lysates contained material which reacted with antiserum, but it was heterogeneous and mainly larger than 5 x 10(6) daltons. Material from nonvacuolated mutants reacted weakly with antiserum, but the amino acid composition of the precipitated antigen was different from that of vesicles and of soluble cross-reacting material from vacuolated cells.
The isolated cell envelope of Halobacterium salinarium strain 1 contained 15 to 20 proteins that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All but one of these proteins had molecular weights of 130,000 or less and together accounted for 50 to 60% of the total envelope protein. The remaining 40 to 50% of the envelope protein was accounted for by a single protein with an apparent molecular weight of approximately 194,000 that stained for carbohydrate with periodate-Schiff reagent. The proteolytic enzymes trypsin and Pronase were used to show that the carbohydrate is covalently bound to the protein. Separation of amino sugar- and hexose-containing tryptic peptides by gel filtration indicated that all of the nonlipid carbohydrate of the cell envelope is covalently bound to protein. The results of partial purification by phenol extraction indicated that both the amino sugar and hexose are bound to the 194,000-molecular-weight protein. Exposure of isolated cell envelopes to low salt concentration resulted in solubilization of a majority of the envelope proteins. A relatively small number of proteins, including the high-molecular-weight, carbohydrate-containing protein, remained bound to the sedimentable cell membrane fraction.
Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.
Halobacterium halobium exhibits an extraordinary degree of spontaneous variability. Mutants which are defective in the formation of gas vacuoles (vac) arise at a frequency of 10(-2). Other easily detectable phenotypes, like the synthesis of bacterioruberin (Rub) or the synthesis of retinal (Ret) and bacterio-opsin (Ops), the two components which form the purple membrane (Pum) of H. halobium, are lost at a frequency of about 10(-4). With the same frequency a mutant type appears which exhibits an extremely high variability in these phenotypes. With the exception of the ret mutants, all spontaneously arising mutants show alterations, i.e., insertions, rearrangements, or deletions, in the plasmid pHH1. It appears that the introduction of one insertion into pHH1 triggers further insertions, which makes the identification of relationships between phenotypic and genotypic alterations rather difficult. From the analysis of a large number of spontaneous vac mutants and their vac+ revertants it can be concluded that the formation of the gas vacuoles is determined or controlled by plasmid genes. No such conclusion is yet possible for the rub mutants, although all mutants of this type so far analyzed exhibit a defined insertion. pum mutants which have lost the capability of forming bacterio-opsin carry insertions in the plasmid which are distributed over a rather large region of the plasmid. No strains of H. halobium could be obtained which had lost plasmid pHH1 completely.
Pure extracellular serine protease was isolated from the culture filtrate of Halobacterium halobium by bacitracin-Sepharose affinity chromatography. The enzyme activity was completely and irreversibly lost if the NaCl concentration fell below 2 M. The protease consists of one polypeptide chain with a molecular weight of 41,000. It is characteristically enriched in Asx and Glx content, whereas the level of basic amino acids in the enzyme molecule is unusually low. The protease shows a preference for leucine in the carboxylic side of the scissile bond of the substrate, cleaving the B-chain of oxidized bovine insulin only at the Leu15-Tyr16 bond and liberating p-nitroaniline from L-pyroglutamyl-L-alanyl-L-alanyl-L-leucine-p-nitroanilide.
Auxotrophic mutants of Halobacterium volcanii generated by chemical mutagenesis were used to demonstrate a native genetic transfer system in this extremely halophilic member of the class Archaeobacteria.
Data from plating experiments indicated that Halobacterium cutirubrum NRC34001 has at least two separate restriction-modification systems. A spontaneous or induced loss of one or both systems resulted in four restriction-modification phenotypes. There was a positive correlation between changes in gas vacuolation phenotypes and either restriction-modification system.
A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.
Restoration of bacterioopsin (bop) gene expression in a revertant of Halobacterium halobium was investigated. The phenotype of the revertant is the result of a translocation of the 588-base-pair (bp) sequence "ISH25", adjacent to an ISH24 insertion found in the parental mutant IV-4. These insertions are located about 1,400 bp upstream of the bop gene within the coding region of the putative brp (bacterioopsin-related protein) gene. The level at which the brp gene affects bop gene expression is unknown. Analysis of bop and brp gene transcription in the wild type, mutant IV-4, and the revertant supports the hypothesis that transcription of the putative brp gene is necessary for bop gene expression in the revertant. Eight insertion mutants of the Bop revertant were analyzed to further elucidate restoration of bop gene expression in the revertant. Bop mutants of the revertant were recovered with a frequency of about 10(-4) and were found to contain insertion elements in addition to ISH24 and "ISH25". Six-eighths of these mutants have the insertion element ISH2, and two mutants have previously uncharacterized insertion elements (ISH27 [1,400 bp] and ISH28 [1,000 bp]). ISH27 and ISH28 are confined to the more A + T-rich fraction of the H. halobium genome, as are most copies of other halobacterial insertion elements. The insertion sites in the Bop mutants of the revertant mapped within the coding region of the bop gene (three mutants), immediately upstream of the bop gene presumably in the bop promoter region (two mutants), or within a region from 241 to 449 bp upstream of the bop gene (three mutants). This distribution of insertion sites suggests that the integrity of the 526-bp region between the bop and the brp genes is important for bop gene expression in the revertant.
13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.
Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.
Halobacterium halobium spontaneously reverses its swimming direction with a frequency of about 0.1 s-1. The self-sustained periodicity can be entrained by rhythmic light stimuli within a limited range between 0.13 and 0.3 s-1. Increase of temperature increases the reversal frequency and shortens the time to reach maximal responsiveness to attractant stimuli during a cycle. Also, the period of absolute refractoriness to repellent stimuli is shortened. The results illustrate characteristic properties of biological oscillators.
Stimulation of Halobacterium halobium through its sensory photosystems, PS 370 and PS 565, leads either to a prolonged or to a shortened interval between two reversals of the swimming direction of the cell, the attractant or repellent response. Stimuli are integrated to yield the same response regardless through which photosystem they are given. Simultaneously elicited attractant and repellent signals cancel each other at any time during a reversal interval, even in the period of refractoriness shortly after a reversal, when the cell is insensitive to repellent stimuli. Successively applied stimuli are less completely integrated. The net response depends on the moment of stimulation during the interval, on the sequence of stimuli, and on the delay between them. Integration of successively applied effective stimuli (after refractoriness) is to a great extent explained in terms of a cellular oscillator (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985) which is changed in opposite directions by attractant and repellent signals. Some conclusions on the shape of the oscillator after its disturbance by a stimulus can be made. Integration of signals during refractoriness leads us to postulate an additional step before the oscillator in the sensory pathway. Cancelling of simultaneous opposite signals is thought to proceed at this integrator. It also takes part in the integration of successively evoked signals. At this step signals rapidly decline within 10 ms, and the total life time (at least of repellent signals) does not exceed 1.2 s.
Chloride uptake in intact cells of Halobacterium halobium was characterized by rates of influx and efflux of 36Cl- under conditions of light, respiration, or both. Halobacterial mutant strains with and without retinal transport proteins allowed study of the effects of halorhodopsin and bacteriorhodopsin under illumination. Two structurally independent chloride transport systems could be distinguished: halorhodopsin, the already known light-driven chloride pump, and a newly described secondary uptake system, which was energized by respiration or by light via bacteriorhodopsin.
Under certain conditions Halobacterium halobium organisms respond to a weak attractant light stimulus with a repellent response and to a weak repellent stimulus with an attractant response. The appearance of inverse responses depends on the stimulus strength, on the interval length between spontaneous reversals, and on the moment of stimulation during the interval. Although the cells are absolutely refractory to repellent stimuli for 500 ms after a reversal, repellent responses can be evoked even during that period if they are inverse responses to weak attractant stimuli. Simultaneous attractant and repellent stimuli cancel each other even when one of them leads to an inverse response, indicating that normal cellular signals occur at the site of signal integration. We postulate that the inverse responses are caused by certain properties of a cellular oscillator for which we previously postulated a role in response regulation and sensory control in halobacteria (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985).
Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II).