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At least 253 records · Page 14Linked to original sources
PubMed1990

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium.

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.

Archaeal Proteins↗
PubMed1995

Photocycle of halorhodopsin from Halobacterium salinarium.

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.

Bacteriorhodopsins↗
PubMed1996

Effects of sequential stimuli on Halobacterium salinarium photobehavior.

We analyzed the motor photoresponses of Halobacterium salinarium to different test stimuli applied after a first photophobic response produced by a step-down of red-orange light (prestimulus). We observed that pulses given with a suitable delay after the prestimulus produced unusual responses. Pulses of blue, green, or red-orange light, each eliciting no response when applied alone, produced a secondary photophobic response when applied several seconds after the prestimulus; the same occurred with a negative blue pulse (rapid shut-off and turning on of a blue light). Conversely, no secondary photophobic response was observed when the test stimulus was a step (a step-up for red-orange light, a step-down for blue light) of the same wavelength and intensity. When the delay was varied, different results were obtained with different wavelengths; red-orange pulses were typically effective in producing a secondary photophobic response, even with a delay of 2 s, whereas the response to a blue pulse was suppressed when the test stimulus was applied within 5 s after the prestimulus. The secondary photophobic response to pulses was abolished by reducing the intensity of the prestimulus without affecting the primary photophobic response. These results, some of which were previously reported in the literature as inverse effects, must be produced by a facilitating mechanism depending on the prestimulus itself, the occurrence of reversals being per se ineffective. The fact that red-orange test stimuli are facilitated even at the shortest delay, whereas those of different wavelengths become effective only after several seconds, suggests that the putative mechanism of the facilitating effect is specific for different signaling pathways.

Biophysical Phenomena↗
PubMed1998

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.

Biophysical Phenomena↗
PubMed2001

NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt.

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Amino Acid Sequence↗
PubMed1998

Heterologous coexpression of the blue light receptor psRII and its transducer pHtrII from Natronobacterium pharaonis in the Halobacterium salinarium strain Pho81/w restores negative phototaxis.

The photophobic receptor (psRII) and its transducer pHtrII from Natronobacterium pharaonis were heterologously coexpressed in the phototaxis-deficient Halobacterium salinarium strain Pho81/w which lacks all four bacterial rhodopsins, i.e. the two ion pumps bacteriorhodopsin and halorhodopsin as well as the two sensory pigments SRI and SRII. This genetically transformed Pho81/w strain showed a photophobic response upon illumination with blue light. The action spectrum of the psRII/pHtrII mediated phototactic behavior was determined in the range of 420-600 nm. The shape of the action spectrum was similar to the absorption spectrum of psRII, clearly indicating that the psRII-specific photophobic response in Pho81/w was restored. These results suggest that the pharaonis photoreceptor-transducer complex (psRII/pHtrII) is functionally competent to substitute the corresponding salinarium receptor system. Although the two archaea are phylogenetically quite distant from each other the two signal transduction chains are homologous systems which can replace each other.

Archaeal Proteins↗
PubMed2002

The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili.

Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin. The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament. Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is no evidence for a central channel. On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.

Bacterial Proteins↗
PubMed1990

Quantitation of photochromism of sensory rhodopsin-I by computerized tracking of Halobacterium halobium cells.

The swimming behavior of Halobacterium halobium is controlled by light which acts through retinal photoreceptor proteins. The sensing of near-ultraviolet (u.v.) was proposed to be mediated by the thermally metastable intermediate SR-I373 that is formed upon orange light absorption by sensory rhodopsin-I (SR-I). In order to test the validity of this proposal, we analyzed the photochromic behavior of the functional near-u.v. receptor in situ by use of an automated cell tracking system. The system was specifically designed for detection of swimming reversals in individual cells and calibrated with a straight-swimming mutant of H. halobium. Quantitative analysis of the response of the cells to near-u.v. revealed that orange background light increased the number of active near-u.v. receptor molecules. The intensity-dependence of this effect fitted into the kinetic scheme of a photochromic receptor pigment. The half-life of the functional near-u.v. receptor species was determined under continuous orange background light and found to be similar to that of the SR-I373 intermediate of sensory rhodopsin-I in intact cells. These results clearly support the assignment of the near-u.v. receptor to SR-I373. The kind of kinetic analysis described here, might be a useful tool in assigning spectroscopic data of pigments to photoreceptor function also in other organisms.

Bacteriorhodopsins↗
PubMed2002

The DpsA-homologue of the archaeon Halobacterium salinarum is a ferritin.

An iron-rich protein, DpsA(Hsal), was isolated from the archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC7942. It consists of 20-kDa subunits forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 100 Fe ions per mole of holoprotein. CD spectra and secondary structure calculations are consistent with an alpha-helical contribution of 60%. The UV/VIS spectrum provides no evidence for the presence of heme groups. This protein exhibits features of a non-heme type bacterial ferritin (Ftn) although it shares only little sequence homology with Ftn. Molecular modelling disclosed a high structural similarity to E. coli Dps.

Amino Acid Sequence↗
PubMed2001

Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium.

Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant. The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ([H(2)O]/[surfactant]), salt concentration, with and without Mn(+2). At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C). The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

1-Butanol↗
PubMed2000

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum.

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.

Bacteriorhodopsins↗
PubMed1989

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase.

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.

Glutamate Dehydrogenase↗
PubMed1997

Photosensory transduction in Halobacterium salinarium: evidence for a non-linear network of cross-talking pathways.

Transduction of light stimuli in Halobacterium salinarium is studied by behavioural experiments. Selected patterns of sequential stimuli (impinging on couples of the signalling states of its photoreceptors) show that a simple model integrating different stimuli is inadequate and that non linear interactions between different pathways occur through a network with several connections. The experiments reported herein yield rough but clear-cut information on the level of such interactions and shed new light on earlier findings.

Halobacterium↗
PubMed1999

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

Amino Acids↗
PubMed2000

FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase). Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H. cutirubrum has an FKBP-type PPIase or not. In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H. cutirubrum and then sequenced. This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa. Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs. Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp. KS-1. Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP. This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion. The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506. This FKBP suppressed aggregation of the unfolded protein.

Amino Acid Sequence↗
PubMed1997

Gene for a cyclophilin-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum.

A gene encoding a cyclophilin (CyP)-type peptidyl prolyl cis-trans isomerase (PPIase) was cloned from a halophilic archaeum, Halobacterium cutirubrum DSM 669, and sequenced. Although amino-acid residues common to CyPs were conserved, an insertion that showed no homology to other CyPs was found in its N-terminal region. Sequence analysis revealed that the amino-acid sequence of this CyP was 40-45% identical to those of eukaryotes and Bacillus subtilis with high cyclosporin A sensitivity, but 27% identical to those of cyclosporin A-insensitive PPIases of Escherichia coli. The gene was also expressed in E. coli. The activity of purified recombinant CyP-type PPIase was stimulated by the addition of KCl, and was suppressed by cyclosporin A.

Amino Acid Sequence↗
PubMed1997

Responses of Halobacterium halobium and Sulfolobus solfataricus to hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine [correction of N-methyl-N-nitrosoguanidine] exposure.

We have examined the capacity of Halobacterium halobium and Sulfolobus solfataricus to withstand the lethal effect of hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine [corrected]. We tested a variety of pretreatment regimens with both mutagens and all failed to elicit an inducible response to the lethal effects of those compounds in either organism. We have observed AP endonuclease activity in protein extracts from both organisms. In addition, S. solfataricus extracts contain activities that remove 3-methyl-adenine and O(6)-methyl-guanine from methylated DNA. This is the first report of these DNA repair activities in any member of the Archaea.

Carbon-Oxygen Lyases↗