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PubMed1978

Molecular orientation of bacteriorhodopsin within the purple membrane of Halobacterium halobium.

The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.

Bacteriorhodopsins↗
PubMed1982

Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium.

We describe a selection method for mutants altered in the generation and regulation of transmembrane ion flux in Halobacterium halobium. The method is based on experimental control of ion fluxes by a combination of light, ionophore, and external pH to generate an imbalance in the cells' proton circulation through their membranes. The steady-state proton circulation is increased by the introduction of a small inward proton leak with a protonophore. The cells are then illuminated to excite halorhodopsin, which hyperpolarizes the membrane and drives protons into the cells. As a result, wild-type cells suffer cytoplasmic acidification, which causes a dramatic loss of motility and suppresses their growth. These properties can be used to select for mutants that escape cytoplasmic acidification because either they lack halorhodopsin function or they have a greater capacity to eject protons during the illumination. In a popular selected by this method, 97% of the individual cells were demonstrably altered in ion flux properties. Cells were selected with alterations in the halobacterial rhodopsin, specifically with deficiencies in membrane potential generation by halorhodopsin and with increased cellular proton ejection by bacteriorhodopsin. We describe properties of one of the halorhodopsin-deficient strains, Flx37.

Bacteriological Techniques↗
PubMed1982

Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium.

Mutant Halobacterium halobium strains deficient in all previously reported rhodopsin-like pigments show phototaxis responses comparable to those of wild-type strains. Spectroscopic analysis reveals the presence of a third retinal-containing pigment in the cells and their membrane fractions. It undergoes a photoreaction cycle with a half-time of approximately equal to 1 sec at room temperature and at physiological light intensities the photostationary state of the pigment consists of two species, one absorbing in the 580- to 590-nm region and the other at 373 nm, both of which are photoactive. Illumination of the long-wavelength species generates the 373-nm intermediate, which upon photoexcitation reconverts to the long-wavelength form. Therefore, changes in the relative light intensities in the long- and short-wavelength regions of the visible spectrum cause opposing shifts in the photostationary state. The spectral sensitivity of this pigment correlates with the color-discriminating phototaxis sensitivities of this organism and strongly suggests that it is the sensory photoreceptor.

Bacteriorhodopsins↗
PubMed1982

A transposable element from Halobacterium halobium which inactivates the bacteriorhodopsin gene.

We describe the characterization of a transposable element from an archaebacterium. The bacteriorhodopsin genes from the wild-type and two mutant Halobacterium halobium strains have been cloned as BamHI fragments in pBR322. The cloned DNA fragments from the two mutants both contain a 1.1-kilobase-pair insertion sequence (ISH1) near the NH2 terminus of the bacteriorhodopsin coding sequence. ISH1 is present in the two mutants in an identical palindromic site but in opposite orientations. The complete sequence of ISH1 has been determined; it is 1,118 nucleotides long, it has 8-base-pair interrupted inverted repeats at the ends, and it duplicates an 8-base-pair (A-G-T-T-A-T-T-G) target sequence upon insertion. As for most eukaryotic and some prokaryotic transposable elements, the sequence of the ISH1 begins with T-G and ends in C-A. ISH1 contains an open reading frame 810 nucleotides long and codes for an RNA approximately 900 nucleotides long. The copy number of ISH1 ranges from one to five or more in different H. halobium strains. In at least one of the strains, one copy of ISH1 is present also on a plasmid DNA.

Bacteriorhodopsins↗
PubMed1983

High-frequency spontaneous mutation in the bacterio-opsin gene in Halobacterium halobium is mediated by transposable elements.

We have recently characterized a transposable element, ISH1, which inactivates the bacterio-opsin (BO) gene in two purple membrane-deficient (Pum-) mutants of Halobacterium halobium. Examination of nine additional Pum- mutants now shows that in all of these the BO gene has been inactivated by insertion of one of two types of transposable elements. Four Pum- strains contain ISH1 within the BO gene, probably at the same site that we have previously characterized. A second element, ISH2, which is present in four more strains, inserts at multiple sites within the BO coding sequence. Significantly, another Pum- strain contains the ISH2 element 102 nucleotides upstream from the initiator codon for BO. ISH2, which is 520 nucleotides long, is the smallest insertion sequence known. Its sequence has been determined: it is A + T-rich (53%), contains a 19-base-pair inverted repeat at its termini, and, interestingly, duplicates either 10 or 20 base pairs at the target site during insertion. ISH2 is present in multiple copy numbers in the genome and contains several relatively short open reading frames.

Apoproteins↗
PubMed1987

Biochemical and spectroscopic characterization of the blue-green photoreceptor in Halobacterium halobium.

Spectroscopic evidence indicates the presence of a second sensory receptor sR-II in Halobacterium halobium, which causes a repellent response to blue-green light. Reactions with hydroxylamine and NaCNBH3 and reconstitution of the bleached pigment with retinal show that it is very similar to the other retinylidene pigments bacteriorhodopsin, halorhodopsin, and especially the earlier-discovered phototaxis receptor, sensory rhodopsin, renamed sR-I587. The second sensory receptor, sR-II480, has an absorbance maximum at 480 nm and undergoes a cyclic photoreaction with a half-time of approximately 200 msec. Its predominant photocycle intermediate absorbs maximally near 360 nm. The receptor can be detected spectroscopically in the presence of sR-I587 and quantitated through its transient response to 450-nm excitation. It is selectively bleached by low hydroxylamine concentrations that are insufficient to bleach sR-I587 significantly. Its photochemical and phototactic activities can be restored by addition of retinal. The mobility of the receptor, on NaDodSO4/polyacrylamide gels, was similar or identical to that of sR-I587 and slightly faster than bacteriorhodopsin, yielding an apparent molecular mass of 23-24 kDa.

Bacteriorhodopsins↗
PubMed1987

Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium.

We determined the complete nucleotide sequence of the 6354-base-pair plasmid pHV2 of the archaebacterium Halobacterium volcanii. This plasmid is present in approximately six copies per chromosome. We have generated a strain, H. volcanii WFD11, cured of pHV2 by treatment of liquid cultures with ethidium bromide. We describe PEG-mediated transformation of H. volcanii WFD11 with intact pHV2 and with a form of pHV2 marked by a 93-base-pair deletion generated in vitro.

Base Sequence↗
PubMed1989

Shuttle vectors for the archaebacterium Halobacterium volcanii.

Progress in archaebacterial molecular biology requires tools for genetic analysis. We describe vectors that can be selected and maintained in either Halobacterium volcanii or Escherichia coli. A genetic determinant for resistance to the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor mevinolin was isolated by "shotgun cloning" into a derivative of the endogenous H. volcanii plasmid pHV2, to form pWL2, which transforms sensitive H. volcanii to mevinolin resistance at high frequency. The resistance determinant, portions of pHV2, and an ampicillin- and tetracycline-resistance-conferring pBR322 derivative, pAT153, were ligated together to form the shuttle vectors pWL101 and pWL102. We describe conditions for the use of these vectors and provide preliminary definition of regions essential for drug resistance and for plasmid replication and maintenance.

Chromosome Deletion↗
PubMed1989

Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis.

This work demonstrates that phototaxis stimuli in the archaebacterium Halobacterium halobium control a methylation/demethylation system in vivo through photoactivation of sensory rhodopsin I (SR-I) in either its attractant or repellent signaling form as well as through the repellent receptor sensory rhodopsin II (SR-II, also called phoborhodopsin). The effects of positive stimuli that suppress swimming reversals (i.e., an increase in attractant or decrease in repellent light) and negative stimuli that induce swimming reversals (i.e., a decrease in attractant or increase in repellent light) through each photoreceptor were monitored by assaying release of volatile [3H]methyl groups. This assay has been used to measure [3H]methanol produced during the process of adaptation to chemotactic stimuli in eubacteria. In H. halobium positive photostimuli produce a transient increase in the rate of demethylation followed by a decrease below the unstimulated value, whereas negative photostimuli cause an increase followed by a rate similar to that of the unstimulated value. Photoactivation of the SR-I attractant and simultaneous photoactivation of the SR-II repellent receptors cancel in their effects on demethylation, demonstrating the methylation system is regulated by an integrated signal. Analysis of mutants indicates that the source for the volatile methyl groups is intrinsic membrane proteins distinct from the chromoproteins that share the membrane. A methyl-accepting protein (94 kDa) previously correlated in amount with the SR-I chromoprotein (25 kDa) is shown here to be missing in a recently isolated SR-I-SR-II+ mutant (Flx3b), thus confirming the association of this protein with SR-I. Photoactivated SR-II in mutant Flx3b controls demethylation, predicting the existence of a photomodulated methyl-accepting component distinct from the 94-kDa protein of SR-I. We present a model in which the three known phototaxis signaling receptor states (the attractant receptor SR-I587, its repellent form S373, and the repellent receptor SR-II490) are coupled to two distinct transducers the demethylation of which is controlled by one integrated signal.

Archaeal Proteins↗
PubMed1991

Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid.

Bacteriorhodopsin (bR) was expressed in Halobacterium halobium by using a multicopy plasmid containing the bop gene. The plasmid contains pGRB1, a 1.8-kilobase-pair plasmid; a 70-base-pair fragment from ISH11, a recently characterized insertion sequence; and a 1.6-kilobase-pair fragment carrying the bop gene from H. halobium S9. When transformed with this plasmid, a bop- insertion mutant of H. halobium yielded purple (Pum+) colonies. The insertion at the chromosomal bop locus remained intact in transformed cells, indicating that the plasmid bop gene was responsible for the Pum+ phenotype. bR was induced in early stationary phase in both wild-type and transformed cells. The final level of bR in transformed cells was 25-40% of that in wild type. The lower level of expression was presumably due to plasmid instability. Purple membrane purified from transformed strains had absorption and visible CD properties similar to wild type and contained bR in a hexagonal lattice with the same unit-cell dimension as wild type. The structure of bR from wild-type and transformed strains was identical at a resolution of 7.2 A. When reconstituted into vesicles, the purple membrane from wild-type and transformed strains showed similar light-dependent proton-pumping activity.

Bacteriorhodopsins↗
PubMed1993

Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.

A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli.

Amino Acid Sequence↗
PubMed1993

Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium.

We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimized for cassette mutagenesis by incorporating 30 unique restriction sites with uniform spacing throughout the 720-bp coding region. For expression, the coding region was placed downstream of the promoter and translation initiation region of the bacterioopsin gene on a selectable vector. This construct encodes SR-I with an extended N terminus that includes the 13-amino acid leader sequence and the 8-amino acid N terminus of bacterioopsin. To obtain a SR-I- H. halobium strain for expressing the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transformed with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant membranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption difference spectrum and photochemical reaction cycle in membrane envelope vesicles were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.

Bacteriorhodopsins↗
PubMed1994

The bat gene of Halobacterium halobium encodes a trans-acting oxygen inducibility factor.

Oxygen and light affect the expression of the bacterioopsin gene (bop), which encodes a light-driven proton pump in the purple membrane of Halobacterium halobium. This response is thought to be mediated by a set of genes located adjacent to the bop gene. DNA fragments containing either the bop gene or the entire bop gene cluster reversed the phenotype of purple membrane-deficient strains with mutations in the bop gene. Purple membrane synthesis was constitutive in one of these strains transformed with the bop gene alone. The same strain transformed with the bop gene cluster was inducible by low oxygen tension. Moreover, another strain that constitutively expresses purple membrane remained constitutive when transformed with the bop gene alone but the phenotype of the strain changed to inducible when transformed with the bop gene cluster. Additional experiments have confirmed that one of the genes of the bop gene cluster, the bat gene, encodes a trans-acting factor that is necessary and sufficient to confer inducibility of purple membrane synthesis by low oxygen tension.

Bacteriorhodopsins↗
PubMed1996

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Amino Acid Sequence↗
PubMed1996

The primary structures of the Archaeon Halobacterium salinarium blue light receptor sensory rhodopsin II and its transducer, a methyl-accepting protein.

Recently, a large family of transducer proteins in the Archaeon Halobacterium salinarium was identified. On the basis of the comparison of the predicted structural domains of these transducers, three distinct subfamilies of transducers were proposed. Here we report isolation, complete gene sequences, and analysis of the encoded primary structures of transducer gene htrII, a member of family B, and its blue light receptor gene (sopII) of sensory rhodopsin II (SRII). The start codon ATG of the 714-bp sopII gene is one nucleotide beyond the termination codon TGA of the 2298-bp htrII gene. The deduced protein sequence of HtrII predicts a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm. HtrII has a common feature with HtrI, the sensory rhodopsin I transducer; like HtrI, HtrII possesses a hydrophilic loop structure just after the second transmembrane segment. The C-terminal 299 residues (765 amino acid residues total) of HtrII show strong homology to the signaling and methylation domain of eubacterial transducer Tsr. The hydropathy plot of the primary structure of SRII indicates seven membrane-spanning alpha-helical segments, a characteristic feature of retinylidene proteins ("rhodopsins") from a widespread family of photoactive pigments. SRII shows high identity with SRI (42%), bacteriorhodopsin (BR) (32%), and halorhodopsin (24%). The crucial positions for retinal binding sites in these proteins are nearly identical, with the exception of Met-118 (numbering according to the mature BR sequence), which is replaced by Val in SRII. In BR, residues Asp-85 and Asp-96 are crucial in proton pumping. In SRII, the position corresponding to Asp-85 in BR is conserved, but the corresponding position of Asp-96 is replaced by an aromatic Tyr. Coexpression of the htrII and sopII genes restores SRII phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI and insertion in htrII/sopII regions. This paper describes the first example that both HtrI and HtrII exist in the same halobacterial cell, confirming that different sensory rhodopsins SRI and SRII in the same organism have their own distinct transducers.

Amino Acid Sequence↗
PubMed1996

Analysis of left-handed Z-DNA formation in short d(CG)n sequences in Escherichia coli and Halobacterium halobium plasmids. Stabilization by increasing repeat length and DNA supercoiling but not salinity.

To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG)4-6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.

Base Composition↗
PubMed1998

Insights into the molecular basis of salt tolerance from the study of glutamate dehydrogenase from Halobacterium salinarum.

A homology-based modeling study on the extremely halophilic glutamate dehydrogenase from Halobacterium salinarum has been used to provide insights into the molecular basis of salt tolerance. The modeling reveals two significant differences in the characteristics of the surface of the halophilic enzyme that may contribute to its stability in high salt. The first of these is that the surface is decorated with acidic residues, a feature previously seen in structures of halophilic enzymes. The second is that the surface displays a significant reduction in exposed hydrophobic character. The latter arises not from a loss of surface-exposed hydrophobic residues, as has previously been proposed, but from a reduction in surface-exposed lysine residues. This is the first report of such an observation.

Amino Acid Sequence↗
PubMed1999

Bacterioopsin-triggered retinal biosynthesis is inhibited by bacteriorhodopsin formation in Halobacterium salinarium.

Factors regulating retinal biosynthesis in halobacteria are not clearly understood. In halobacteria, events leading to the biosynthesis of bacteriorhodopsin have been proposed to participate in stringent regulation of retinal biosynthesis. The present study describes a novel approach of in vivo introductions of mRNA and membrane proteins via liposome fusion to test their role in cellular metabolism. Both the bacterioopsin-encoding mRNA and the liposome-encapsulated bacterioopsin (apoprotein) are independently introduced in spheroplasts of the purple membrane-negative strain Halobacterium salinarium that initially contain neither bacterioopsin nor retinal. Isoprenoid analyses of these cells indicate that the expression/presence of bacterioopsin triggers retinal biosynthesis from lycopene, and its subsequent binding to opsin generates bacteriorhodopsin. When bacteriorhodopsin and excess retinal were independently introduced into spheroplasts of purple membrane-negative cells, the introduction of bacteriorhodopsin resulted in an accumulation of lycopene, indicating an inhibition of retinal biosynthesis. These results provide direct evidence that the formation of bacterioopsin acts as a trigger for lycopene conversion to beta-carotene in retinal biosynthesis. The trigger for this event does not lie with either transcription or translation of the bop gene. It is clearly associated with the folded and the membrane-integrated state of bacterioopsin. On the other hand, the trigger signaling inhibition of retinal biosynthesis does not lie with the presence of excess retinal but with the correctly folded, retinal-bound form, bacteriorhodopsin.

Amino Acid Sequence↗