We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

SummaryTo facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-58-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-¯uoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Dura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-58-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Dbop strain was constructed by transforming a Dura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Dbop recombinants at a frequency of > 10 À2 in both approaches. These studies establish an ef®cient new genetic strategy towards the systematic knockout of genes in an archaeon.

Bacteriorhodopsin was selectively spin labeled at residues 72, 101, or 105 after replacement of the native amino acids by cysteine. Only the electron paramagnetic resonance spectrum of the label at 101 was time-dependent during the photocycle. The spectral change rose with the decay of the M intermediate and fell with recovery of the ground state. The transient signal is interpreted as the result of movement in the C-D or E-F interhelical loop, or in both, coincident with protonation changes at the key aspartate 96 residue. These results link the optically characterized intermediates with localized conformational changes in bacteriorhodopsin during the photocycle.

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9-or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased ϳ3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9-or 11-cis-retinal had blueshifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.

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The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.

Site-specific mutagenesis has identified amino acids involved in bR proton transport. Biophysical studies of the mutants have elucidated the roles of two membrane-embedded residues: Asp-85 serves as the acceptor for the proton from the isomerized retinylidene Schiff base, and Asp-96 participates in reprotonation of this group. The functions of Arg-82, Leu-93, Asp-212, Tyr-185, and other residues that affect bR properties when substituted are not as well understood. Structural characterization of the mutant proteins will clarify the effects of substitutions at these positions. Current efforts in the field remain directed at understanding how retinal isomerization is coupled to proton transport. In particular, there has been more emphasis on determining the structures of bR and its photointermediates. Since well-ordered crystals of bR have not been obtained, continued electron diffraction studies of purple membrane offer the best opportunity for structure refinement. Other informative techniques include solid-state nuclear magnetic resonance of isotopically labeled bR (56) and electron paramagnetic resonance of bR tagged with nitroxide spin labels (2, 3, 13, 15). Site-directed mutagenesis will be essential in these studies to introduce specific sites for derivatization with structural probes and to slow the decay of intermediates. Thus, combining molecular biology and biophysics will continue to provide solutions to fundamental problems in bR.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the ⌬brp strain, bacteriorhodopsin levels are decreased ϳ4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, ␤-carotene, is increased ϳ3.8-fold, whereas retinal is decreased by ϳ3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the ⌬brp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of ␤-carotene increased ϳ5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of ␤-carotene to retinal.Rhodopsins are integral membrane proteins containing seven transmembrane ␣-helices and a covalently bound molecule of retinal. Two distinct rhodopsin families are known: the visual rhodopsins, which bind 11-cis retinal or related compounds and function as photoreceptors in vertebrates (1) and invertebrates (2), and the archaeal rhodopsins, which bind all-trans-retinal and function as light-driven ion pumps and phototaxis receptors in archaea (3). Archaeal rhodopsin orthologs have been found recently in bacteria (4) and fungi (5), suggesting that retinal-based pigments are of widespread significance. Despite their importance, the biogenesis of these molecules is not fully understood. In particular, relatively little is known about how retinal is assembled with the opsin apoprotein in vivo. Thus, a goal in elucidating rhodopsin biogenesis is to identify the cellular factors that mediate the biosynthesis or uptake of retinal, the transport of retinal in the cell, and the binding of retinal to the corresponding opsin.To this end, we have studied the biogenesis of bacteriorhodopsin (BR), 1 a light-driven proton pump in the archaeon Halobacterium salinarum. BR consists of the membrane protein bacterioopsin (BO) and all-trans-retinal. Under microaerobic conditions, BR is induced ϳ50-fold (6) and forms a twodimensional crystal known as the purple membrane. This system has served as a model for studying key steps in membrane protein biogenesis, including protein insertion into the membrane (7,8) and the assembly of protein-lipid complexes (9, 10). H. salinarum is genetically tractable, and the genome sequence of a closely related organism, Halo...