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Results for “Rhodopsins, Microbial”

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PubMed2006

H+ -pumping rhodopsin from the marine alga Acetabularia.

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.

Acetabularia↗

PubMed2004

Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A.

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.

Anabaena↗

PubMed2003

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Algal Proteins↗

PubMed2006

Structure, function, and wavelength selection in blue-absorbing proteorhodopsin.

The absorption maximum of blue proteorhodopsin (BPR) is the most blue-shifted of all retinal proteins found in archaea or bacteria, with the exception of sensory rhodopsin II (SRII). The absorption spectrum also exhibits a pH dependence larger than any other retinal protein. We examine the structural origins of these two properties of BPR by using optical spectroscopy, homology modeling, and molecular orbital theory. Bacteriorhodopsin (BR) and SRII are used as homology parents for comparative purposes. We find that the tertiary structure of BPR based on SRII is more realistic with respect to free energy, dynamic stability, and spectroscopic properties. Molecular orbital calculations including full single- and double-configuration interaction within the chromophore pi-electron system provide perspectives on the wavelength regulation in this protein and indicate that Arg-95, Gln-106, Glu-143, and Asp-229 play important, and in some cases pH-dependent roles. A possible model for the 0.22 eV red shift of BPR at low pH is examined, in which Glu-143 becomes protonated and releases Arg-95 to rotate up into the binding site, altering the electrostatic environment of the chromophore. At high pH, BPR has spectroscopic properties similar to SRII, but at low pH, BPR has spectroscopic properties more similar to BR. Nevertheless, SRII is a significantly better homology model for BPR and opens up the question of whether this protein serves as a proton pump, as commonly believed, or is a light sensor with structure-function properties more comparable to those of SRII. The function of BPR in the native organism is discussed with reference to the results of the homology model.

Amino Acid Sequence↗

PubMed2003

Novel Proteorhodopsin variants from the Mediterranean and Red Seas.

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently found in the widespread uncultured SAR86 bacterial group in oceanic surface waters. To survey proteorhodopsin diversity, new degenerate sets of proteorhodopsin primers were designed based on a genomic proteorhodopsin gene sequence originating from an Antarctic fosmid library. New proteorhodopsin variants were identified in Red Sea samples that were most similar to the original green-light absorbing proteorhodopsins found in Monterey Bay California. Unlike green-absorbing proteorhodopsins however, these new variants contained a glutamine residue at position 105, the same site recently shown to control spectral tuning in naturally occurring proteorhodopsins. Different proteorhodopsin variants were also found in the Mediterranean Sea. These proteorhodopsins formed new and distinctive proteorhodopsin groups. Phylogenetic analyses show that some of the new variants were very different from previously characterized proteorhodopsins, and formed the deepest branching groups found so far among marine proteorhodopsins. The existence of these varied proteorhodopsin sequences suggests that this class of proteins has undergone substantial evolution. These variants could represent functionally divergent paralogous genes, derived from the same or similar species, or orthologous proteorhodopsins that are distributed amongst divergent planktonic microbial taxa.

Amino Acid Sequence↗

PubMed2004

Metagenomics: genomic analysis of microbial communities.

Uncultured microorganisms comprise the majority of the planet's biological diversity. Microorganisms represent two of the three domains of life and contain vast diversity that is the product of an estimated 3.8 billion years of evolution. In many environments, as many as 99% of the microorganisms cannot be cultured by standard techniques, and the uncultured fraction includes diverse organisms that are only distantly related to the cultured ones. Therefore, culture-independent methods are essential to understand the genetic diversity, population structure, and ecological roles of the majority of microorganisms. Metagenomics, or the culture-independent genomic analysis of an assemblage of microorganisms, has potential to answer fundamental questions in microbial ecology. This review describes progress toward understanding the biology of uncultured Bacteria, Archaea, and viruses through metagenomic analyses.

Archaea↗

PubMed2005

New insights into metabolic properties of marine bacteria encoding proteorhodopsins.

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Bacterial Proteins↗

PubMed2006

Genomics and marine microbial ecology.

Genomics has brought about a revolution in all fields of biology. Before the development of microbial ecology in the 1970s, microbes were not even considered in marine ecological studies. Today we know that half of the total primary production of the planet must be credited to microorganisms. This and other discoveries have changed dramatically the perspective and the focus of marine microbial ecology. The application of genomics-based approaches has provided new challenges and has allowed the discovery of novel functions, an appreciation of the great diversity of microorganisms, and the introduction of controversial ideas regarding the concepts of species, genome, and niche. Nevertheless, thorough knowledge of the traditional disciplines of biology is necessary to explore the possibilities arising from these new insights. This work reviews the different genomic techniques that can be applied to marine microbial ecology, including both sequencing of the complete genomes of microorganisms and metagenomics, which, in turn, can be complemented with the study of mRNAs (transcriptomics) and proteins (proteomics). The example of proteorhodopsin illustrates the type of information that can be gained from these approaches. A genomics perspective constitutes a map that will allow microbiologists to focus their research on potentially more productive aspects.

Bacteria↗

PubMed2006

Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea.

Planktonic Bacteria, Archaea and Eukarya reside and compete in the ocean's photic zone under the pervasive influence of light. Bacteria in this environment were recently shown to contain photoproteins called proteorhodopsins, thought to contribute to cellular energy metabolism by catalysing light-driven proton translocation across the cell membrane. So far, proteorhodopsin genes have been well documented only in proteobacteria and a few other bacterial groups. Here we report the presence and distribution of proteorhodopsin genes in Archaea affiliated with the order Thermoplasmatales, in the ocean's upper water column. The genomic context and phylogenetic relationships of the archaeal and proteobacterial proteorhodopsins indicate its probable lateral transfer between planktonic Bacteria and Archaea. About 10% of the euryarchaeotes in the photic zone contained the proteorhodopsin gene adjacent to their small-subunit ribosomal RNA. The archaeal proteorhodopsins were also found in other genomic regions, in the same or in different microbial lineages. Although euryarchaeotes were distributed throughout the water column, their proteorhodopsins were found only in the photic zone. The cosmopolitan phylogenetic distribution of proteorhodopsins reflects their significant light-dependent fitness contributions, which drive the photoprotein's lateral acquisition and retention, but constrain its dispersal to the photic zone.

Archaea↗

PubMed1988

Isolation of a prokaryotic photoreceptor: sensory rhodopsin from halobacteria.

The photoreceptor sensory rhodopsin was isolated from halobacterial cell membranes solubilized in laurylmaltoside. In the presence of retinal, detergent and salt the native protein was obtained in pure form by sucrose density gradient centrifugation, hydroxyapatite chromatography and gel filtration. The apparent mol. wt of the molecule was 24 kd if analyzed by SDS gel electrophoresis, and 49 kd by sedimentation and size-exclusion chromatographic analysis. The chromoprotein had an absorption maximum at 580 nm which was 8 nm blue-shifted compared to the membrane-bound state. The molecule was photochemically active and the action spectrum for formation of SR380, the long-lived intermediate, coincided with the absorption spectrum.

Centrifugation, Density Gradient↗

PubMed2005

Proteorhodopsin in the ubiquitous marine bacterium SAR11.

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 ('Pelagibacter ubique'), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

Alphaproteobacteria↗

PubMed2005

Channelrhodopsins: directly light-gated cation channels.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial type chromophores, i.e. all-trans-retinal in the ground state. The green alga Chlamydomonas reinhardtii was recently completely sequenced and the EST (expressed sequence tag) database was made public. We and others detected overlapping partial cDNA sequences that encode two proteins which we termed channelopsins (Chops). The N-terminal half of chop1 (approximately 300 of 712 amino acids) comprises hypothetical seven-transmembrane segments with sequence similarity to the proton pump bacteriorhodopsin and the chloride pump halorhodopsin. Even though the overall sequence homology is low, several amino acids are conserved that define the retinal-binding site and the H+-transporting network in BR (bacteriorhodopsin). Expression of Chop1, or only the hydrophobic core, in Xenopus laevis oocytes, enriched with retinal, produced a light-gated conductance (maximum at approx. 500 nm), which shows characteristics of a channel [ChR1 (channelrhodopsin-1)] that is selectively permeable for protons. Also ChR2 (737 amino acids) is an ion channel that is switched directly by light and also here the hydrophobic N-terminal half of the protein is sufficient to enable light-sensitive channel activity. The action spectrum is blue-shifted (maximum at approx. 460 nm) with respect to ChR1. In addition to protons, ChR2 is permeable to univalent and bivalent cations. We suggest that ChRs are involved in phototaxis of green algae. We show that heterologous expression of ChR2 is useful to manipulate intracellular pCa or membrane potential of animal cells, simply by illumination.

Amino Acid Sequence↗

PubMed2003

Exploring potential energy surfaces for chemical reactions: an overview of some practical methods.

Potential energy surfaces form a central concept in the application of electronic structure methods to the study of molecular structures, properties, and reactivities. Recent advances in tools for exploring potential energy surfaces are surveyed. Methods for geometry optimization of equilibrium structures, searching for transition states, following reaction paths and ab initio molecular dynamics are discussed. For geometry optimization, topics include methods for large molecules, QM/MM calculations, and simultaneous optimization of the wave function and the geometry. Path optimization methods and dynamics based techniques for transition state searching and reaction path following are outlined. Developments in the calculation of ab initio classical trajectories in the Born-Oppenheimer and Car-Parrinello approaches are described.

Mathematical Computing↗

PubMed2002

Modeling light-induced currents in the eye of Chlamydomonas reinhardtii.

Rhodopsin-mediated electrical events in green algae have been recorded in the past from the eyes of numerous micro-algae like Haematococcus pluvialis, Chlamydomonas reinhardtii and Volvox carteri. However, the electrical data gathered by suction-pipette techniques could be interpreted in qualitative terms only. Here we present two models that allow a quantitative analysis of such results: First, an electrical analog circuit for the cell in suction pipette configuration is established. Applying this model to experimental data from unilluminated cells of C. reinhardtii yields a membrane conductance of about 3 Sm(-2). Furthermore, an analog circuit allows the determination of the photocurrent fraction that is recorded under experimental conditions. Second, a reaction scheme of a rhodopsin-type photocycle with an early Ca(2+) conductance and a later H(+) conductance is presented. The combination of both models provides good fits to light-induced currents recorded from C. reinhardtii. Finally, it allowed the calculation of the impact of each model parameter on the time courses of observable photocurrent and of inferred transmembrane voltage. The reduction of the flash-to-peak times at increasing light intensities are explained by superposition of two kinetically distinct rhodopsins and by assuming that the Ca(2+)-conducting state decays faster at more positive membrane voltages.

Algorithms↗

PubMed2004

New insights into the evolutionary history of type 1 rhodopsins.

Type 1 (archaeal) rhodopsins and related rhodopsin-like proteins had been described in a few halophile archaea, gamma-proteobacteria, a single cyanobacteria, some fungi, and a green alga. In exhaustive database searches, we detected rhodopsin-related sequences derived not only from additional fungal species but also from organisms belonging to three groups in which opsins had hitherto not been described: the alpha-proteobacterium Magnetospirillum magnetotacticum, the cryptomonad alga Guillardia theta, and the dinoflagellate Pyrocystis lunula. Putative plant and human type 1 rhodopsin sequences found in the databases are demonstrated to be contaminants of fungal origin. However, a highly diverged sequence supposedly from the plant Oryza sativa was found that is, together with the Pyrocystis sequence, quite similar to gamma-proteobacterial rhodopsins. These close relationships suggest that at least one horizontal gene transfer event involving rhodopsin genes occurred between prokaryotes and eukaryotes. Alternative hypotheses to explain the current phylogenetic range of type 1 rhodopsins are suggested. The broader phylogenetic range found is compatible with an ancient origin of type 1 rhodopsins, their patchy distribution being caused by losses in multiple lineages. However, the possibility of ancient horizontal transfer events between distant relatives cannot be dismissed.

Amino Acid Sequence↗

PubMed2005

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin.

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp(97) occurs with a pK(a) of 8.2+/-0.1. R94C mutation reduces this slightly to 7.0+/-0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK(a) of Asp(85) by approximately 5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg(94) and Asp(97) in the unphotolyzed state of pR, in comparison to Arg(82) and Asp(85) in bR. Therefore, while fast light-driven H(+) release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.

Arginine↗

PubMed2003

Proteorhodopsin in living color: diversity of spectral properties within living bacterial cells.

Proteorhodopsin is a family of over 50 proteins that provide phototrophic capability to marine bacteria by acting as light-powered proton pumps. The potential importance of proteorhodopsin to global ocean ecosystems and the possible applications of proteorhodopsin in optical data storage and optical signal processing have spurred diverse research in this new family of proteins. We show that proteorhodopsin expressed in Escherichia coli is functional and properly inserted in the membrane. At high expression levels, it appears to self-associate. We present a method for determining spectral properties of proteorhodopsin in intact E. coli cells that matches results obtained with detergent-solubilized, purified proteins. Using this method, we observe distinctly different spectra for protonated and deprotonated forms of 21 natural proteorhodopsin proteins in intact E. coli cells. Upon protonation, the wavelength maxima red shifts between 13 and 53 nm. We find that pKa values between 7.1 and 8.5 describe the pH-dependent spectral shift for all of the 21 natural variants of proteorhodopsin. The wavelength maxima of the deprotonated forms of the 21 natural proteorhodopsins cluster in two sequence-related groups: blue proteorhodopsins (B-PR) and green proteorhodopsins (G-PR). The site-directed substitution Leu105Gln in Bac31A8 proteorhodopsin shifts this G-PR's wavelength maximum to a wavelength maximum the same as that of the B-PR Hot75m1 proteorhodopsin. The site-directed substitution Gln107Leu in Hot75m1 proteorhodopsin shifts this B-PR's wavelength maximum to a wavelength maximum as that of Bac31A8 proteorhodopsin.

Bacterial Outer Membrane Proteins↗

PubMed2006

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas.

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.

Algal Proteins↗