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Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.

The appearance of oxygenic photosynthesis in cyanobacteria is a major event in evolution. It had an irreversible impact on the Earth, promoting the Great Oxygenation Event (GOE) ~2.4 billion years ago. Ancient cyanobacteria predating the GOE were Gloeobacter-type cells lacking thylakoids, which hosted photosystems in their cytoplasmic membrane. The driver of the GOE was proposed to be the transition from unicellular to filamentous cyanobacteria. However, the appearance of thylakoids expanded the photosynthetic surface to such an extent that it introduced a multiplier effect, which would be more coherent with an impact on the atmosphere. Primitive thylakoids self-organize as concentric parietal uninterrupted multilayers. There is no robust evidence for an origin of thylakoids via a vesicular-based scenario. This review reports studies supporting that hexagonal II-forming glucolipids and galactolipids at the periphery of the cytosolic membrane could be turned, within nanoseconds and without any external source of energy, into membrane multilayers. Comparison of lipid biosynthetic pathways shows that ancient cyanobacteria contained only one anionic lamellar-forming lipid, phosphatidylglycerol. The acquisition of sulfoquinovosyldiacylglycerol biosynthesis correlates with thylakoid emergence, possibly enabling sufficient provision of anionic lipids to trigger a hexagonal II-to-lamellar phase transition. With this non-vesicular lipid-phase transition, a framework is also available to re-examine the role of companion proteins in thylakoid biogenesis.

Photosystem I (PSI) of photosynthetic organisms is a multi-subunit pigment-protein complex and functions in light harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the site of low-energy Chls is still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure showed absence of some subunits commonly found in other cyanobacteria, confirming the primitive nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 does not exist in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. Since Gloeobacter is susceptible to light, this indicates that Low1 serves as a main photoprotection site in most oxyphototrophs, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs. Thus, these findings provide insights into not only the functional significance of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls responsible for the photoprotection machinery from photosynthetic prokaryotes to eukaryotes.

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

12 páginas, 6 figuras, Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeplyrooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments., This work was supported by the Spanish Ministry of Science and Innovation—State Research Agency (MICINN) Grants [PID2019-110900GB-I00 to M.B., PID2019-109671GB-I00 to R.M.B., BFU-2016-78232-P to A.V.-C., and PID2019-103901GB-I00 to M.M.], and the Government of Aragón FEDER [Grupo de Referencia Biología Estructural (E35_20R to M.M., and E45_17R to A.V.-C.)]. D.F.J. was supported by a predoctoral contract from the Junta de Castilla y León. A.G.- L. acknowledges support from CSIC Jae-Intro program.

Domination of certain aerophytic phototrophic group or specific taxon in biofilms is connected with biofilm features recognised in situ. Well-developed, gelatinous, olive to dark-green biofilms are composed mostly of coccoid cyanobacterial forms. The same features, characterised biofilms dominated by one coccoid taxon, except the latter were vividly coloured. Gloeobacter caused the appearance of purple, Gloeocapsa representatives yellow and Chroococcidiopsis black biofilm. The brown to the dark colour of heterocytous biofilms was mainly caused by Nostoc. Simple trichal Cyanobacteria were occasionally present in biofilm, except in one blue-coloured sample. According to the principal component analysis (PCA), well-developed and gelatinous biofilms were correlated with Cyanobacteria, while scanning electron microscopy (SEM) revealed richness of extracellular polymeric substances (EPS) in such biofilms. Biofilm with calcified cyanobacterium (Geitleria cf. calcarea) was also found. Chlorophyta-abundant biofilms (many rich in Desmococcus), thinner than cyanobacterial, were predominantly green and occasionally yellow and blue. Many were dry when observed in situ (confirmed with PCA), with few being moistened (i.e. Klebsormidium-dominant). Diatom biofilms were usually developed on sediment, mosses or near seeping water (demonstrated by PCA) and were also thinner than cyanobacterial ones. Compared to cyanobacterial biofilms, SEM showed less developed EPS in those rich in diatoms and green algae, where microorganisms are more exposed to the environment. The study demonstrates an easy method for biofilm assessment based on visual characterisation and provides encouragement for more frequent biofilm investigation in caves that can be important from an ecological, biological, biotechnological point of view and which assessment can have an irreplaceable role in potential monitoring and protection.

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops intoGloeobacterrhodopsin, thereby generatingGloeobacterand human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.One Sentence SummaryOptogenetic therapy with Gloeobacter and human chimeric rhodopsin resulted in highly sensitive visual restoration and protection effects.

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR’s hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR’s oligomerization state using double electron–electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

Contains fulltext : 205161.pdf (Publisher’s version ) (Open Access) The approach of providing an oxygenic photosynthetic organism with a cyclic electron transfer system, i.e., a far-red light-driven proton pump, is widely proposed to maximize photosynthetic efficiency via expanding the absorption spectrum of photosynthetically active radiation. As a first step in this approach, Gloeobacter rhodopsin was expressed in a PSI-deletion strain of Synechocystis sp. PCC6803. Functional expression of Gloeobacter rhodopsin, in contrast to Proteorhodopsin, did not stimulate the rate of photoheterotrophic growth of this Synechocystis strain, analyzed with growth rate measurements and competition experiments. Nevertheless, analysis of oxygen uptake and-production rates of the Gloeobacter rhodopsin-expressing strains, relative to the DeltaPSI control strain, confirm that the proton-pumping Gloeobacter rhodopsin provides the cells with additional capacity to generate proton motive force. Significantly, expression of the Gloeobacter rhodopsin did modulate levels of pigment formation in the transgenic strain.

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins.

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of Thermus thermophilus bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 °C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump Gloeobacter rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump-probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed.

International audience; Cyanobacteria are widely-diverse prokaryotes that colonize our planet. They use solar energy to assimilates huge amounts of atmospheric CO2 and produce a large part of the biomass and oxygen that sustain most life forms. Hence cyanobacteria are increasingly studied for basic research objective, as well as for the photosynthetic production of chemicals of industrial interests. One potential approach to reduce the cost of the future bioproduction processes will be to couple them with the treatment of wastewaters that are often polluted with urea, which in any case is cheaper than nitrate. As yet, however, research has mostly focused on a very small number of model cyanobacteria growing on nitrate. Thus, the genetic inventory of the cyanobacterial phylum is still insufficiently employed to meaningfully select the right host for the right purpose. This review reports what is known about urea transport and catabolism in cyanobacteria, and what can be inferred from the comparative analysis of the publicly available genome sequence of 308 cyanobacteria. We found that most cyanobacteria harbor the genes encoding the urea catabolytic enzymes urease (ureABCDEFG) mostly, but not systematically, together with the urea transport (urtABCDE). These findings are consistent with the capacity of the few tested cyanobacteria to grow on urea as the sole nitrogen source. They also indicate that urease is important for the detoxification of internally generated urea (re-cycling its carbon and nitrogen). In contrast, several cyanobacteria have urtABCDE but not ureABCDEFG, suggesting that urtABCDE could operate in the transport of not only urea but also of other nutrients. Only four cyanobacteria appeared to have the genes encoding the urea carboxylase (uc) and allophanate hydrolase (ah) enzymes that sequentially catabolize urea. Three of these cyanobacteria belongs to the genera Gloeobacter and Gloeomargarita that have likely diverged early from other cyanobacteria, suggesting that the urea carboxylase and allophanate hydrolase enzymes appeared in cyanobacteria before urease.

The oldest species of bacteria capable of oxygenic photosynthesis today are the freshwater Cyanobacteria Gloeobacter spp., belonging to the class Oxyphotobacteria. Several modern molecular evolutionary studies support the freshwater origin of cyanobacteria during the Archaean and their subsequent acquisition of salt tolerance mechanisms necessary for their expansion into the marine environment. This study investigated the effect of a sudden washout event from a freshwater location into either a brackish or marine environment on the photosynthetic efficiency of two unicellular freshwater cyanobacteria: the salt-tolerant Chroococcidiopsis thermalis PCC7203 and the cyanobacterial phylogenetic root species, Gloeobacter violaceus PCC7421. Strains were cultured under present atmospheric levels (PAL) of CO2 or an atmosphere containing elevated levels of CO2 and reduced O2 (eCO2 rO2 ) in simulated shallow water or terrestrial environmental conditions. Both strains exhibited a reduction in growth rates and gross photosynthesis, accompanied by significant reductions in chlorophyll a content, in brackish water, with only C. thermalis able to grow at marine salinity levels. While the experimental atmosphere caused a significant increase in gross photosynthesis rates in both strains, it did not increase their growth rates, nor the amount of O2 released. The differences in growth responses to increasing salinities could be attributed to genetic differences, with C. thermalis carrying additional genes for trehalose synthesis. This study demonstrates that, if cyanobacteria did evolve in a freshwater environment, they would have been capable of withstanding a sudden washout into increasingly saline environments. Both C. thermalis and G. violaceus continued to grow and photosynthesise, albeit at diminished rates, in brackish water, thereby providing a route for the evolution of open ocean-dwelling strains, necessary for the oxygenation of the Earth's atmosphere.

Cys-loop receptors play important roles in signal transduction. The Gloeobacter ligand-gated ion channel (GLIC) pore binds similar compounds to Cys-loop receptor pores, but has the advantage of known structures in open and closed states. GLIC is activated by protons with a pEC50 of 5.4, and has a histidine residue (His 11') in its pore-forming α-helix (M2) which is involved in gating. Here we explore the role of this His and other M2 residues using two-electrode voltage clamp of mutant receptors expressed in oocytes. We show that 11'His is very sensitive to substitution; replacement with a range of amino acids ablates function. Similarly altering its location in M2 to the 8', 9', 10', 12', 13' or 14' positions ablated function. Most substitutions of Ser6' or Ile9' were also non-functional, although not Ile9'Leu and Ile9'Val. Unexpectedly, an Ile9'His substitution was constitutively active at pH 7, but closed as [H+] increased, with a pIC50 of 5.8. Substitution at 2', 5' and 7' had little effect on pEC50. Overall the data show Ser6' and His11' are critical for the function of the receptor, and thus distinguish the roles of these M2 residues from those of Cys-loop receptors, where substitutions are mostly well tolerated. These data suggest modellers should be aware of these atypical features when using the GLIC pore as a model for Cys-loop receptor pores.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pKa 3.5 and another at the higher pKa 8.4, that affect the pigment’s formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of t...

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S2 of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H+ pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H+ transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H+ pumps but replaced by Met in an archaeal H+ pump. We found that the H87M mutation removes the “monomerization effects”: Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H+ transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H+ transfer reactions through adjusting the positions of key residues.

Contains fulltext : 169812.pdf (Publisher’s version ) (Open Access) Proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) are retinal-based light-driven proton pumps that absorb visible light (maxima at 520-540 nm). Shifting the action spectra of these proton pumps beyond 700 nm would generate new prospects in optogenetics, membrane sensor technology, and complementation of oxygenic phototrophy. We therefore investigated the effect of red-shifting analogues of retinal, combined with red-shifting mutations, on the spectral properties and pump activity of the resulting pigments. We investigated a variety of analogues, including many novel ones. One of the novel analogues we tested, 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR), produced exciting results. This analogue red-shifted all of the rhodopsin variants tested, accompanied by a strong broadening of the absorbance band, tailing out to 850-950 nm. In particular, MMAR showed a strong synergistic effect with the PR-D212N,F234S double mutant, inducing an astonishing 200 nm red shift in the absorbance maximum. To our knowledge, this is by far the largest red shift reported for any retinal protein. Very importantly, all MMAR-containing holoproteins are the first rhodopsins retaining significant pump activity under near-infrared illumination (730 nm light-emitting diode). Such MMAR-based rhodopsin variants present very promising opportunities for further synthetic biology modification and for a variety of biotechnological and biophysical applications.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABAA receptor antagonist, with an IC50 of 0.2 μM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9′ or between the 0′ and 2′ residues. Similar experiments with picrotoxinin (IC50 = 2.6 μM) and rimantadine (IC50 = 2.6 μM) reveal interactions with 2′Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2′S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.

Great efforts in using non-photosynthetic bacteria as light-utilizing bacteria for producing biomaterials have been developed recently as increasing interest in renewable resources such as light energy. With respect to producing bio-materials industrially such as food ingredients and amino acids, huge amount of adenosine-5'-triphosphate (ATP) is required. In this work, we developed a bio-ATP-synthesis system using ATP synthase of Escherichia coil as a biocatalyst and a microbial rhodopsin which is from primitive cyanobacteria, Gloeobacter violaceus. Gloeobacter rhodopsin (GR) is a light-driven proton pump. Besides electro-chemical gradient produced by cellular respiration system, GR produces a proton gradient using light illumination which is used in additional driving force of synthesizing ATP by ATP synthase. Inverted membrane vesicle was prepared so that it could be incorporated with both of GR and ATP synthase and produced ATP in the exterior side of the vesicle in the presence of light. Since inverted membrane vesicle does not contain precursors for ATP, we added ADP and inorganic phosphate (P(i)). Then, we measured the amounts of ATP produced by ATP synthase in the presence of light. As the average value of 6 samples, 4.79 x 10(-2) micromole of ATP produced for 1 microg of GR per minute. Also, we measured again after 7 days and 65 days, respectively, in order to check the stability of the bio-ATP-synthesis system. Amount of ATP produced decayed double-exponentially and an expected value of half-life of the system was 1.5 days and 39.7 days. Our results demonstrate that ATP was regenerated successfully by using GR and ATP synthase. However, the stability of ATP synthase should be increased to use this system industrially in the near future.

In previous work, we reconstituted salinixanthin, the C(40)-carotenoid acyl glycoside that serves as a light-harvesting antenna to the light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in Escherichia coli, and demonstrated that it transfers energy to the retinal chromophore [Imasheva, E. S., et al. (2009) Biochemistry 48, 10948]. The key to binding of salinixanthin was the accommodation of its ring near the retinal β-ionone ring. Here we examine two questions. Do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding? There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group but lacking the acyl glycoside, is present in addition to β-carotene and oscillol. We show that β-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for carotenoid binding. Further evidence of this is the fact that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. A similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around the C6-C7 bond and probably is engaged in an interaction that locks the carotenoid in the binding site.

We show that salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin, can be reconstituted into the retinal protein from Gloeobacter violaceus expressed in Escherichia coli. Reconstitution of gloeobacter rhodopsin with the carotenoid is accompanied by characteristic absorption changes and the appearance of CD bands similar to those observed for xanthorhodopsin that indicate immobilization and twist of the carotenoid in the binding site. As in xanthorhodopsin, the carotenoid functions as a light-harvesting antenna. The excitation spectrum for retinal fluorescence emission shows that ca. 36% of the energy absorbed by the carotenoid is transferred to the retinal. From excitation anisotropy, we calculate the angle between the two chromophores as being ca. 50 degrees , similar to that in xanthorhodopsin. The results indicate that gloeobacter rhodopsin binds salinixanthin in a manner similar to that of xanthorhodopsin and suggest that it might bind a carotenoid also in vivo. In the crystallographic structure of xanthorhodopsin, the conjugated chain of the carotenoid lies on the surface of helices E and F, and the 4-keto ring is immersed in the protein at van der Waals distance from the ionone ring of the retinal. The 4-keto ring is in the space occupied by a tryptophan in bacteriorhodopsin, which is replaced by the smaller glycine in xanthorhodopsin and gloeobacter rhodopsin. Specific binding of the carotenoid and its light-harvesting function are eliminated by a single mutation of the gloeobacter protein that replaces this glycine with a tryptophan. This indicates that the 4-keto ring is critically involved in carotenoid binding and suggests that a number of other recently identified retinal proteins, from a diverse group of organisms, could also contain carotenoid antenna since they carry the homologous glycine near the retinal.

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu132, the homolog of Asp96 of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp85 and Asp96 (Asp121 and Glu132). We assigned a pair of FTIR bands (positive at 1749 cm−1 and negative at 1734 cm−1) to the protonation and deprotonation, respectively, of these carboxylic acids.

Fil: Barrantes, Francisco José. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas. Laboratorio de Neurobiología Molecular; Argentina Fil: Barrantes, Francisco José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Fantini, Jacques. Aix-Marseille Université. Interactions Moléculaires et Systèmes Membranaires; Francia Abstract: Pentameric ligand-gated ion channels (pLGICs) and their lipid microenvironments appear to have acquired mutually adaptive traits along evolution: 1) the three-ring architecture of their transmembrane (TM) region; 2) the ability of the outermost TM ring to convey lipid signals to the middle ring, which passes them on to the central pore ring, and 3) consensus motifs for sterol recognition in all pLGICs. Hopanoids are triterpenoid fossil lipids that constitute invaluable biomarkers for tracing evolution at the molecular scale. The cyanobacterium Gloeobacter violaceus is the oldest known living organism in which the X-ray structure of its pLGIC, GLIC, reveals the presence of the above attributes and, as discussed in this review, the ability to bind hopanoids. ELIC, the pLGIC from the bacillus Erwinia chrysanthemi is the only other known case to date. Both prokaryotes lack cholesterol but their pLGICs exhibit the same sterol motifs as mammalian pLGIC. This remarkable conservation suggests that the association of sterols and hopanoid surrogate molecules arose from the early need in prokaryotes to stabilize pLGIC TM regions by means of relatively rigid lipid molecules. The conservation of these phenotypic traits along such a long phylogenetic span leads us to suggest the possible co-evolution of these sterols with pLGICs.

Contains fulltext : 153410.pdf (Publisher’s version ) (Closed access) Proteorhodopsins are heptahelical membrane proteins which function as light-driven proton pumps. They use all-trans-retinal A1 as a ligand and chromophore and absorb visible light (520-540 nm). In the present paper, we describe modulation of the absorbance band of the proteorhodopsin from Monterey Bay SAR 86 gammaproteobacteria (PR), its red-shifted double mutant PR-D212N/F234S (PR-DNFS) and Gloeobacter rhodopsin (GR). This was approached using three analogues of all-trans-retinal A1, which differ in their electronic and conformational properties: all-trans-6,7-s-trans-locked retinal A1, all-trans-phenyl-retinal A1 and all-trans-retinal A2. We further probed the effect of these retinal analogues on the proton pump activity of the proteorhodopsins. Our results indicate that, whereas the constraints of the retinal-binding pocket differ for the proteorhodopsins, at least two of the retinal analogues are capable of shifting the absorbance bands of the pigments either bathochromically or hypsochromically, while maintaining their proton pump activity. Furthermore, the shifts implemented by the analogues add up to the shift induced by the double mutation in PR-DNFS. This type of chromophore substitution may present attractive applications in the field of optogenetics, towards increasing the flexibility of optogenetic tools or for membrane potential probes.

Cyanophyte-like prokaryotes are widely presumed to be the progenitors of eukaryote plastids. A few rare protistan species bearing cyanophyte-like cyanelles may represent intermediate stages in the evolution of true organelles. Cyanophyte DNA disposition in the cell, so far as is known from electron microscopy, seems uniform within the group and distinctly different from the several known arrangements of DNA in plastids. Therefore a survey of representative cyanophytes and protistan cyanelles was undertaken to determine whether forms reminiscent of plastids could be found. DNA-specific fluorochromes were utilized, along with epifluorescent microscopy, to study the DNA arrangement in situ in whole cells. Only the endospore (baeocyte)-forming Cyanophyta contained more than one, centrally located DNA skein per cell, and then only for the period just preceding visible baeocyte formation. Such forms might, with modification, presage the “scattered nucleoid” DNA disposition found in plastids of several groups, including Rhodophytes, Cryptophytes, Chlorophytes and higher plants. The DNA arrangement in cyanelles of two protists, Cyanophora and Glaucocystis, appear different from each other and possibly related to, respectively, the cyanophytes Gloeobacter and Synechococcus. Cyanelles of the third protist, Glaucosphaera, like the cells of the unique prokaryote Prochloron, appear to have multiple sites of DNA, somewhat similar to those of the “scattered nucleoid” line of plastid evolution. No obvious precursor of the “ring nucleoid” or other types of plastid DNA conformation was found.

GR is directly shown by ultrafast pump-probe measurements to bind the carotenoid Salinixanthin, which acts as an efficient light harvesting antenna. Along with Xanthorhodopsin, This proves light harvesting to be a prevalent strategy in retinal proteins.

A homologue of type I rhodopsin was found in the unicellular Gloeobacter violaceus PCC7421, which is believed to be primitive because of the lack of thylakoids and peculiar morphology of phycobilisomes. The Gloeobacter rhodopsin (GR) gene encodes a polypeptide of 298 amino acids. This gene is localized alone in the genome unlike cyanobacterium Anabaena opsin, which is clustered together with 14 kDa transducer gene. Amino acid sequence comparison of GR with other type I rhodopsin shows several conserved residues important for retinal binding and H+ pumping. In this study, the gene was expressed in Escherichia coli and bound all-trans retinal to form a pigment (lambda max = 544 nm at pH 7). The pK(a) of proton acceptor (Asp121) for the Schiff base, is approximately 5.9, so GR can translocate H+ under physiological conditions (pH 7.4). In order to prove the functional activity in the cell, pumping activity was measured in the sphaeroplast membranes of E. coli and one of Gloeobacter whole cell. The efficient proton pumping and rapid photocycle of GR strongly suggests that Gloeobacter rhodopsin functions as a proton pumping in its natural environment, probably compensating the shortage of energy generated by chlorophyll-based photosynthesis without thylakoids.

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light.

We studied the photocurrents of a cyanobacterial rhodopsin Gloeobacter violaceus (GR) in Xenopus laevis oocytes and HEK-293 cells. This protein is a light-driven proton pump with striking similarities to marine proteorhodopsins, including the D121-H87 cluster of the retinal Schiff base counterion and a glutamate at position 132 that acts as a proton donor for chromophore reprotonation during the photocycle. Interestingly, at low extracellular pHo and negative voltage, the proton flux inverted and directed inward. Using electrophysiological measurements of wild-type and mutant GR, we demonstrate that the electrochemical gradient limits outward-directed proton pumping and converts it into a purely passive proton influx. This conclusion contradicts the contemporary paradigm that at low pH, proteorhodopsins actively transport H+ into cells. We identified E132 and S77 as key residues that allow inward directed diffusion. Substitution of E132 with aspartate or S77 with either alanine or cysteine abolished the inward-directed current almost completely. The proton influx is likely caused by the pKa of E132 in GR, which is lower than that of other microbial ion pumping rhodopsins. The advantage of such a low pKa is an acceleration of the photocycle and high pump turnover at high light intensities.

Gloeobacter violaceus sp. PCC 7421 is an unusual cyanobacterium with only one cellular membrane, which lacks the thylakoid membranes found in other oxygenic photosynthetic organisms. The cell membrane lipids in G. violaceus sp. PCC 7421 are monogalactosyl diacylglycerol, digalactosyl diacylglycerol, phosphatidyl glycerol and phosphatidic acid in the molar proportion of 51, 24, 18 and 4% respectively. This lipid composition resembles that of the cell membrane from other cyanobacteria, but completely lacks sulfoquinovosyl diacylglycerol. This lack of sulfoquinovosyl diacylglycerol is exceptional for a photosynthetic membrane. The membrane lipids are esterified to 14:0, 16:0, 16:1, 18:0, 18:1, 18:2 and α18:3 fatty acids.

The deeply purple cyanobacterium Gloeobacter violaceus is subject of this investigation. It does not contain thylakoids, and the photosynthetic apparatus is located in the only membrane of the cell, the plasma membrane. Upon excitation with blue light, the 77 K fluorescence emission spectra of neither intact cells (excited with 427 nm) nor of the isolated plasma membrane (excited with 430 nm), show the expected long wavelength photosystem I emission characteristic for low energy chlorophylls. Maximal fluorescence emission was observed at 688 nm, independent on the excitation wavelength, 427 (430) nm blue light, exciting mainly chlorophyll, or 550 nm green light, exciting mainly phycoerythin. The ratio of P700 to chlorophyll was 175. O 2 -evolution was 160 μmol mg -1 chlorophyll h -1 in saturating white light ; the compensation point was reached at 6 μmol m -2 s -1 in cultures grown at 25 μmol m -2 s -1 . Dark O 2 uptake was 50 μmol mg -1 chlorophyll h -1 . During adaptation to increasing white light intensities Gloeobacter reduces the amount of phycocyanin and chlorophyll per cell and strongly increases the concentration of carotenoids relative to chlorophyll. The carotenoid concentration per cell increases with increasing light intensity. Apparently, part of the carotenoids is not located in the plasma membrane.

Gloeobacter rhodopsin (GR) is a eubacterial proton pump having a highly conserved histidine near the retinal Schiff base counter-ion, aspartate. Various interactions between His and Asp of the eubacterial proton pump have been reported. Here, we showed the pH-dependent trimer/monomer transition of GR in the presence of dodecyl-β-d-maltoside by size-exclusion chromatography. The pH dependence was closely related to the protonation state of the counter-ion, Asp121. For the H87M mutant, pH dependence disappeared and a monomer became dominant. We concluded that the formation or breaking of the salt bridge between His87 and Asp121 inside the protein changes the quaternary structure.Structured summary of protein interactionsRhodopsin and Rhodopsin bind by molecular sieving (View interaction)Rhodopsin and Rhodopsin bind by molecular sieving (View interaction: 1, 2)

Xanthorhodopsin, a proton pump from the eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light-harvesting antenna. The key factors affecting binding and function of the antenna were established and examined. The first is the binding of the carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4-keto group in the carotenoid ring. A close analogue of salinixanthin, salinixanthol, in which the 4-keto group (C=O) is reduced to hydroxyl (C-OH), does not bind. Third, the binding site itself. Another protein capable of carotenoid binding was found. Gloeobacter rhodopsin, was shown to bind salinixanthin and echinenone but not beta-carotene or salinixanthol. Using femtosecond absorption spectroscopy, a large transient electrochromic shift of the salinixanthin band was detected 150 fs after excitation of the retinal chromophore. It indicates that excitation of retinal is accompanied by charge separation and a strong electrostatic field. It partially remains in the first photoproduct K. Thermal conversion of the latter involves at least two substates which were characterized with FTIR spectroscopy. The kinetics of photocycle and proton transport by xanthorhodopsin was examined.

Gloeobacter rhodopsin (GR) is a light-driven proton-pump protein similar to bacteriorhodopsin (BR), found in Gloeobacter violaceus PCC 7421, a primitive cyanobacterium. In this paper, structural changes of GR following retinal photoisomerization are studied by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. The initial motivation was to test our hypothesis that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules in the active center. Water O-D stretching vibrations at2400 cm(-1) in D(2)O have been regarded as coming from such strongly hydrogen-bonded water, and there is a strong correlation between the proton-pumping activity and the presence of such water molecule. Since GR pumps protons, we expected that GR also possesses strongly hydrogen-bonded water molecule(s), and the FTIR results clearly show that this is indeed the case. In addition, another unexpected finding was gained from the frequency region of protonated carboxylic acids in the GR(K) minus GR spectra at 77 K, where we observed the unique bands of a protonated carboxylic acid at 1735 (+)/1730 (-) cm(-1). Comprehensive mutation study revealed that the vibrational bands originate from the carboxylic C=O stretch of Glu132 at the position corresponding to Asp96 in BR. Glu132 presumably functions as an internal proton donor for the retinal Schiff base, but they may be located far apart (ca. 12 A in BR). The present study demonstrates the long-range structural changes of GR along the proton pathway, even though the protein matrix is frozen at 77 K.

Summary In angiosperms, chlorophyll biosynthesis is light dependent. A key factor in this process is protochlorophyllide oxidoreductase (POR), which requires light to catalyze the reduction of protochlorophyllide to chlorophyllide. It is believed that this protein originated from an ancient cyanobacterial enzyme that was introduced into proto-plant cells during the primary symbiosis. Here we report that PORs from the cyanobacteria Gloeobacter violaceus PCC7421 and Synechocystis sp. PCC6803 function in plastids. First, we found that the G. violaceus POR shows a higher affinity to its substrate protochlorophyllide than the Synechocystis POR but a similar affinity to plant PORs. Secondly, the reduced size of prolamellar bodies caused by a knockdown mutation of one of the POR genes, PORA, in Arabidopsis could be complemented by heterologous expression of the cyanobacterial PORs. Photoactive protochlorophyllide in the etioplasts of the complementing lines, however, was retained at a low level as in the parent PORA knockdown mutant, indicating that the observed formation of prolamellar bodies was irrelevant to the assembly of photoactive protochlorophyllide. This work reveals a new view on the formation of prolamellar bodies and provides new clues about the function of POR in the etioplast–chloroplast transition.

NADPH-dependent protochlorophyllide oxidoreductase (POR) catalyzes a light-dependent reduction of protochlorophyllide that is a key regulatory step in the biosynthesis of chlorophyll in all oxygenic phototrophic organisms. In eukaryotic phototrophs, PORs are localized in plastids, suggesting that it originated by endosymbiosis of an ancestral cyanobacterium. In light of the evolutionary considerations, we studied the POR from the cyanobacterium Gloeobacter violaceus PCC 7421 that has been suggested to retain the ancestral properties of cyanobacteria, since on the phylogenetic tree based on 16S rRNA sequences it branches off at the earliest stage within the cyanobacterial linage. We found that the recombinant POR from G. violaceus overexpressed in E. coli possessed a light-dependent catalytic activity of protochlorophyllide reduction. Modification of Cys residues by N-ethyl maleimide (NEM) treatment results in the inhibition of the enzymatic activity, suggesting that several conserved Cys residues are involved in the activity.

Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. β-Carotene and (2S,2′S)-oscillol 2,2′-di(α-l-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.

Se estudiaron las comunidades estratificadas de microorganismos fotosintéticos que se encuentran en el Salar de Llamará ubicado en el desierto de Atacama, norte de Chile, mediante métodos microscópicos y espectrofotométricos. El espesor de la zona fótica de los tapetes descritos varió entre 8 y 30 mm lo cual podría atribuirse a la granulometría y la composición mineralógica de los sedimentos. Se diferencian tres tipos de tapetes. El primero con una única capa pigmentada de color verde; el segundo con capas de colores verde y naranja y un tercero en el que se observa, además de las capas verde y naranja, una de color púrpura. En uno de los sitios muestreados no se encontraron capas pigmentadas. Debajo de la zona pigmentada el sedimento es de color blanco, excepto en uno de los sectores donde se observó una coloración negra atribuible a sulfuro de hierro. Los microorganismos predominantes de la capa naranja fueron diatomeas y cianobacterias unicelulares principalmente de los grupos Cyanothece y Synechococcus. Las cianobacterias filamentosas Microccoleus sp. y Oscillatoria sp. fueron las más abundantes en la capa verde. No se observaron diatomeas en los sitios estudiados donde la salinidad del agua intersticial osciló entre 12 y 33 %. En la capa verde de estos sitios predominaron las cianobacterias cocoides, de los grupos Synechococcus, Cyanothece y Gloeocapsa y del género Gloeobacter, sobre las cianobacterias filamentosas. La capa púrpura estuvo compuesta principalmente por bacterias fototróficas anoxigénicas similares a células de los géneros Chromatium y Thiocapsa. Los espectros de absorción revelaron que la clorofila a es el pigmento más abundante en la mayoría de las muestras analizadas. Los valores integrados de clorofila a y bacterioclorofila a alcanzaron 230 y 144 mg m-2 en el espesor de la zona pigmentada, respectivamente. También se detectaron abundantes microorganismos no fotosintéticos en los tapetes incluyendo cocos y bacilos no identificados. En todos los tapetes muestreados en el Salar se encontraron bacterias reductoras de sulfato.

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.

The possibility that cyanobacterial carboxysomes may contain DNA was investigated using Chlorogloeopsis 6912, Gloeobacter 7421 and Anabaena 7120. No evidence of extrachromosomal DNA was obtained in whole-cell lysates or isolated disrupted carboxysomal fractions of Chlorogloeopsis or Gloeobacter. A plasmid of about 5 kb was found in Anabaena 7120 lysates, although this was localized in the supernatants of cell extracts and was not associated with particulate carboxysomal fractions.

Gloebacter violaceus gen. and sp. n. is a unicellular photosynthetic prokaryote of unusual cellular structure. The only unit membrane in the small, rod-shaped cells is the cytoplasmic membrane, which has a simple contour, without intrusions. Immediately underlying it is an electron-dense layer 80 nm thick. Gloeobacter is an aerobic photoautotroph which contains chlorophyll α, β-carotene and other carotenoids, allophycocyanin, phycocyanin and phycoerythrin. Chlorophyll and carotenoids are associated with the particulate fraction of cell-free extracts, and are thus probably localized in the cytoplasmic membrane. The phycobiliproteins may be associated with the electron-dense 80 nm layer. The DNA contains 64.4 moles percent GC. The cellular lipids have a high content of polyunsaturated fatty acids, largely linoleate and γ-linolenate. Despite its atypical fine structure, Gloeobacter is evidently a cyanobacterium, sufficiently different from other unicellular cyanobacteria to be placed in a new genus.

Cys-loop receptors play important roles in signal transduction in multicellular organisms, but similar proteins exist in prokaryotes, the best studied of which is the Gloeobacter ligand-gated ion channel (GLIC). GLIC is activated by protons with 50% activation (pH50) at pH 5.5, and while a histidine residue in its pore-forming α-helix (M2) is known to be involved in gating, there is also evidence of a proton-sensitive region in the extracellular domain. However, this proton-sensitive region does not appear to be located in the region of GLIC equivalent to the agonist binding site in related proteins. Here we explore functional effects of a range of compounds that could bind to this site and show that some GABA analogues, the most potent of which is crotonic acid, inhibit GLIC function. Mutagenesis and docking studies suggest crotonic acid can bind to this region of the protein and, when bound, can allosterically inhibit GLIC function. These data therefore suggest that there is a transduction pathway from ...

Prokaryotic homologues of Cys-loop receptors have proven to be useful in understanding their eukaryotic counterparts, but even the best studied of these, Gloeobacter ligand-gated ion channel (GLIC), is still not yet fully understood. GLIC is activated by protons with a pH50 between 5 and 6, implicating a histidine residue in its activation, but although a histidine residue (His11′) in the pore-forming α-helix (M2) is known to be involved in gating, the His in the extracellular domain (ECD), His127, is not. Nevertheless, there is evidence from a GLIC–glycine chimera for a proton sensitive residue or region in the GLIC extracellular domain. Here we create a novel chimeric receptor with the ECD of GLIC and the transmembrane domain of ELIC (GELIC). Expression of this receptor in oocytes reveals proton activation, although the pH50 (6.7) differs from that of GLIC (5.4). Exploration of protonatable residues in the ECD reveals that the pKas of five Asp residues (31, 49, 91, 136, and 178) differ between the open ...

In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3–5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

The Gloeobacter ligand-gated ion channel, GLIC, has up to 28% sequence identity with eukaryotic Cys-loop receptors, and many key residues are conserved, especially in the 2nd trans-membrane pore lining region, M2. The M2 region is responsible for ion selectivity, ion flux, and binding a wide range of non-competitive inhibitors (Alqazzaz et al., 2011). The aim of this work was to investigate the GLIC M2 region, especially His235 (or 11'), which has been proposed as essential in proton sensing linked to channel opening (Wang et al., 2012), and residues that are conserved across the Cys-loop receptor family. To probe the roles of specific amino acids in GLIC M2 trans-membrane domain, we substituted M2 residues lining the ion pore and M2 residues facing M3 trans-membrane domains. We generated over 40 mutations at various positions including those at Glu 222(-2'), Thr 226 (2'), Ser 230 (6'), Leu 232 (8'), Ile 233 (9'), Ala 234 (10'), Ile 236 (12'), Ala 237 (13') and Phe 238 (14') using site-directed mutagenesis, and tested their function using two-electrode voltage clamp as previously described (Alqazzaz et al., 2011). Our results showed that Ser6', Ile9' and His11' residues are very sensitive to subsitution with all substituents resulting in non-functional receptors. We conclude that His 11' has a role in channel opening/closing, and the residues Thr226, Ser230, and Ile233 also play an important role in receptor function.Alqazzaz M, Thompson AJ, Price KL, Breitinger HG, Lummis SC (2011). Cys-loop receptor channel blockers also block GLIC. Biophysical journal 101(12): 2912-2918.Wang HL, Cheng X, Sine SM (2012). Intramembrane proton binding site linked to activation of bacterial pentameric ion channel. The Journal of biological chemistry 287(9): 6482-6489.

Xanthorhodopsin is a light-driven proton pump like bacteriorhodopsin, but contains a second chromophore, salinixanthin, a carotenoid. Action spectra for transport and fluorescence of the retinal upon excitation of salinixanthin, as well as femtosecond kinetics of carotenoid fluorescence, indicate that the carotenoid functions as an antenna to the retinal, with ca. 50% efficiency for excited-state energy transfer. The resulting charge redistribution in the retinal and the local electric field produced, in the excited state and in the primary stable photoproduct K, in turn, causes distinct electrochromic shifts in the carotenoid spectrum. The close interaction of carotenoid and retinal suggests that the two chromophores are in proximity. Tight binding of the carotenoid, as indicated by its sharpened vibration bands and intense induced circular dichroism in the visible, is removed in the absence of the retinal, and restored upon reconstitution with retinal or retinal analogues. The crystallographic structure of the xanthorhodopsin at 1.9 A resolution identifies the location of the chromophores. The structural model from x-ray diffraction reveals that the ring moieties of the retinal and the carotenoid are at nearly van der Waals distance from one another. Salinixanthin and one of the native carotenoids of Gloeobacter, echinenone, bind to gloeorhodopsin expressed in E. coli, and the complex shows excited-state energy exchange much like xanthorhodopsin. Protein sequences and mutational studies in Gloeobacter indicate that binding of the carotenoid depends on a conserved glycine at the keto-ring, which is a tryptophan in bacteriorhodopsin and other archaeal rhodopsins. On this basis, numerous otherwise unrelated eubacterial rhodopsins are predicted to contain light-harvesting carotenoids.