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Sample records for anabaena sensory rhodopsin

  1. Anabaena sensory rhodopsin is a light-driven unidirectional rotor.

    PubMed

    Strambi, Angela; Durbeej, Bo; Ferré, Nicolas; Olivucci, Massimo

    2010-12-14

    The implementation of multiconfigurational quantum chemistry methods into a quantum-mechanics/molecular-mechanics protocol has allowed the construction of a realistic computer model for the sensory rhodopsin of the cyanobacterium Anabaena PCC 7120. The model, which reproduces the absorption spectra of both the all-trans and 13-cis forms of the protein and their associated K and L intermediates, is employed to investigate the light-driven steps of the photochromic cycle exhibited by the protein. It is found that the photoisomerizations of the all-trans and 13-cis retinal chromophores occur through unidirectional, counterclockwise 180° rotations of the =C14-C15= moiety with respect to the Lys210-linked end of the chromophore axis. Thus, the sequential interconversions of the all-trans and 13-cis forms during a single photochromic cycle yield a complete (360°) unidirectional rotation of the =C14-C15= moiety. This finding implies that Anabaena sensory rhodopsin is a biological realization of a light-driven molecular rotor. PMID:21098308

  2. Ultrafast photochemistry of anabaena sensory rhodopsin: experiment and theory.

    PubMed

    Schapiro, Igor; Ruhman, Sanford

    2014-05-01

    Light induced isomerization of the retinal chromophore activates biological function in all retinal protein (RP) driving processes such as ion-pumping, vertebrate vision and phototaxis in organisms as primitive as archea, or as complex as mammals. This process and its consecutive reactions have been the focus of experimental and theoretical research for decades. The aim of this review is to demonstrate how the experimental and theoretical research efforts can now be combined to reach a more comprehensive understanding of the excited state process on the molecular level. Using the Anabaena Sensory Rhodopsin as an example we will show how contemporary time-resolved spectroscopy and recently implemented excited state QM/MM methods consistently describe photochemistry in retinal proteins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

  3. Quantum yields for the light adaptations in Anabaena sensory rhodopsin and bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Wada, Yoichiro; Kawanabe, Akira; Furutani, Yuji; Kandori, Hideki; Ohtani, Hiroyuki

    2008-02-01

    Archael-type rhodopsin has an all- trans or a 13- cis retinal. The light-induced interconversion between these two forms has been found in Anabaena sensory rhodopsin, even though only the photoreaction from the 13- cis form to the all- trans form exists in bacteriorhodopsin. In this study, we obtained the quantum yields for the 13- cis → all- trans and all- trans → 13- cis reactions of Anabaena sensory rhodopsin (0.24 ± 0.03 and 0.38 ± 0.07, respectively) and concluded that these values were independent of the wavelength of the excitation light as well as bacteriorhodopsin. In other words, no excess energy effects can be found in these reactions.

  4. DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy.

    PubMed

    Kim, Sung Hyun; Kim, So Young; Jung, Kwang-Hwan; Kim, Doseok

    2015-01-01

    Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K(D), of ASRT to 20 bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.

  5. A role of Anabaena sensory rhodopsin transducer (ASRT) in photosensory transduction.

    PubMed

    Kim, So Young; Yoon, Sa Ryong; Han, SongI; Yun, Yuna; Jung, Kwang-Hwan

    2014-08-01

    In 2003, Anabaena sensory rhodopsin (ASR), a membrane-bound light sensor protein, was discovered in cyanobacteria. Since then, a large number of functions have been described for ASR, based on protein biochemical and biophysical studies. However, no study has determined the in vivo mechanism of photosensory transduction for ASR and its transducer protein (ASRT). Here, we aimed to determine the role of ASRT in physiological photo-regulation. ASRT is known to be related to photochromism, because it regulates the expression of phycocyanin (cpc-gene) and phycoerythrocyanin (pec gene), two major proteins of the phycobilisome in cyanobacteria. By examining wild type and knockout mutant Anabaena cells, we showed that ASRT repressed the expression of these two genes. We also demonstrated physical interactions between ASRT, ASR, and the promoter regions of cpc, pec, kaiABC (circadian clock gene) and the asr operon, both in vitro and in vivo. Binding assays indicated that ASRT had different sites of interaction for binding to ASR and DNA promoter regions. ASRT also influenced the retinal re-isomerization rate in dark through a physical interaction with ASR, and it regulated reporter gene expression in vivo. These results suggested that ASRT relayed the photosignal from ASR and directly regulated gene expression.

  6. 100 fs photo-isomerization with vibrational coherences but low quantum yield in Anabaena Sensory Rhodopsin.

    PubMed

    Cheminal, Alexandre; Léonard, Jérémie; Kim, So-Young; Jung, Kwang-Hwan; Kandori, Hideki; Haacke, Stefan

    2015-10-14

    Anabaena Sensory Rhodopsin (ASR) stands out among the microbial retinal proteins in that, under light-adaptation (LA) conditions, it binds both the 13-cis isomer and the all-trans isomer of the protonated Schiff base of retinal (PSBR). In the dark-adapted (DA) state, more than 95% of the proteins bear all-trans PSBR, and the protein environment adopts a different equilibrium state. We report the excited state and photo-isomerization kinetics of ASR under different LA conditions. The full data set allows confirming that the photoisomerization of the 13C isomer occurs within 100 fs and indications of an excited and ground state wavepacket launched by the ultrafast non-adiabatic reaction are reported. Even though this recalls the record isomerization time and the coherent reaction scenario of 11-cis PSBR in rhodopsin, the photoisomerization quantum yield (QY) is much lower, actually the lowest value ever reported for retinal proteins (<15%). Noticeably, in ASR the excited state lifetime (ESL) is at least five times larger and the QY is more than twice as large for AT PSBR as compared to 13C PSBR. We argue that ESL and QY cannot be expected to be correlated at all, but that the latter is decided on, as often anticipated, by the wavepacket pathways leading to the conical intersection seam.

  7. In situ structural studies of Anabaena sensory rhodopsin in the E. coli membrane.

    PubMed

    Ward, Meaghan E; Wang, Shenlin; Munro, Rachel; Ritz, Emily; Hung, Ivan; Gor'kov, Peter L; Jiang, Yunjiang; Liang, Hongjun; Brown, Leonid S; Ladizhansky, Vladimir

    2015-04-01

    Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids. We find that ASR is organized into trimers in both environments but forms two-dimensional crystal lattices of different symmetries. It favors hexagonal packing in liposomes, but may form a square lattice in the E. coli membrane. To examine possible changes in structure site-specifically, we perform two- and three-dimensional SSNMR experiments and analyze the differences in chemical shifts and peak intensities. Overall, this analysis reveals that the structure of ASR is largely conserved in the inner membrane of E. coli, with many of the important structural features of rhodopsins previously observed in ASR in proteoliposomes being preserved. Small, site-specific perturbations in protein structure that occur as a result of the membrane changes indicate that the protein can subtly adapt to its environment without large structural rearrangement.

  8. In Situ Structural Studies of Anabaena Sensory Rhodopsin in the E. coli Membrane

    PubMed Central

    Ward, Meaghan E.; Wang, Shenlin; Munro, Rachel; Ritz, Emily; Hung, Ivan; Gor’kov, Peter L.; Jiang, Yunjiang; Liang, Hongjun; Brown, Leonid S.; Ladizhansky, Vladimir

    2015-01-01

    Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids. We find that ASR is organized into trimers in both environments but forms two-dimensional crystal lattices of different symmetries. It favors hexagonal packing in liposomes, but may form a square lattice in the E. coli membrane. To examine possible changes in structure site-specifically, we perform two- and three-dimensional SSNMR experiments and analyze the differences in chemical shifts and peak intensities. Overall, this analysis reveals that the structure of ASR is largely conserved in the inner membrane of E. coli, with many of the important structural features of rhodopsins previously observed in ASR in proteoliposomes being preserved. Small, site-specific perturbations in protein structure that occur as a result of the membrane changes indicate that the protein can subtly adapt to its environment without large structural rearrangement. PMID:25863060

  9. Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights.

    PubMed

    Dong, Bamboo; Sánchez-Magraner, Lissete; Luecke, Hartmut

    2016-09-01

    Microbial rhodopsins are light-activated, seven-α-helical, retinylidene transmembrane proteins that have been identified in thousands of organisms across archaea, bacteria, fungi, and algae. Although they share a high degree of sequence identity and thus similarity in structure, many unique functions have been discovered and characterized among them. Some function as outward proton pumps, some as inward chloride pumps, whereas others function as light sensors or ion channels. Unique among the microbial rhodopsins characterized thus far, Anabaena sensory rhodopsin (ASR) is a photochromic sensor that interacts with a soluble 14-kDa cytoplasmic transducer that is encoded on the same operon. The sensor itself stably interconverts between all-trans-15-anti and 13-cis-15-syn retinal forms depending on the wavelength of illumination, although only the former participates in a photocycle with a signaling M intermediate. A mutation in the cytoplasmic half-channel of the protein, replacing Asp217 with Glu (D217E), results in the creation of a light-driven, single-photon, inward proton transporter. We present the 2.3 Å structure of dark-adapted D217E ASR, which reveals significant changes in the water network surrounding Glu217, as well as a shift in the carbon backbone near retinal-binding Lys210, illustrating a possible pathway leading to the protonation of Glu217 in the cytoplasmic half-channel, located 15 Å from the Schiff base. Crystallographic evidence for the protonation of nearby Glu36 is also discussed, which was described previously by Fourier transform infrared spectroscopy analysis. Finally, two histidine residues near the extracellular surface and their possible role in proton uptake are discussed. PMID:27602724

  10. Crystallization, X-ray diffraction analysis and SIRAS/molecular-replacenent phasing of three crystal forms of Anabaena sensory rhodopsin transducer

    SciTech Connect

    Vogeley, Lutz; Luecke, Hartmut

    2006-04-01

    Crystals of Anabaena sensory rhodopsin transducer, the transducer for the cyanobacterial photosensor Anabaena sensory rhodopsin, obtained in the space groups P4, C2 and P2{sub 1}2{sub 1}2{sub 1} diffract to 1.8, 2.1 and 2.0 Å, respectively. Phases for these crystal forms were obtained by SIRAS phasing using an iodide quick-soak derivative (P4) and molecular replacement (C2 and P2{sub 1}2{sub 1}2{sub 1}). Anabaena sensory rhodopsin transducer (ASRT) is a 14.7 kDa soluble signaling protein associated with the membrane-embedded light receptor Anabaena sensory rhodopsin (ASR) from Anabaena sp., a freshwater cyanobacterium. Crystals of ASRT were obtained in three different space groups, P4, C2 and P2{sub 1}2{sub 1}2{sub 1}, which diffract to 1.8, 2.1 and 2.0 Å, respectively. Phases for one of these crystal forms (P4) were obtained by SIRAS phasing using an iodide quick-soak derivative and a partial model was built. Phases for the remaining crystal forms were obtained by molecular replacement using the partial model from the P4 crystal form.

  11. Steady state emission of the fluorescent intermediate of Anabaena Sensory Rhodopsin as a function of light adaptation conditions

    NASA Astrophysics Data System (ADS)

    Cheminal, A.; Léonard, J.; Kim, S. Y.; Jung, K.-H.; Kandori, H.; Haacke, S.

    2013-11-01

    Steady-state fluorescence measurements of the first excited state of the anabaena sensory rhodopsin (ASR), and Bacteriorhodopsin are reported for different light stabilization conditions, including the dark-adapted state. We determine the fluorescence spectra of both all-trans (AT), and 13-cis (13C) protonated Schiff base of retinal, and compare the effect of the proteins. Referenced against the fluorescence quantum yield of AT-bR (2.5 × 10-4) we find for AT-ASR, 13C-ASR, and 13C-bR the values of 3.3 × 10-4, 0.8 × 10-4, and 1.7 × 10-4, respectively. Using reported excited state lifetimes, the radiative rates are deduced, and their differences discussed on the basis of a configuration-dependent oscillator strength.

  12. Primary structural response in tryptophan residues of Anabaena sensory rhodopsin to photochromic reactions of the retinal chromophore

    NASA Astrophysics Data System (ADS)

    Inada, Seisuke; Mizuno, Misao; Kato, Yoshitaka; Kawanabe, Akira; Kandori, Hideki; Wei, Zhengrong; Takeuchi, Satoshi; Tahara, Tahei; Mizutani, Yasuhisa

    2013-06-01

    Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin found in eubacteria and functions as a photosensor. The photoreaction of ASR is photochromic between all-trans, 15-anti (ASRAT), and 13-cis, 15-syn (ASR13C) isomers. To understand primary protein dynamics in the photoreaction starting in ASRAT and ASR13C, picosecond time-resolved ultraviolet resonance Raman spectra were obtained. In the intermediate state appearing in the picosecond temporal region, spectral changes of Trp bands were observed. For both ASRAT and ASR13C, the intensities of the Trp bands were bleached within the instrumental response time and recovered with a time constant of 30 ps. This suggests that the rates of structural changes in the Trp residue in the vicinity of the chromophore do not depend on the direction of the isomerization of retinal. A comparison between spectra of the wild-type and Trp mutants indicates that the structures of Trp76 and Trp46 change upon the primary photoreaction of retinal.

  13. pH dependence of Anabaena sensory rhodopsin: retinal isomer composition, rate of dark adaptation, and photochemistry.

    PubMed

    Rozin, Rinat; Wand, Amir; Jung, Kwang-Hwan; Ruhman, Sanford; Sheves, Mordechai

    2014-07-31

    Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory rhodopsin (ASR), a microbial rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two pKa values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional pKa value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than under acidic conditions. (d) Primary photochemical events of ASR at pH 5 are recorded using VIS hyperspectral pump-probe spectroscopy with <100 fs resolution and compared with the previously recorded results at pH 7.5. For AT-ASR, these are shown to be almost pH-independent. However, photochemistry of 13C-ASR is pH-dependent and slowed down in acidic environments.

  14. Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin

    NASA Astrophysics Data System (ADS)

    Ward, Meaghan E.; Brown, Leonid S.; Ladizhansky, Vladimir

    2015-04-01

    Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic α-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination. In this Perspective article we describe our recent efforts directed at determining the structure and functional dynamics of Anabaena Sensory Rhodopsin, a heptahelical transmembrane (7TM) protein. We review some of the established and emerging methods which can be utilized for SSNMR-based structure determination, with a particular focus on those used for ASR, a bacterial protein which shares its 7TM architecture with G-protein coupled receptors.

  15. Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: application to Anabaena Sensory Rhodopsin.

    PubMed

    Ward, Meaghan E; Brown, Leonid S; Ladizhansky, Vladimir

    2015-04-01

    Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic α-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination. In this Perspective article we describe our recent efforts directed at determining the structure and functional dynamics of Anabaena Sensory Rhodopsin, a heptahelical transmembrane (7TM) protein. We review some of the established and emerging methods which can be utilized for SSNMR-based structure determination, with a particular focus on those used for ASR, a bacterial protein which shares its 7TM architecture with G-protein coupled receptors.

  16. Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

    PubMed

    Good, Daryl B; Wang, Shenlin; Ward, Meaghan E; Struppe, Jochem; Brown, Leonid S; Lewandowski, Józef R; Ladizhansky, Vladimir

    2014-02-19

    The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions. In contrast, the R1ρ rates, which were measured under fast magic angle spinning conditions, vary by an order of magnitude between the TM and exposed regions and suggest the presence of intermediate time scale motions. Using a simple model, which assumes a single exponential autocorrelation function, we estimated the time scales of dominant stochastic motions to be on the order of low tens of nanoseconds for most residues within the TM helices and tens to hundreds of nanoseconds for the extracellular B-C and F-G loops. These relatively slow time scales could be attributed to collective anisotropic motions. We used the 3D Gaussian axial fluctuations model to estimate amplitudes, directions, and time scales of overall motions for helices and the extracellular B-C and F-G loops. Within this model, the TM helices A,B,C,D,E,F undergo rigid body motions on a time scale of tens of nanoseconds, while the time scale for the seventh helix G approaches 100 ns. Similar time scales of roughly 100-200 ns are estimated for the B-C and F-G loops. PMID:24467417

  17. A Schiff base connectivity switch in sensory rhodopsin signaling.

    PubMed

    Sineshchekov, Oleg A; Sasaki, Jun; Phillips, Brian J; Spudich, John L

    2008-10-21

    Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

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

    SciTech Connect

    Spudich, E.N.; Takahashi, T.; Spudich, J.L. )

    1989-10-01

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

  19. The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II.

    PubMed

    Seidel, R; Scharf, B; Gautel, M; Kleine, K; Oesterhelt, D; Engelhard, M

    1995-03-28

    The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II). Upstream of both sopII gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II were recognized. In N. pharaonis, psopII and phtrII are transcribed as a single transcript. Comparison of the amino acid sequences of vHtr-II and pHtr-II with Htr-I and the chemotactic methyl-accepting proteins from Escherichia coli revealed considerable identities in the signal domain and methyl-accepting sites. Similarities with Htr-I in Halobacterium salinarium suggest a common principle in the phototaxis of extreme halophiles. Alignment of all known retinal protein sequences from Archaea identifies both SR-IIs as an additional subgroup of the family. Positions defining the retinal binding site are usually identical with the exception of Met-118 (numbering is according to the bacteriorhodopsin sequence), which might explain the typical blue color shift of SR-II to approximately 490 nm. In archaeal retinal proteins, the function can be deduced from amino acids in positions 85 and 96. Proton pumps are characterized by Asp-85 and Asp-96; chloride pumps by Thr-85 and Ala-96; and sensors by Asp-85 and Tyr-96 or Phe-96.

  20. The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II.

    PubMed Central

    Seidel, R; Scharf, B; Gautel, M; Kleine, K; Oesterhelt, D; Engelhard, M

    1995-01-01

    The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II). Upstream of both sopII gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II were recognized. In N. pharaonis, psopII and phtrII are transcribed as a single transcript. Comparison of the amino acid sequences of vHtr-II and pHtr-II with Htr-I and the chemotactic methyl-accepting proteins from Escherichia coli revealed considerable identities in the signal domain and methyl-accepting sites. Similarities with Htr-I in Halobacterium salinarium suggest a common principle in the phototaxis of extreme halophiles. Alignment of all known retinal protein sequences from Archaea identifies both SR-IIs as an additional subgroup of the family. Positions defining the retinal binding site are usually identical with the exception of Met-118 (numbering is according to the bacteriorhodopsin sequence), which might explain the typical blue color shift of SR-II to approximately 490 nm. In archaeal retinal proteins, the function can be deduced from amino acids in positions 85 and 96. Proton pumps are characterized by Asp-85 and Asp-96; chloride pumps by Thr-85 and Ala-96; and sensors by Asp-85 and Tyr-96 or Phe-96. Images Fig. 2 Fig. 3 PMID:7708770

  1. Quantum chemical modeling of rhodopsin mutants displaying switchable colors.

    PubMed

    Melaccio, Federico; Ferré, Nicolas; Olivucci, Massimo

    2012-09-28

    We look at the possibility to compute and understand the color change occurring upon mutation of a photochromic protein. Accordingly, ab initio multiconfigurational quantum chemical methods are used to construct basic quantum-mechanics/molecular-mechanics (QM/MM) models for a small mutant library of the sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120 cyanobacterium. Together with the wild-type forms, a set of 26 absorption maxima spanning a ca. 80 nm range is obtained. We show that these models can be used to capture the electrostatic change controlling the computed color variation and the change in the ionization of specific side chains. PMID:22699180

  2. Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

    PubMed Central

    McCain, D A; Amici, L A; Spudich, J L

    1987-01-01

    Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II). PMID:3654583

  3. Molecular bases for the selection of the chromophore of animal rhodopsins.

    PubMed

    Luk, Hoi Ling; Melaccio, Federico; Rinaldi, Silvia; Gozem, Samer; Olivucci, Massimo

    2015-12-15

    The functions of microbial and animal rhodopsins are triggered by the isomerization of their all-trans and 11-cis retinal chromophores, respectively. To lay the molecular basis driving the evolutionary transition from the all-trans to the 11-cis chromophore, multiconfigurational quantum chemistry is used to compare the isomerization mechanisms of the sensory rhodopsin from the cyanobacterium Anabaena PCC 7120 (ASR) and of the bovine rhodopsin (Rh). It is found that, despite their evolutionary distance, these eubacterial and vertebrate rhodopsins start to isomerize via distinct implementations of the same bicycle-pedal mechanism originally proposed by Warshel [Warshel A (1976) Nature 260:678-683]. However, by following the electronic structure changes of ASR (featuring the all-trans chromophore) during the isomerization, we find that ASR enters a region of degeneracy between the first and second excited states not found in Rh (featuring the 11-cis chromophore). We show that such degeneracy is modulated by the preorganized structure of the chromophore and by the position of the reactive double bond. It is argued that the optimization of the electronic properties of the chromophore, which affects the photoisomerization efficiency and the thermal isomerization barrier, provided a key factor for the emergence of the striking amino acid sequence divergence observed between the microbial and animal rhodopsins. PMID:26607446

  4. Molecular bases for the selection of the chromophore of animal rhodopsins

    PubMed Central

    Luk, Hoi Ling; Melaccio, Federico; Rinaldi, Silvia; Gozem, Samer; Olivucci, Massimo

    2015-01-01

    The functions of microbial and animal rhodopsins are triggered by the isomerization of their all-trans and 11-cis retinal chromophores, respectively. To lay the molecular basis driving the evolutionary transition from the all-trans to the 11-cis chromophore, multiconfigurational quantum chemistry is used to compare the isomerization mechanisms of the sensory rhodopsin from the cyanobacterium Anabaena PCC 7120 (ASR) and of the bovine rhodopsin (Rh). It is found that, despite their evolutionary distance, these eubacterial and vertebrate rhodopsins start to isomerize via distinct implementations of the same bicycle-pedal mechanism originally proposed by Warshel [Warshel A (1976) Nature 260:678–683]. However, by following the electronic structure changes of ASR (featuring the all-trans chromophore) during the isomerization, we find that ASR enters a region of degeneracy between the first and second excited states not found in Rh (featuring the 11-cis chromophore). We show that such degeneracy is modulated by the preorganized structure of the chromophore and by the position of the reactive double bond. It is argued that the optimization of the electronic properties of the chromophore, which affects the photoisomerization efficiency and the thermal isomerization barrier, provided a key factor for the emergence of the striking amino acid sequence divergence observed between the microbial and animal rhodopsins. PMID:26607446

  5. Probing the Photodynamics of Rhodopsins with Reduced Retinal Chromophores.

    PubMed

    Manathunga, Madushanka; Yang, Xuchun; Luk, Hoi Ling; Gozem, Samer; Frutos, Luis Manuel; Valentini, Alessio; Ferrè, Nicolas; Olivucci, Massimo

    2016-02-01

    While the light-induced population dynamics of different photoresponsive proteins has been investigated spectroscopically, systematic computational studies have not yet been possible due to the phenomenally high cost of suitable high level quantum chemical methods and the need of propagating hundreds, if not thousands, of nonadiabatic trajectories. Here we explore the possibility of studying the photodynamics of rhodopsins by constructing and investigating quantum mechanics/molecular mechanics (QM/MM) models featuring reduced retinal chromophores. In order to do so we use the sensory rhodopsin found in the cyanobacterium Anabaena PCC7120 (ASR) as a benchmark system. We find that the basic mechanistic features associated with the excited state dynamics of ASR QM/MM models are reproduced using models incorporating a minimal (i.e., three double-bond) chromophore. Furthermore, we show that ensembles of nonadiabatic ASR trajectories computed using the same abridged models replicate, at both the CASPT2 and CASSCF levels of theory, the trends in spectroscopy and lifetimes estimated using unabridged models and observed experimentally at room temperature. We conclude that a further expansion of these studies may lead to low-cost QM/MM rhodopsin models that may be used as effective tools in high-throughput in silico mutant screening. PMID:26640959

  6. Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga.

    PubMed

    Wada, Takashi; Shimono, Kazumi; Kikukawa, Takashi; Hato, Masakatsu; Shinya, Naoko; Kim, So Young; Kimura-Someya, Tomomi; Shirouzu, Mikako; Tamogami, Jun; Miyauchi, Seiji; Jung, Kwang-Hwan; Kamo, Naoki; Yokoyama, Shigeyuki

    2011-09-01

    Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the "late proton release".

  7. Rhodopsin 7-The unusual Rhodopsin in Drosophila.

    PubMed

    Senthilan, Pingkalai R; Helfrich-Förster, Charlotte

    2016-01-01

    Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1-Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a "vertebrate-melanopsin-type"-cluster, and Rh3, Rh4 and Rh5 form an "insect-type"-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins. PMID:27651995

  8. The trafficking of bacterial type rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated

    PubMed Central

    Awasthi, Mayanka; Ranjan, Peeyush; Sharma, Komal; Veetil, Sindhu Kandoth; Kateriya, Suneel

    2016-01-01

    The bacterial type rhodopsins are present in all the three domains of life. In contrast to the animal type rhodopsin that performs mainly sensory functions in higher eukaryotes, the bacterial type rhodopsin could function as ion channel, pumps and as sensory proteins. The functioning of rhodopsin in higher eukaryotes requires the transport of rhodopsin from its site of synthesis to the ciliated outer segment of the photoreceptive cells. However, the trafficking of bacterial type rhodopsin from its site of synthesis to the position of action is not characterized. Here we present the first report for the existence of an IFT-interactome mediated trafficking of the bacterial type rhodopsins into eyespot and flagella of the Chlamydomonas. We show that there is a light-dependent, dynamic localization of rhodopsins between flagella and eyespot of Chlamydomonas. The involvement of IFT components in the rhodopsin trafficking was elucidated by the use of conditional IFT mutants. We found that rhodopsin can be co-immunoprecipitated with the components of IFT machinery and with other protein components required for the IFT-cargo complex formation. These findings show that light-regulated localization of rhodopsin is not restricted to animals thereby suggesting that rhodopsin trafficking is an IFT dependent ancient process. PMID:27694882

  9. Rhodopsin 7–The unusual Rhodopsin in Drosophila

    PubMed Central

    2016-01-01

    Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins.

  10. Rhodopsin 7–The unusual Rhodopsin in Drosophila

    PubMed Central

    2016-01-01

    Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins. PMID:27651995

  11. Cyanobacterial Light-Driven Proton Pump, Gloeobacter Rhodopsin: Complementarity between Rhodopsin-Based Energy Production and Photosynthesis

    PubMed Central

    Choi, Ah Reum; Shi, Lichi; Brown, Leonid S.; Jung, Kwang-Hwan

    2014-01-01

    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 (λmax  = 544 nm at pH 7). The pKa 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. PMID:25347537

  12. The rhodopsins: structure and function. Introduction

    NASA Technical Reports Server (NTRS)

    Lanyi, J. K.

    1992-01-01

    Nature makes use of the propensity of retinal for light-dependent double-bond isomerization in a number of systems and in a variety of ways. The common theme for light receptors based on this kind of chemistry is that (1) the retinal is bound in most cases to a small membrane protein via a protonated lysine-retinal Schiff base, (2) the absorption maximum in the visible is tuned to a suitable wavelength largely by electrostatic interaction with polar protein residues, and (3) the light-induced bond rotations and strains in the retinal set off reaction chains during which at least part of the excess free energy acquired is transferred to the protein and causes pK shifts of acidic residues and/or backbone conformational changes. The physiological consequence of the process initiated by absorption of light is either the activation of an information transfer chain (sensory and visual rhodopsins) or energy transduction which drives the electrogenic movement of ions across the membrane (ion-motive rhodopsins). Rhodopsins with these functions occur in bacteria and in higher organisms; from an evolutionary standpoint they are not related to one another. Nevertheless, all of these proteins are remarkably similar and form a distinct family.

  13. Algal sensory photoreceptors.

    PubMed

    Hegemann, Peter

    2008-01-01

    Only five major types of sensory photoreceptors (BLUF-proteins, cryptochromes, phototropins, phytochromes, and rhodopsins) are used in nature to regulate developmental processes, photosynthesis, photoorientation, and control of the circadian clock. Sensory photoreceptors of algae and protists are exceptionally rich in structure and function; light-gated ion channels and photoactivated adenylate cyclases are unique examples. During the past ten years major progress has been made with respect to understanding the function, photochemistry, and structure of key sensory players of the algal kingdom.

  14. Torsion potential works in rhodopsin.

    PubMed

    Yamada, Atsushi; Yamato, Takahisa; Kakitani, Toshiaki; Yamamoto, Shigeyoshi

    2004-05-01

    We investigate the role of protein environment of rhodopsin and the intramolecular interaction of the chromophore in the cis-trans photoisomerization of rhodopsin by means of a newly developed theoretical method. We theoretically produce modified rhodopsins in which a force field of arbitrarily chosen part of the chromophore or the binding pocket of rhodopsin is altered. We compare the equilibrium conformation of the chromophore and the energy stored in the chromophore of modified rhodopsins with those of native rhodopsins. This method is called site-specific force field switch (SFS). We show that this method is most successfully applied to the torsion potential of rhodopsin. Namely, by reducing the twisting force constant of the C11=C12 of 11-cis retinal chromophore of rhodopsin to zero, we found that the equilibrium value of the twisting angle of the C11=C12 bond is twisted in the negative direction down to about -80 degrees. The relaxation energy obtained by this change amounts to an order of 10 kcal/mol. In the case that the twisting force constant of the other double bond is reduced to zero, no such large twisting of the bond happens. From these results we conclude that a certain torsion potential is applied specifically to the C11=C12 bond of the chromophore in the ground state of rhodopsin. This torsion potential facilitates the bond-specific cis-trans photoisomerization of rhodopsin. This kind of the mechanism is consistent with our torsion model proposed by us more than a quarter of century ago. The origin of the torsion potential is analyzed in detail on the basis of the chromophore structure and protein conformation, by applying the SFS method extensively.

  15. Light-Promoted Rhodopsin Expression and Starvation Survival in the Marine Dinoflagellate Oxyrrhis marina

    PubMed Central

    Guo, Zhiling; Zhang, Huan; Lin, Senjie

    2014-01-01

    The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria. PMID:25506945

  16. Light-promoted rhodopsin expression and starvation survival in the marine dinoflagellate Oxyrrhis marina.

    PubMed

    Guo, Zhiling; Zhang, Huan; Lin, Senjie

    2014-01-01

    The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.

  17. The Azolla, Anabaena azollae Relationship

    PubMed Central

    Peters, Gerald A.; Mayne, Berger C.

    1974-01-01

    Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source. Morphological aspects of the symbiotic association show the confinement of the Anabaena azollae within the leaf cavity of the Azolla. Procedures were established for the isolation of pure preparations of Anabaena azollae and Azolla chloroplasts. It has not yet been possible to grow the isolated alga in independent culture. Photochemical activities of the isolated alga and fern chloroplasts were measured by spectrophotometric assays for photosystems I and II as well as by P700-content (photosystem I) and delayed light emission (photosystem II). In the algal fraction, both photosystems were repressed when compared to freeliving Anabaena cylindrica, but the relative ratio of photosystem I to photosystem II may be appreciably greater in Anabaena azollae. Azolla chloroplasts were generally comparable to spinach chloroplasts. A comparison of the chlorophyll a and b content of Azolla fronds with and without the symbiotic alga resulted in an estimate that in the symbiotic association, the Anabaena azollae accounts for from 7.5 to 15% of the total chlorophyll. Images PMID:16658796

  18. Rhodopsin Molecular Evolution in Mammals Inhabiting Low Light Environments

    PubMed Central

    Zhao, Huabin; Ru, Binghua; Teeling, Emma C.; Faulkes, Christopher G.; Zhang, Shuyi; Rossiter, Stephen J.

    2009-01-01

    The ecological radiation of mammals to inhabit a variety of light environments is largely attributed to adaptive changes in their visual systems. Visual capabilities are conferred by anatomical features of the eyes as well as the combination and properties of their constituent light sensitive pigments. To test whether evolutionary switches to different niches characterized by dim-light conditions coincided with molecular adaptation of the rod pigment rhodopsin, we sequenced the rhodopsin gene in twenty-two mammals including several bats and subterranean mole-rats. We compared these to thirty-seven published mammal rhodopsin sequences, from species with divergent visual ecologies, including nocturnal, diurnal and aquatic groups. All taxa possessed an intact functional rhodopsin; however, phylogenetic tree reconstruction recovered a gene tree in which rodents were not monophyletic, and also in which echolocating bats formed a monophyletic group. These conflicts with the species tree appear to stem from accelerated evolution in these groups, both of which inhabit low light environments. Selection tests confirmed divergent selection pressures in the clades of subterranean rodents and bats, as well as in marine mammals that live in turbid conditions. We also found evidence of divergent selection pressures among groups of bats with different sensory modalities based on vision and echolocation. Sliding window analyses suggest most changes occur in transmembrane domains, particularly obvious within the pinnipeds; however, we found no obvious pattern between photopic niche and predicted spectral sensitivity based on known critical amino acids. This study indicates that the independent evolution of rhodopsin vision in ecologically specialised groups of mammals has involved molecular evolution at the sequence level, though such changes might not mediate spectral sensitivity directly. PMID:20016835

  19. Rhodopsin photochemistry is vibrationally coherent

    SciTech Connect

    Mathies, R.A.; Wang, Q.; Peteanu, L.A.

    1995-12-31

    Visual excitation is initiated by the absorption of a photon by the 11-cis retinal chromophore bound within the pigment called rhodopsin. We have used a variety of vibrational spectroscopies to obtain information about the vibrational nuclear dynamics that lead to this efficient photochemical isomerization. The cis-trans isomerization in rhodopsin is complete in only 200 fs. The extreme speed of this process, which is consistent with the {approximately}50 fs lifetime indicated by the spontaneous emission yield, suggests that the photochemistry involves non-stationary states or vibrational coherence. Recent studies have in fact observed vibrationally coherent oscillations of the ground state photoproduct called bathorhodopsin following impulsive excitation of the rhodopsin reactant. This conclusively demonstrates that the isomerization process in rhodopsin is vibrationally coherent. These observations further suggest that the isomerization quantum yield is directly dependent on the excited-state torsional velocity and can be thought of as a Landau-Zener tunneling process. This work establishes a vibrationally coherent paradigm for the photochemistry of vision that may be relevant for many other photochemical and photobiological processes including photosynthesis and proton pumping in bacteriorhodopsin.

  20. Methyl-accepting protein associated with bacterial sensory phodopsin I

    SciTech Connect

    Spudich, E.N.; Hasselbacher, C.A. ); Spudich, J.L. )

    1988-09-01

    In vivo radiolabeling of Halaobacterium halobium phototaxis mutants and revertants with L-(methyl-{sup 3}H) methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in photoaxis. The lability of the radiolabeled bands to mild base treatment indicated the the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing photoaxis receptors in H. halobium. It was absent in a strain the contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based in the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By ({sup 3}H)retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced ({sup 3}H) retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the siginal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.

  1. The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin*

    PubMed Central

    Kazmin, Roman; Rose, Alexander; Szczepek, Michal; Elgeti, Matthias; Ritter, Eglof; Piechnick, Ronny; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W.; Bartl, Franz J.

    2015-01-01

    Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences. PMID:26105054

  2. The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin.

    PubMed

    Kazmin, Roman; Rose, Alexander; Szczepek, Michal; Elgeti, Matthias; Ritter, Eglof; Piechnick, Ronny; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W; Bartl, Franz J

    2015-08-14

    Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (Gt). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences. PMID:26105054

  3. A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus.

    PubMed

    Fabczak, Hanna; Sobierajska, Katarzyna; Fabczak, Stanisław

    2008-09-01

    Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota. PMID:18754050

  4. A rhodopsin immunoanalog in the related photosensitive protozoans Blepharisma japonicum and Stentor coeruleus.

    PubMed

    Fabczak, Hanna; Sobierajska, Katarzyna; Fabczak, Stanisław

    2008-09-01

    Immunoblotting of isolated cell membrane fractions from ciliates Blepharisma japonicum and Stentor coeruleus with a polyclonal antibody raised against rhodopsin revealed one strong protein band of about 36 kDa, thought to correspond to protozoan rhodopsin. Inspection of both ciliates labeled with the same antibody using a confocal microscope confirmed the immunoblotting result and demonstrated the presence of these rhodopsin-like molecules localized within the cell membrane area. Immunoblot analysis of the ciliate membrane fractions resolved by two-dimensional gel electrophoresis identified two distinct 36 kDa spots at pIs of 4.5 and 7.0 for Blepharisma, and three spots at pIs of 4.4, 5.0 and 6.0 for Stentor, indicating a possible mixture of phosphorylated rhodopsin species in these cells. The obtained results suggest that both Blepharisma and the related ciliate Stentor contain within the cell membrane the rhodopsin-like proteins, which may be involved as receptor molecules in the sensory transduction pathway mediating motile photoresponses in these protists as in other species of lower eukaryota.

  5. THE RHODOPSIN SYSTEM OF THE SQUID

    PubMed Central

    Hubbard, Ruth; St. George, Robert C. C.

    1958-01-01

    Squid rhodopsin (λmax 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λmax 500 mµ) near neutrality, yellow (λmax 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H+ ⇌acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions. PMID:13491819

  6. Structural elements of the signal propagation pathway in squid rhodopsin and bovine rhodopsin.

    PubMed

    Sugihara, Minoru; Fujibuchi, Wataru; Suwa, Makiko

    2011-05-19

    Squid and bovine rhodopsins are G-protein coupled receptors (GPCRs) that activate Gq- and Gt-type G-proteins, respectively. To understand the structural elements of the signal propagation pathway, we performed molecular dynamics (MD) simulations of squid and bovine rhodopsins plus a detailed sequence analysis of class A GPCRs. The computations indicate that although the geometry of the retinal is similar in bovine and squid rhodopsins, the important interhelical hydrogen bond networks are different. In squid rhodopsin, an extended hydrogen bond network that spans ∼13 Å to Tyr315 on the cytoplasmic site is present regardless of the protonation state of Asp80. In contrast, the extended hydrogen bond network is interrupted at Tyr306 in bovine rhodopsin. Those differences in the hydrogen bond network may play significant functional roles in the signal propagation from the retinal binding site to the cytoplasmic site, including transmembrane helix (TM) 6 to which the G-protein binds. The MD calculations demonstrate that the elongated conformation of TM6 in squid rhodopsin is stabilized by salt bridges formed with helix (H) 9. Together with the interhelical hydrogen bonds, the salt bridges between TM6 and H9 stabilize the protein conformation of squid rhodopsin and may hinder the occurrence of large conformational changes that are observed upon activation of bovine rhodopsin.

  7. FTIR difference and resonance Raman spectroscopy of rhodopsins with applications to optogenetics

    NASA Astrophysics Data System (ADS)

    Saint Clair, Erica C.

    The major aim of this thesis is to investigate the molecular basis for the function of several types of rhodopsins with special emphasis on their application to the new field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7 alpha-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a light driven proton pump similar to the extensively studied bacteriorhodopsin (BR); channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII), a light-sensing protein that modulates phototaxis used in archaebacteria; and squid rhodopsins (sRho), the major photopigment in squid vision and a model for human melanopsin, which controls circadian rhythms. The primary techniques used in these studies were FTIR difference spectroscopy and resonance Raman spectroscopy. These techniques, in combination with site directed mutagenesis and other biochemical methodologies produced new knowledge regarding the structural changes of the retinal chromophore, the location and function of internal water molecules as well as specific amino acids and peptide backbone. Specialized techniques were developed that allowed rhodopsins to be studied in intact membrane environments and in some cases in vivo measurements were made on rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting proteins and membrane potential to be investigated. Evidence was found that the local environment of one or more internal water molecules in SRII is altered by interaction with its cognate transducer, HtrII, and is also affected by the local lipid environment. In the case of AR3, many of the broad IR continuum absorption changes below 3000 cm -1, assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions in BR, were found to be very similar to BR. Bands assigned to water molecules near the Schiff base postulated to be involved in proton transport were, however, shifted

  8. Mechanism of colour discrimination by a bacterial sensory rhodopsin

    NASA Technical Reports Server (NTRS)

    Spudich, J. L.; Bogomolni, R. A.

    1984-01-01

    A photosensitive protein resembling the visual pigments of invertebrates enables phototactic archaebacteria to distinguish color. This protein exists in two spectrally-distinct forms, one of which is a transient photoproduct of the other and each of which undergoes photochemical reactions controlling the cell's swimming behaviour. Activation of a single pigment molecule in the cell is sufficient to signal the flagellar motor. This signal-transduction mechanism makes evident a color-sensing capability inherent in the retinal/protein chromophore.

  9. Towards Understanding the Ultrafast Dynamics of Rhodopsin

    NASA Astrophysics Data System (ADS)

    Aalberts, Daniel; Vos, Fernando; van Saarloos, Wim

    1997-03-01

    The photoisomerization of rhodopsin in 200 femtoseconds is among the fastest and most efficient photochemical reactions known. We have developed a microscopic model to study rhodopsin's dynamics which retains the collective quantum mechanics of the π electrons in the conjugated system. Our model is a generalization to three dimensions of Su, Schrieffer, and Heeger's model for polyacetylene (CH)_x. Model parameters are inferred from comparison with experiments and ab initio calculations. The spatial structure and vibrational modes of the rhodopsin chromophore 11-cis retinal are calculated and shown to agree quite well with NMR and Raman spectroscopy measurements. Dynamics following photoexcitation are studied.

  10. Single-base pair differences in a shared motif determine differential Rhodopsin expression.

    PubMed

    Rister, Jens; Razzaq, Ansa; Boodram, Pamela; Desai, Nisha; Tsanis, Cleopatra; Chen, Hongtao; Jukam, David; Desplan, Claude

    2015-12-01

    The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli.

  11. Single base pair differences in a shared motif determine differential Rhodopsin expression

    PubMed Central

    Rister, Jens; Razzaq, Ansa; Boodram, Pamela; Desai, Nisha; Tsanis, Cleopatra; Chen, Hongtao; Jukam, David; Desplan, Claude

    2016-01-01

    The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11bp activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits unique single bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Novel sensory neuron subtypes can therefore evolve through single base pair changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli. PMID:26785491

  12. The Road to Optogenetics: Microbial Rhodopsins.

    PubMed

    Govorunova, E G; Koppel, L A

    2016-09-01

    Optogenetics technology (using light-sensitive microbial proteins to control animal cell physiology) is becoming increasingly popular in laboratories around the world. Among these proteins, particularly important are rhodopsins that transport ions across the membrane and are used in optogenetics to regulate membrane potential by light, mostly in neurons. Although rhodopsin ion pumps transport only one charge per captured photon, channelrhodopsins are capable of more efficient passive transport. In this review, we follow the history of channelrhodopsin discovery in flagellate algae and discuss the latest addition to the channelrhodopsin family, channels with anion, rather than cation, selectivity. PMID:27682165

  13. Accumulation of Rhodopsin in Late Endosomes Triggers Photoreceptor Cell Degeneration

    PubMed Central

    Chinchore, Yashodhan; Mitra, Amitavo; Dolph, Patrick J.

    2009-01-01

    Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons. PMID:19214218

  14. Functional metagenomic screen reveals new and diverse microbial rhodopsins.

    PubMed

    Pushkarev, Alina; Béjà, Oded

    2016-09-01

    Ion-translocating retinylidene rhodopsins are widely distributed among marine and freshwater microbes. The translocation is light-driven, contributing to the production of biochemical energy in diverse microbes. Until today, most microbial rhodopsins had been detected using bioinformatics based on homology to other rhodopsins. In the past decade, there has been increased interest in microbial rhodopsins in the field of optogenetics since microbial rhodopsins were found to be most useful in vertebrate neuronal systems. Here we report on a functional metagenomic assay for detecting microbial rhodopsins. Using an array of narrow pH electrodes and light-emitting diode illumination, we were able to screen a metagenomic fosmid library to detect diverse marine proteorhodopsins and an actinorhodopsin based solely on proton-pumping activity. Our assay therefore provides a rather simple phenotypic means to enrich our understanding of microbial rhodopsins without any prior knowledge of the genomic content of the environmental entities screened. PMID:26894445

  15. Spectral Tuning of Killer Whale (Orcinus orca) Rhodopsin: Evidence for Positive Selection and Functional Adaptation in a Cetacean Visual Pigment.

    PubMed

    Dungan, Sarah Z; Kosyakov, Alexander; Chang, Belinda S W

    2016-02-01

    Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution. PMID:26486871

  16. Spectral Tuning of Killer Whale (Orcinus orca) Rhodopsin: Evidence for Positive Selection and Functional Adaptation in a Cetacean Visual Pigment.

    PubMed

    Dungan, Sarah Z; Kosyakov, Alexander; Chang, Belinda S W

    2016-02-01

    Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution.

  17. The molecular weight of rhodopsin and the nature of the rhodopsin-digitonin complex.

    PubMed

    HUBBARD, R

    1954-01-20

    The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s(20) of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s(20) of about 9.77 Svedberg units. The s(20) of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.(2) per mole) and the E(1 per cent, 1 cm., 500 mmicro) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 10(6) molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 10(9) molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the

  18. Conservation of molecular interactions stabilizing bovine and mouse rhodopsin

    PubMed Central

    Kawamura, Shiho; Colozo, Alejandro T.; Müller, Daniel J.; Park, Paul S.-H.

    2010-01-01

    Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin is tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disc membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions. PMID:21038881

  19. Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

    PubMed

    Feuerstein, Sophie E; Pulvermüller, Alexander; Hartmann, Rudolf; Granzin, Joachim; Stoldt, Matthias; Henklein, Peter; Ernst, Oliver P; Heck, Martin; Willbold, Dieter; Koenig, Bernd W

    2009-11-17

    Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed. PMID:19835414

  20. The Azolla, Anabaena azollae Relationship

    PubMed Central

    Peters, Gerald A.; Mayne, Berger C.

    1974-01-01

    Anaerobic (microaerophilic) acetylene reduction by Azolla caroliniana Willd. was dependent on light and saturated at approximately 450 foot candles. Maximum rates of acetylene reduction were 60 nmoles/mg chlorophyll minute. However, rates of 25 to 30 nmoles/mg chlorophyll minute were more common. The growth of Azolla for 35 days with nitrate or urea as a nitrogen source decreased the rate of acetylene reduction approximately 30% compared to controls grown on nitrogen. Prolonged growth on nitrate or urea (6-7 months) resulted in a 90% decrease in the rate of acetylene reduction. The inhibition of acetylene reduction by 3 (3,4-dichlorophenol) 1,1-dimethylurea (12 μM) was not pronounced until the Azolla became depleted of the reserves formed during photosynthesis. The interval required for this depletion was dependent upon pretreatment and varied from 2 to more than 12 hours. Oxygen evolution was inhibited 75% in 10 minutes by the same concentration of 3 (3,4-dichlorophenol) 1,1-dimethylurea. The addition of oxygen, 20% volume per volume, resulted in a 30 to 40% decrease in the rate of acetylene reduction and the onsetof 3(3,4-dichlorophenol) 1,1-dimethylurea inhibition was more rapid then under microaerophilic conditions. The aerobic dark reduction of acetylene was from 10 to 30% of the rate of aerobic reduction in the light. Acetylene reduction activity was absent in fronds freed ofthe symbiotic algae and present in isolated Anabaena azollae. This study shows that the alga is the agent of acetylene reduction and suggests that there is considerable transport of metabolites between the fern and the blue-green alga. PMID:16658797

  1. Chapter 64: Targeting the proteostasis network in rhodopsin retinitis pigmentosa

    PubMed Central

    Parfitt, David A.; Cheetham, Michael E.

    2016-01-01

    Mutations in rhodopsin are one of the most common causes of retinitis pigmentosa (RP). Misfolding of rhodopsin can result in disruptions in cellular protein homeostasis, or proteostasis. There is currently no available treatment for RP. In this review, we discuss the different approaches currently being investigated for treatment of rhodopsin RP, focusing on the potential of manipulation of the proteostasis network as a therapeutic approach to combat retinal degeneration. PMID:26427449

  2. On the disulphide bonds of rhodopsins.

    PubMed Central

    Al-Saleh, S; Gore, M; Akhtar, M

    1987-01-01

    Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones). PMID:3675552

  3. Crystallographic Study of the LUMI Intermediate of Squid Rhodopsin.

    PubMed

    Murakami, Midori; Kouyama, Tsutomu

    2015-01-01

    Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.

  4. Complete Genome Sequence of the Cyanobacterium Anabaena sp. 33047

    PubMed Central

    2016-01-01

    This study presents the complete nucleotide sequence of Anabaena sp. ATCC 33047 (Anabaena CA), a filamentous, nitrogen-fixing marine cyanobacterium, which under salt stress conditions accumulates sucrose internally. The elucidation of the genome will contribute to the understanding of cyanobacterial diversity. PMID:27516507

  5. Retinal Conformation Changes Rhodopsin's Dynamic Ensemble.

    PubMed

    Leioatts, Nicholas; Romo, Tod D; Danial, Shairy Azmy; Grossfield, Alan

    2015-08-01

    G protein-coupled receptors are vital membrane proteins that allosterically transduce biomolecular signals across the cell membrane. However, the process by which ligand binding induces protein conformation changes is not well understood biophysically. Rhodopsin, the mammalian dim-light receptor, is a unique test case for understanding these processes because of its switch-like activity; the ligand, retinal, is bound throughout the activation cycle, switching from inverse agonist to agonist after absorbing a photon. By contrast, the ligand-free opsin is outside the activation cycle and may behave differently. We find that retinal influences rhodopsin dynamics using an ensemble of all-atom molecular dynamics simulations that in aggregate contain 100 μs of sampling. Active retinal destabilizes the inactive state of the receptor, whereas the active ensemble was more structurally homogenous. By contrast, simulations of an active-like receptor without retinal present were much more heterogeneous than those containing retinal. These results suggest allosteric processes are more complicated than a ligand inducing protein conformational changes or simply capturing a shifted ensemble as outlined in classic models of allostery.

  6. Archaebacterial rhodopsin sequences: Implications for evolution

    NASA Technical Reports Server (NTRS)

    Lanyi, J. K.

    1991-01-01

    It was proposed over 10 years ago that the archaebacteria represent a separate kingdom which diverged very early from the eubacteria and eukaryotes. It follows that investigations of archaebacterial characteristics might reveal features of early evolution. So far, two genes, one for bacteriorhodopsin and another for halorhodopsin, both from Halobacterium halobium, have been sequenced. We cloned and sequenced the gene coding for the polypeptide of another one of these rhodopsins, a halorhodopsin in Natronobacterium pharaonis. Peptide sequencing of cyanogen bromide fragments, and immuno-reactions of the protein and synthetic peptides derived from the C-terminal gene sequence, confirmed that the open reading frame was the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences of this gene, as well as those of other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences were calculated. These indicate very considerable evolutionary distance between each pair of genes, even in the dame organism. In spite of this, three protein sequences show extensive similarities, indicating strong selective pressures.

  7. Free backbone carbonyls mediate rhodopsin activation.

    PubMed

    Kimata, Naoki; Pope, Andreyah; Sanchez-Reyes, Omar B; Eilers, Markus; Opefi, Chikwado A; Ziliox, Martine; Reeves, Philip J; Smith, Steven O

    2016-08-01

    Conserved prolines in the transmembrane helices of G-protein-coupled receptors (GPCRs) are often considered to function as hinges that divide the helix into two segments capable of independent motion. Depending on their potential to hydrogen-bond, the free C=O groups associated with these prolines can facilitate conformational flexibility, conformational switching or stabilization of the receptor structure. To address the role of conserved prolines in family A GPCRs through solid-state NMR spectroscopy, we focus on bovine rhodopsin, a GPCR in the visual receptor subfamily. The free backbone C=O groups on helices H5 and H7 stabilize the inactive rhodopsin structure through hydrogen-bonds to residues on adjacent helices. In response to light-induced isomerization of the retinal chromophore, hydrogen-bonding interactions involving these C=O groups are released, thus facilitating repacking of H5 and H7 onto the transmembrane core of the receptor. These results provide insights into the multiple structural and functional roles of prolines in membrane proteins. PMID:27376589

  8. Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

    SciTech Connect

    Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

    1986-03-05

    The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with ..gamma..-/sup 32/P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity.

  9. Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin.

    PubMed

    Smitienko, O A; Mozgovaya, M N; Shelaev, I V; Gostev, F E; Feldman, T B; Nadtochenko, V A; Sarkisov, O M; Ostrovsky, M A

    2010-01-01

    The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400-720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin(570) with vibrationally-excited all-trans-retinal and rhodopsin(498) in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm(-1) and 44, 142 cm(-1) for photorhodopsin(570) and for rhodopsin(498), respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.

  10. Projection structure of frog rhodopsin in two crystal forms.

    PubMed Central

    Schertler, G F; Hargrave, P A

    1995-01-01

    Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with a p2 lattice (a = 32 A, b = 83 A, gamma = 91 degrees) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger p22(1)2(1) lattices (a = 40 A, b = 146 A, gamma = 90 degrees). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-A resolution for the p22(1)2(1) form and to 6-A resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-A map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved. Images Fig. 1 Fig. 2 Fig. 6 PMID:8524807

  11. Microbial rhodopsins: wide distribution, rich diversity and great potential

    PubMed Central

    Kurihara, Marie; Sudo, Yuki

    2015-01-01

    One of the major topics in biophysics and physicobiology is to understand and utilize biological functions using various advanced techniques. Taking advantage of the photoreactivity of the seven-transmembrane rhodopsin protein family has been actively investigated by a variety of methods. Rhodopsins serve as models for membrane-embedded proteins, for photoactive proteins and as a fundamental tool for optogenetics, a new technology to control biological activity with light. In this review, we summarize progress of microbial rhodopsin research from the viewpoint of distribution, diversity and potential. PMID:27493861

  12. Atomic-force microscopy: Rhodopsin dimers in native disc membranes

    NASA Astrophysics Data System (ADS)

    Fotiadis, Dimitrios; Liang, Yan; Filipek, Slawomir; Saperstein, David A.; Engel, Andreas; Palczewski, Krzysztof

    2003-01-01

    In vertebrate retinal photoreceptors, the rod outer-segment disc membranes contain densely packed rhodopsin molecules for optimal light absorption and subsequent amplification by the visual signalling cascade, but how these photon receptors are organized with respect to each other is not known. Here we use infrared-laser atomic-force microscopy to reveal the native arrangement of rhodopsin, which forms paracrystalline arrays of dimers in mouse disc membranes. The visualization of these closely packed rhodopsin dimers in native membranes gives experimental support to earlier inferences about their supramolecular structure and provides insight into how light signalling is controlled.

  13. Crystal structure of constitutively active rhodopsin: How an agonist can activate its GPCR

    PubMed Central

    Standfuss, Jörg; Edwards, Patricia C.; D’Antona, Aaron; Fransen, Maikel; Xie, Guifu; Oprian, Daniel D.; Schertler, Gebhard F. X.

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters, and sensory stimuli. While some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists1. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state2,3. However, these structures are for the apoprotein or opsin form that does not contain the agonist all-trans retinal. We present here a crystal structure for the constitutively active rhodopsin mutant E113Q4-6 in complex with a peptide derived from the C-terminus of the G protein transducin (the GαCT peptide). Importantly, the protein appears to be in an active conformation, and retinal is retained in the binding pocket after photoactivation. Comparison with the structure of ground state rhodopsin7 suggests how translocation of the retinal β-ionone ring leads to a rotational tilt of transmembrane helix 6 (TM6), the critical conformational change upon activation8. A key feature of this conformational change is a reorganization of water mediated hydrogen-bonding networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. For the first time we thus show how an agonist ligand can activate its GPCR. PMID:21389983

  14. Vitamin A activates rhodopsin and sensitizes it to ultraviolet light.

    PubMed

    Miyazono, Sadaharu; Isayama, Tomoki; Delori, François C; Makino, Clint L

    2011-11-01

    The visual pigment, rhodopsin, consists of opsin protein with 11-cis retinal chromophore, covalently bound. Light activates rhodopsin by isomerizing the chromophore to the all-trans conformation. The activated rhodopsin sets in motion a biochemical cascade that evokes an electrical response by the photoreceptor. All-trans retinal is eventually released from the opsin and reduced to vitamin A. Rod and cone photoreceptors contain vast amounts of rhodopsin, so after exposure to bright light, the concentration of vitamin A can reach relatively high levels within their outer segments. Since a retinal analog, β-ionone, is capable of activating some types of visual pigments, we tested whether vitamin A might produce a similar effect. In single-cell recordings from isolated dark-adapted salamander green-sensitive rods, exogenously applied vitamin A decreased circulating current and flash sensitivity and accelerated flash response kinetics. These changes resembled those produced by exposure of rods to steady light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments and binding of vitamin A to rhodopsin was confirmed in in vitro assays. In addition, vitamin A improved the sensitivity of photoreceptors to ultraviolet (UV) light. Apparently, the energy of a UV photon absorbed by vitamin A transferred by a radiationless process to the 11-cis retinal chromophore of rhodopsin, which subsequently isomerized. Therefore, our results suggest that vitamin A binds to rhodopsin at an allosteric binding site distinct from the chromophore binding pocket for 11-cis retinal to activate the rhodopsin, and that it serves as a sensitizing chromophore for UV light. PMID:22192505

  15. Recent Advances in Engineering Microbial Rhodopsins for Optogenetics

    PubMed Central

    Arnold, Frances H.

    2015-01-01

    Protein engineering of microbial rhodopsins has been successful in generating variants with improved properties for applications in optogenetics. Members of this membrane protein family can act as both actuators and sensors of neuronal activity. Chimeragenesis, structure-guided mutagenesis, and directed evolution have proven effective strategies for tuning absorption wavelength, altering ion specificity and increasing fluorescence. These approaches facilitate the development of useful optogenetic tools and, in some cases, have yielded insights into rhodopsin structure-function relationships. PMID:26038227

  16. Recent advances in engineering microbial rhodopsins for optogenetics.

    PubMed

    McIsaac, R Scott; Bedbrook, Claire N; Arnold, Frances H

    2015-08-01

    Protein engineering of microbial rhodopsins has been successful in generating variants with improved properties for applications in optogenetics. Members of this membrane protein family can act as both actuators and sensors of neuronal activity. Chimeragenesis, structure-guided mutagenesis, and directed evolution have proven effective strategies for tuning absorption wavelength, altering ion specificity and increasing fluorescence. These approaches facilitate the development of useful optogenetic tools and, in some cases, have yielded insights into rhodopsin structure-function relationships.

  17. TRP and Rhodopsin Transport Depends on Dual XPORT ER Chaperones Encoded by an Operon.

    PubMed

    Chen, Zijing; Chen, Hsiang-Chin; Montell, Craig

    2015-10-20

    TRP channels and G protein-coupled receptors (GPCRs) play critical roles in sensory reception. However, the identities of the chaperones that assist GPCRs in translocating from the endoplasmic reticulum (ER) are limited, and TRP ER chaperones are virtually unknown. The one exception for TRPs is Drosophila XPORT. Here, we show that the xport locus is bicistronic and encodes unrelated transmembrane proteins, which enable the signaling proteins that initiate and culminate phototransduction, rhodopsin 1 (Rh1) and TRP, to traffic to the plasma membrane. XPORT-A and XPORT-B are ER proteins, and loss of either has a profound impact on TRP and Rh1 targeting to the light-sensing compartment of photoreceptor cells. XPORT-B complexed in vivo with the Drosophila homolog of the mammalian HSP70 protein, GRP78/BiP, which, in turn, associated with Rh1. Our work highlights a coordinated network of chaperones required for the biosynthesis of the TRP channel and rhodopsin in Drosophila photoreceptor cells. PMID:26456832

  18. The rhodopsin-like pigments of halobacteria - Light-energy and signal transducers in an archaebacterium

    NASA Technical Reports Server (NTRS)

    Stoeckenius, W.

    1985-01-01

    Three, small retinylidene proteins observed in halobacteria are described. The characteristics of bacteriorhodopsin (bR), which is synthesized during low O2 tension and intense illumination, and the role of bR in the cyclic photoreactions that translocate protons are examined. The detected light-driven chloride influx pigment, halorhodopsin (hR), is also capable of light-driven ion translocation; the hR transport reactions which are chloride dependent and involve isomerization are studied. The sensory photosystem of halobacteria and the receptor functions of the retinal pigment slow rhodopsin are discussed. The similarity of the choromphore structure and photoreactions, and the evolutionary relation between halobacteria and animal pigments are considered.

  19. Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype

    PubMed Central

    Han, Zongchao; Banworth, Marcellus J.; Makkia, Rasha; Conley, Shannon M.; Al-Ubaidi, Muayyad R.; Cooper, Mark J.; Naash, Muna I.

    2015-01-01

    Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.— Han, Z., Banworth, M. J., Makkia, R., Conley, S. M., Al-Ubaidi, M. R., Cooper, M. J., Naash, M. I. Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype. PMID:25713057

  20. Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts

    SciTech Connect

    Pande, C.; Pande, A.; Yue, K.T.; Callender, R.; Ebrey, T.G.; Tsuda, M.

    1987-08-11

    The authors report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 /sup 0/C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approx. 1660 cm/sup -1/ in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a proteonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with the previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.

  1. THE ACCESSIBILITY OF BOVINE RHODOPSIN IN PHOTORECEPTOR MEMBRANES

    PubMed Central

    Saari, John C.

    1974-01-01

    Bovine photoreceptor membranes have been treated with proteases to determine the accessibility of rhodopsin to these large, water soluble molecules. The polypeptides that remain associated with the membranous structure after proteolysis were detected by sodium dodecyl sulfate gel electrophoresis. Thermolysin and chymotrypsin degraded rhodopsin (apparent mol wt 35,000–36,000) to fragments of 29,000 and 23,000 apparent mol wt, respectively, without affecting the chromophoric absorption of the molecule or removing the region of the polypeptide carrying carbohydrate. The two fragments were isolated and their amino acid compositions were determined. They do not appear to be more hydrophobic than rhodopsin. Subtilisin, at low concentration and temperature, produced a fragment with the same molecular weight as that produced by thermolysin. At higher concentrations, subtilisin yields major fragments of mol wt 23,000 and 20,000 without affecting the chromophoric absorption. Two intermediate fragments of apparent mol wt 29,000 and 26,000 were detected during the course of this degradation. Carbohydrate is retained by all but the smallest fragment. Bleaching of the photoreceptor pigment did not appreciably alter any of the fragmentation patterns. Trypsin did not alter the molecular weight of rhodopsin under the conditions of this study. Approximately 35–45% of rhodopsin appears to be accessible to the aqueous environment and can be removed without affecting the chromophoric properties of the retinaldehyde-carrying region which remains bound to the membrane. PMID:4417532

  2. Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

    PubMed Central

    Ploier, Birgit; Caro, Lydia N.; Morizumi, Takefumi; Pandey, Kalpana; Pearring, Jillian N.; Goren, Michael A.; Finnemann, Silvia C.; Graumann, Johannes; Arshavsky, Vadim Y.; Dittman, Jeremy S.; Ernst, Oliver P.; Menon, Anant K.

    2016-01-01

    Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5. PMID:27694816

  3. Atomistic insights into rhodopsin activation from a dynamic model.

    PubMed

    Tikhonova, Irina G; Best, Robert B; Engel, Stanislav; Gershengorn, Marvin C; Hummer, Gerhard; Costanzi, Stefano

    2008-08-01

    Rhodopsin, the light sensitive receptor responsible for blue-green vision, serves as a prototypical G protein-coupled receptor (GPCR). Upon light absorption, it undergoes a series of conformational changes that lead to the active form, metarhodopsin II (META II), initiating a signaling cascade through binding to the G protein transducin (G(t)). Here, we first develop a structural model of META II by applying experimental distance restraints to the structure of lumi-rhodopsin (LUMI), an earlier intermediate. The restraints are imposed by using a combination of biased molecular dynamics simulations and perturbations to an elastic network model. We characterize the motions of the transmembrane helices in the LUMI-to-META II transition and the rearrangement of interhelical hydrogen bonds. We then simulate rhodopsin activation in a dynamic model to study the path leading from LUMI to our META II model for wild-type rhodopsin and a series of mutants. The simulations show a strong correlation between the transition dynamics and the pharmacological phenotypes of the mutants. These results help identify the molecular mechanisms of activation in both wild type and mutant rhodopsin. While static models can provide insights into the mechanisms of ligand recognition and predict ligand affinity, a dynamic model of activation could be applicable to study the pharmacology of other GPCRs and their ligands, offering a key to predictions of basal activity and ligand efficacy.

  4. Distribution of rhodopsin and retinochrome in the squid retina

    PubMed Central

    1976-01-01

    The cephalopod retina contains two kinds of photopigments, rhodopsin and retinochrome. For many years retinochrome has been thought to be localized in the inner segments of the visual cells, whereas rhodopsin is in the outer segments. However, it is now clear that retinochrome can be extracted also from fragments of outer segments. In the dark- adapted retina of Loligo pealei retinochrome is distributed half-and- half in the inner and outer segments. Todarodes pacificus contains much more retinochrome than Loligo, and it is more abundant in the outer than in the inner segments. The outer segments of Loligo contain retinochrome and metarhodopsin in addition to rhodopsin, whether squids are kept in the dark or in the light. But there is extremely little metarhodopsin (about 3% of rhodopsin) even in light-adapted eyes. The inner segments contain only retinochrome, and much less in the light than in the dark. On the other hand, retinochrome in the outer segments increases markedly during light adaptation. These facts suggest the possibility that some retinochrome moves forward from the inner to the outer segments during light adaptation and there reacts with metarhodopsin to promote regeneration of rhodopsin. PMID:6620

  5. Microbial rhodopsins on leaf surfaces of terrestrial plants.

    PubMed

    Atamna-Ismaeel, Nof; Finkel, Omri M; Glaser, Fabian; Sharon, Itai; Schneider, Ron; Post, Anton F; Spudich, John L; von Mering, Christian; Vorholt, Julia A; Iluz, David; Béjà, Oded; Belkin, Shimshon

    2012-01-01

    The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10(26) microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.

  6. Photometer for measuring intensity and rhodopsin distributions in intact eyes

    NASA Astrophysics Data System (ADS)

    Williams, Theodore P.; Webbers, Jacob P. P.

    1995-09-01

    We describe a photometer that measures light transmitted through excised eyes. The instrument, an ocular transmission photometer, employs sensitive single photon-counting techniques, and its usefulness has been tested by the study of the absorbance of rhodopsin in retinal rod cells in situ. We find that absorbances of rat rods agree well with those predicted by microspectrophotometry without making corrections for cellular mosaics. Additional tests of the ocular transmission photometer show that (a) the instrument is sensitive to subtle differences in rhodopsin absorbance, known to exist in specific locations in the rat retina, and (b) using the rate of rhodopsin bleaching as the measure of intensity, we can determine the intensity distribution at several locations across the rat retina.

  7. Rhodopsin photoactivation dynamics revealed by quasi-elastic neutron scattering

    DOE PAGESBeta

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchithranga M.d.c.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael F.; Chu, Xiang -Qiang

    2015-01-27

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision under dim light conditions. During rhodopsin photoactivation, the chromophore retinal undergoes cis-trans isomerization, and subsequently dissociates from the protein yielding the opsin apoprotein [1]. What are the changes in protein dynamics that occur during the rhodopsin photoactivation process? Here, we studied the microscopic dynamics of the dark-state rhodopsin and the ligand-free opsin using quasi-elastic neutron scattering (QENS). The QENS technique tracks the individual hydrogen atom motions in the protein molecules, because the neutron scattering cross-section of hydrogen is much higher than other atoms [2-4]. We used protein (rhodopsin/opsin) samples with CHAPSmore » detergent hydrated with heavy water. The solvent signal is suppressed due to the heavy water, so that only the signals from proteins and detergents are detected. The activation of proteins is confirmed at low temperatures up to 300 K by the mean-square displacement (MSD) analysis. Our QENS experiments conducted at temperatures ranging from 220 K to 300 K clearly indicate that the protein dynamic behavior increases with temperature. The relaxation time for the ligand-bound protein rhodopsin was longer compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which forms a band around the protein molecule in the micelle. Unlike the protein, the CHAPS detergent manifests localized motions that are the same as in the bulk empty micelles. Furthermore QENS provides unique understanding of the key dynamics involved in the activation of the GPCR involved in the visual process.« less

  8. Rhodopsin photoactivation dynamics revealed by quasi-elastic neutron scattering

    SciTech Connect

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchithranga M.d.c.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael F.; Chu, Xiang -Qiang

    2015-01-27

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision under dim light conditions. During rhodopsin photoactivation, the chromophore retinal undergoes cis-trans isomerization, and subsequently dissociates from the protein yielding the opsin apoprotein [1]. What are the changes in protein dynamics that occur during the rhodopsin photoactivation process? Here, we studied the microscopic dynamics of the dark-state rhodopsin and the ligand-free opsin using quasi-elastic neutron scattering (QENS). The QENS technique tracks the individual hydrogen atom motions in the protein molecules, because the neutron scattering cross-section of hydrogen is much higher than other atoms [2-4]. We used protein (rhodopsin/opsin) samples with CHAPS detergent hydrated with heavy water. The solvent signal is suppressed due to the heavy water, so that only the signals from proteins and detergents are detected. The activation of proteins is confirmed at low temperatures up to 300 K by the mean-square displacement (MSD) analysis. Our QENS experiments conducted at temperatures ranging from 220 K to 300 K clearly indicate that the protein dynamic behavior increases with temperature. The relaxation time for the ligand-bound protein rhodopsin was longer compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which forms a band around the protein molecule in the micelle. Unlike the protein, the CHAPS detergent manifests localized motions that are the same as in the bulk empty micelles. Furthermore QENS provides unique understanding of the key dynamics involved in the activation of the GPCR involved in the visual process.

  9. The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

    PubMed Central

    Corley, Scott C.; Sprangers, Peter; Albert, Arlene D.

    2011-01-01

    Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (Tm) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the Tm as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (Eact) was calculated from the scan rate dependence of the Tm and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The Eact as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the Eact to rhodopsin denaturation. PMID:21689528

  10. Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration

    PubMed Central

    Chiang, Wei-Chieh; Kroeger, Heike; Sakami, Sanae; Messah, Carissa; Yasumura, Douglas; Matthes, Michael T.; Coppinger, Judith A.; Palczewski, Krzysztof; LaVail, Matthew M.; Lin, Jonathan H.

    2014-01-01

    Rhodopsin is a G protein-coupled receptor essential for vision and rod photoreceptor viability. Disease-associated rhodopsin mutations, such as P23H rhodopsin, cause rhodopsin protein misfolding and trigger endoplasmic reticulum (ER) stress, activating the Unfolded Protein Response (UPR). The pathophysiologic effects of ER stress and UPR activation on photoreceptors are unclear. Here, by examining a P23H rhodopsin knock-in mouse, we found that the UPR IRE1 signaling pathway is strongly activated in misfolded rhodopsin-expressing photoreceptors. IRE1 significantly upregulated ER-associated protein degradation (ERAD), triggering pronounced P23H rhodopsin degradation. Rhodopsin protein loss occurred as soon as photoreceptors developed, preceding photoreceptor cell death. By contrast, IRE1 activation did not affect JNK signaling or rhodopsin mRNA levels. Interestingly, pro-apoptotic signaling from the PERK UPR pathway was also not induced. Our findings reveal that an early and significant pathophysiologic effect of ER stress in photoreceptors is the highly efficient elimination of misfolded rhodopsin protein. We propose that early disruption of rhodopsin protein homeostasis in photoreceptors could contribute to retinal degeneration. PMID:25270370

  11. Depth-resolved rhodopsin molecular contrast imaging for functional assessment of photoreceptors

    PubMed Central

    Liu, Tan; Wen, Rong; Lam, Byron L.; Puliafito, Carmen A.; Jiao, Shuliang

    2015-01-01

    Rhodopsin, the light-sensing molecule in the outer segments of rod photoreceptors, is responsible for converting light into neuronal signals in a process known as phototransduction. Rhodopsin is thus a functional biomarker for rod photoreceptors. Here we report a novel technology based on visible-light optical coherence tomography (VIS-OCT) for in vivo molecular imaging of rhodopsin. The depth resolution of OCT allows the visualization of the location where the change of optical absorption occurs and provides a potentially accurate assessment of rhodopsin content by segmentation of the image at the location. Rhodopsin OCT can be used to quantitatively image rhodopsin distribution and thus assess the distribution of functional rod photoreceptors in the retina. Rhodopsin OCT can bring significant impact into ophthalmic clinics by providing a tool for the diagnosis and severity assessment of a variety of retinal conditions. PMID:26358529

  12. Is rhodopsin isomerization correlated to astronauts' phosphene perceptions in space?

    NASA Astrophysics Data System (ADS)

    Narici, L.; Altea-Biophys Team

    Anomalous Phosphene Perception APP phoenomenon may just be a first example of how microgravity and particle radiation may modify the normal behaviour of the Central Nervous System CNS and also is an evidence that space environment may indeed influence the correct functioning of the visual system The ALTEA program is going to provide i an assesment of the CNS functional hazard due to microgravity and particle radiation during long space human permanence ii a definition for the needed shielding optimized for reducing these risks and iii a survey of ISS radiation environment aimed at the validation of spacecrafts computer models As known Rhodopsin is at the start of the photo-transduction cascade and its involvement in the phosphene perception would suggest a possible physiological pathway The bleaching of few molecules in the retina is sufficient to start the process of vision A very preliminary measurements on rhodopsin irradiation has been conducted in April 2003 Irradiation of 17 vials containing a solution of suine rhodopsine has been performed with 12 C ions at 200 MeV n - total dose has been varied from 10 9 to 10 13 ions over each vial New and more complete data from most recent measurements are now available Preparation and purification of bovine rhodopsin and regenerations of bleached molecules was carried out using reproducible procedures The samples was irradiated with controlled 12 C ion beams and with different amount of light radiation in order 1 to understand if the molecules have been

  13. H+ -pumping rhodopsin from the marine alga Acetabularia.

    PubMed

    Tsunoda, Satoshi P; Ewers, David; Gazzarrini, Sabrina; Moroni, Anna; Gradmann, Dietrich; Hegemann, Peter

    2006-08-15

    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.

  14. Comparative FTIR study of a new fungal rhodopsin.

    PubMed

    Ito, Hiroyasu; Sumii, Masayo; Kawanabe, Akira; Fan, Ying; Furutani, Yuji; Brown, Leonid S; Kandori, Hideki

    2012-10-01

    Bacteriorhodopsin (BR) is a light-driven proton pump of halophilic Archaea , and BR-like proton-pumping rhodopsins have been discovered in Bacteria and Eucarya as well. Leptosphaeria rhodopsin (LR) and Phaeosphaeria Rhodopsin 2 (PhaeoRD2) are both fungal rhodopsins in such a functional class, even though they belong to different branches of the phylogenetic tree. In this study, we compared light-induced structural changes in the K, L, and M photointermediates for PhaeoRD2, LR, and BR using low-temperature Fourier transform infrared (FTIR) spectroscopy. We observed a strongly hydrogen-bonded water molecule in PhaeoRD2 (water O-D stretch in D(2)O at 2258 cm(-1)) as well as in LR and BR. This observation provided additional experimental evidence to the concept that strongly hydrogen-bonded water molecule is the functional determinant of light-driven proton pumping. The difference FTIR spectra for all the K, L, and M states are surprisingly similar between PhaeoRD2 and LR, but not for BR. PhaeoRD2 is more homologous to LR than to BR, but the difference is small. The amino acid identities between PhaeoRD2 and LR, and between PhaeoRD2 and BR are 34.5% and 30.2%, respectively. In addition, the amino acids uniquely identical for the fungal rhodopsins are located rather far from the retinal chromophore. In fact, the amino acid identities within 4 Å from retinal are the same among PhaeoRD2, LR, and BR. For more than 100 amino acids located within 12 Å from retinal, the identities are 48.7% between PhaeoRD2 and LR, 46.0% between PhaeoRD2 and BR, and 47.8% between LR and BR. These results suggest that protein core structures are equally different among the three rhodopsins. Thus, the identical FTIR spectra between PhaeoRD2 and LR (but not BR), even for the K state, indicate that fungal rhodopsins possess some common structural motif and dynamics not obvious from the amino acid sequences. PMID:22973982

  15. Resonance raman spectroscopy of an ultraviolet-sensitive insect rhodopsin

    SciTech Connect

    Pande, C.; Deng, H.; Rath, P.; Callender, R.H.; Schwemer, J.

    1987-11-17

    The authors present the first visual pigment resonance Raman spectra from the UV-sensitive eyes of an insect, Ascalaphus macaronius (owlfly). This pigment contains 11-cis-retinal as the chromophore. Raman data have been obtained for the acid metarhodopsin at 10/sup 0/C in both H/sub 2/O and D/sub 2/O. The C=N stretching mode at 1660 cm/sup -1/ in H/sub 2/O shifts to 1631 cm/sup -1/ upon deuteriation of the sample, clearly showing a protonated Schiff base linkage between the chromophore and the protein. The structure-sensitive fingerprint region shows similarities to the all-trans-protonated Schiff base of model retinal chromophores, as well as to the octopus acid metarhodopsin and bovine metarhodopsin I. Although spectra measured at -100/sup 0/C with 406.7-nm excitation, to enhance scattering from rhodopsin (lambda/sub max/ 345 nm), contain a significant contribution from a small amount of contaminants (cytochrome(s) and/or accessory pigment) in the sample, the C=N stretch at 1664 cm/sup -1/ suggests a protonated Schiff base linkage between the chromophore and the protein in rhodopsin as well. For comparison, this mode also appears at approx. 1660 cm/sup -1/ in both the vertebrate (bovine) and the invertebrate (octopus) rhodopsins. These data are particularly interesting since the absorption maximum of 345 nm for rhodopsin might be expected to originate from an unprotonated Schiff base linkage. That the Schiff base linkage in the owlfly rhodopsin, like in bovine and in octopus, is protonated suggests that a charged chromophore is essential to visual transduction.

  16. Molecular, enzymatic and functional properties of rhodopsin kinase from rat pineal gland.

    PubMed

    Palczewski, K; Carruth, M E; Adamus, G; McDowell, J H; Hargrave, P A

    1990-01-01

    Rhodopsin kinase activity from rat pineal gland and from rat retina are indistinguishable, based upon determination of a variety of enzymatic and molecular properties. Both activities are independent of calcium, cyclic nucleotides, and calmodulin. Both are activated by spermine and inhibited by adenosine and some rhodopsin kinase specific adenosine derivatives such as sangivamycin. The Km's for rhodopsin, ATP, and GTP are indistinguishable for the protein kinase in extracts from the retina and from the pineal gland. The apparent molecular weight of the kinase from both sources, as determined by gel filtration and autoradiography of the 32P-labeled autophosphorylated kinase, is about 70 kDa. Rhodopsin kinase activity from pineal binds in a light-dependent manner to rhodopsin in rod outer segments as does the enzyme from retina. Monoclonal antibodies against bovine rhodopsin were used in an immunochemical study that identified a rhodopsin-immunoreactive protein in rat pineal gland and retina. Using an ELISA we demonstrated the presence of a rhodopsin-immunoreactive protein in rat pineal gland equivalent to 0.075 pmol rhodopsin per gland. Frog pineal organ (Rana catesbiana) contains 33 times more of this rhodopsin-like protein than does rat pineal gland. PMID:2402884

  17. Rhodopsin molecular contrast imaging by optical coherence tomography for functional assessment of photoreceptors (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nafra, Zahra; Liu, Tan; Jiao, Shuliang

    2016-03-01

    Rhodopsin, the light-sensing molecule in the outer segments of rod photoreceptors, is responsible for converting light into neuronal signals in a process known as phototransduction. Rhodopsin is thus a functional biomarker for rod photoreceptors. We developed a novel technology based on visible-light optical coherence tomography (VIS-OCT) for in vivo molecular imaging of rhodopsin. The depth resolution of OCT allows the visualization of the location where the change of optical absorption occurs and provides a potentially accurate assessment of rhodopsin content by segmentation of the image at the location. A broadband supercontinuum laser, whose filtered output was centered at 520 nm, was used as the illuminating light source. To test the capabilities of the system on rhodopsin mapping we imaged the retina of albino rats. The rats were dark adapted before imaging. An integrated near infrared OCT was used to guide the alignment in dark. VIS-OCT three-dimensional images were then acquired under dark- and light- adapted states sequentially. Rhodopsin distribution was calculated from the differential image. The rhodopsin distributions can be displayed in both en face view and depth-resolved cross-sectional image. Rhodopsin OCT can be used to quantitatively image rhodopsin distribution and thus assess the distribution of functional rod photoreceptors in the retina. Rhodopsin OCT can bring significant impact into ophthalmic clinics by providing a tool for the diagnosis and severity assessment of a variety of retinal conditions.

  18. Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology.

    PubMed

    Pinhassi, Jarone; DeLong, Edward F; Béjà, Oded; González, José M; Pedrós-Alió, Carlos

    2016-12-01

    The recognition of a new family of rhodopsins in marine planktonic bacteria, proton-pumping proteorhodopsin, expanded the known phylogenetic range, environmental distribution, and sequence diversity of retinylidene photoproteins. At the time of this discovery, microbial ion-pumping rhodopsins were known solely in haloarchaea inhabiting extreme hypersaline environments. Shortly thereafter, proteorhodopsins and other light-activated energy-generating rhodopsins were recognized to be widespread among marine bacteria. The ubiquity of marine rhodopsin photosystems now challenges prior understanding of the nature and contributions of "heterotrophic" bacteria to biogeochemical carbon cycling and energy fluxes. Subsequent investigations have focused on the biophysics and biochemistry of these novel microbial rhodopsins, their distribution across the tree of life, evolutionary trajectories, and functional expression in nature. Later discoveries included the identification of proteorhodopsin genes in all three domains of life, the spectral tuning of rhodopsin variants to wavelengths prevailing in the sea, variable light-activated ion-pumping specificities among bacterial rhodopsin variants, and the widespread lateral gene transfer of biosynthetic genes for bacterial rhodopsins and their associated photopigments. Heterologous expression experiments with marine rhodopsin genes (and associated retinal chromophore genes) provided early evidence that light energy harvested by rhodopsins could be harnessed to provide biochemical energy. Importantly, some studies with native marine bacteria show that rhodopsin-containing bacteria use light to enhance growth or promote survival during starvation. We infer from the distribution of rhodopsin genes in diverse genomic contexts that different marine bacteria probably use rhodopsins to support light-dependent fitness strategies somewhere between these two extremes.

  19. Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology.

    PubMed

    Pinhassi, Jarone; DeLong, Edward F; Béjà, Oded; González, José M; Pedrós-Alió, Carlos

    2016-12-01

    The recognition of a new family of rhodopsins in marine planktonic bacteria, proton-pumping proteorhodopsin, expanded the known phylogenetic range, environmental distribution, and sequence diversity of retinylidene photoproteins. At the time of this discovery, microbial ion-pumping rhodopsins were known solely in haloarchaea inhabiting extreme hypersaline environments. Shortly thereafter, proteorhodopsins and other light-activated energy-generating rhodopsins were recognized to be widespread among marine bacteria. The ubiquity of marine rhodopsin photosystems now challenges prior understanding of the nature and contributions of "heterotrophic" bacteria to biogeochemical carbon cycling and energy fluxes. Subsequent investigations have focused on the biophysics and biochemistry of these novel microbial rhodopsins, their distribution across the tree of life, evolutionary trajectories, and functional expression in nature. Later discoveries included the identification of proteorhodopsin genes in all three domains of life, the spectral tuning of rhodopsin variants to wavelengths prevailing in the sea, variable light-activated ion-pumping specificities among bacterial rhodopsin variants, and the widespread lateral gene transfer of biosynthetic genes for bacterial rhodopsins and their associated photopigments. Heterologous expression experiments with marine rhodopsin genes (and associated retinal chromophore genes) provided early evidence that light energy harvested by rhodopsins could be harnessed to provide biochemical energy. Importantly, some studies with native marine bacteria show that rhodopsin-containing bacteria use light to enhance growth or promote survival during starvation. We infer from the distribution of rhodopsin genes in diverse genomic contexts that different marine bacteria probably use rhodopsins to support light-dependent fitness strategies somewhere between these two extremes. PMID:27630250

  20. In silico study of the human rhodopsin and meta rhodopsin II/S-arrestin complexes: impact of single point mutations related to retina degenerative diseases.

    PubMed

    Mokarzel-Falcón, Leonardo; Padrón-García, Juan Alexander; Carrasco-Velar, Ramón; Berry, Colin; Montero-Cabrera, Luis A

    2008-03-01

    We propose two models of the human S-arrestin/rhodopsin complex in the inactive dark adapted rhodopsin and meta rhodopsin II form, obtained by homology modeling and knowledge based docking. First, a homology model for the human S-arrestin was built and validated by molecular dynamics, showing an average root mean square deviation difference from the pattern behavior of 0.76 A. Then, combining the human S-arrestin model and the modeled structure of the two human rhodopsin forms, we propose two models of interaction for the human S-arrestin/rhodopsin complex. The models involve two S-arrestin regions related to the N domain (residues 68-78; 170-182) and a third constituent of the C domain (248-253), with the rhodopsin C terminus (330-348). Of the 22 single point mutations related to retinitis pigmentosa and congenital night blindness located in the cytoplasmatic portion of rhodopsin or in S-arrestin, our models locate 16 in the interaction region and relate two others to possible dimer formation. Our calculations also predict that the light activated complex is more stable than the dark adapted rhodopsin and, therefore, of higher affinity to S-arrestin.

  1. Sensory mononeuropathies.

    PubMed

    Massey, E W

    1998-01-01

    The clinical neurologist frequently encounters patients with a variety of focal sensory symptoms and signs. This article reviews the clinical features, etiologies, laboratory findings, and management of the common sensory mononeuropathies including meralgia paresthetica, cheiralgia paresthetica, notalgia paresthetica, gonyalgia paresthetica, digitalgia paresthetica, intercostal neuropathy, and mental neuropathy. PMID:9608615

  2. THE INTERPLAY OF LIGHT AND HEAT IN BLEACHING RHODOPSIN

    PubMed Central

    St. George, Robert C. C.

    1952-01-01

    Rhodopsin, the pigment of the retinal rods, can be bleached either by light or by high temperature. Earlier work had shown that when white light is used the bleaching rate does not depend on temperature, and so must be independent of the internal energy of the molecule. On the other hand thermal bleaching in the dark has a high temperature dependence from which one can calculate that the reaction has an apparent activation energy of 44 kg. cal. per mole. It has now been shown that the bleaching rate of rhodopsin becomes temperature-dependent in red light, indicating that light and heat cooperate in activating the molecule. Apparently thermal energy is needed for bleaching at long wave lengths where the quanta are not sufficiently energy-rich to bring about bleaching by themselves. The temperature dependence appears at 590 mµ. This is the longest wave length at which bleaching by light proceeds without thermal activation, and corresponds to a quantum energy of 48.5 kg. cal. per mole. This value of the minimum energy to bleach rhodopsin by light alone is in agreement with the activation energy of thermal bleaching in the dark. At wave lengths between 590 and 750 mµ, the longest wave length at which the bleaching rate was fast enough to study, the sum of the quantum energy and of the activation energy calculated from the temperature coefficients remains between 44 and 48.5 kg. cal. This result shows that in red light the energy deficit of the quanta can be made up by a contribution of thermal energy from the internal degrees of freedom of the rhodopsin molecule. The absorption spectrum of rhodopsin, which is not markedly temperature-dependent at shorter wave lengths, also becomes temperature-dependent in red light of wave lengths longer than about 570 to 590 mµ. The temperature dependence of the bleaching rate is at least partly accounted for by the temperature coefficient of absorption. There is some evidence that the temperature coefficient of bleaching is

  3. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

    PubMed

    Rakshit, Tatini; Senapati, Subhadip; Sinha, Satyabrata; Whited, A M; Park, Paul S-H

    2015-01-01

    Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates. PMID:26492040

  4. Rhodopsin Kinase Activity in the Mammalian Pineal Gland and Other Tissues

    NASA Astrophysics Data System (ADS)

    Somers, Robert L.; Klein, David C.

    1984-10-01

    Rhodopsin kinase, an enzyme involved in photochemical transduction in the retina, has been found in the mammalian pineal gland in amounts equal to those in the retina; other tissues had 7 percent of this amount, or less. This finding suggests that, in mammals, rhodopsin kinase functions in the pineal gland and other tissues to phosphorylate rhodopsin-like integral membrane receptors and is thereby involved in signal transduction.

  5. A Photoisomerizing Rhodopsin Mimic Observed at Atomic Resolution.

    PubMed

    Nosrati, Meisam; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

    2016-07-20

    The members of the rhodopsin family of proteins are involved in many essential light-dependent processes in biology. Specific photoisomerization of the protein-bound retinylidene PSB at a specified wavelength range of light is at the heart of all of these systems. Nonetheless, it has been difficult to reproduce in an engineered system. We have developed rhodopsin mimics, using intracellular lipid binding protein family members as scaffolds, to study fundamental aspects of protein/chromophore interactions. Herein we describe a system that specifically isomerizes the retinylidene protonated Schiff base both thermally and photochemically. This isomerization has been characterized at atomic resolution by quantitatively interconverting the isomers in the crystal both thermally and photochemically. This event is accompanied by a large pKa change of the imine similar to the pKa changes observed in bacteriorhodopsin and visual opsins during isomerization. PMID:27310917

  6. Rhodopsin Photoactivation Dynamics Revealed by Quasi-Elastic Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Bhowmik, Debsindhu; Shrestha, Utsab; Perera, Suchhithranga M. C. D.; Chawla, Udeep; Mamontov, Eugene; Brown, Michael; Chu, Xiang-Qiang

    2015-03-01

    Rhodopsin is a G-protein-coupled receptor (GPCR) responsible for vision. During photoactivation, the chromophore retinal dissociates from protein yielding the opsin apoprotein. What are the changes in protein dynamics that occur during the photoactivation process? Here, we studied the microscopic dynamics of dark-state rhodopsin and the ligand-free opsin using quasielastic neutron scattering (QENS). The QENS technique tracks individual hydrogen atom motion because of the much higher neutron scattering cross-section of hydrogen than other atoms. We used protein with CHAPS detergent hydrated with heavy water. The activation of proteins is confirmed at low temperatures up to 300 K by mean-square displacement (MSD) analysis. The QENS experiments at temperatures ranging from 220 K to 300 K clearly indicate an increase in protein dynamic behavior with temperature. The relaxation time for the ligand-bound protein rhodopsin is faster compared to opsin, which can be correlated with the photoactivation. Moreover, the protein dynamics are orders of magnitude slower than the accompanying CHAPS detergent, which unlike protein, manifests localized motions.

  7. The effects of octanol on the late photointermediates of rhodopsin.

    PubMed

    Mah, T L; Szundi, I; Lewis, J W; Jäger, S; Kliger, D S

    1998-11-01

    Membrane suspensions of unperturbed rhodopsin and rhodopsin perturbed with 2.5 mM octanol were photolyzed with 477 nm laser pulses at 20 degrees C and 35 degrees C. Changes in absorbance were monitored at times ranging from 1 microsecond to 80 ms after excitation. The data were analyzed using singular value decomposition, global exponential fitting and kinetic modeling. A recently proposed model involving the photointermediate Meta-I380 (T. E. Thorgeirsson, J. W. Lewis, S. E. Wallace-Williams, and D. S. Kliger, Biochemistry 32, 13861-13872, 1993) fits data for samples with and without octanol. Comparison of the microscopic rates shows this alcohol accelerates the formation of Meta-II via Meta-I380. Activation and equilibrium thermodynamic parameters obtained from Arrhenius plots suggest that octanol reduces the entropy increase in forming both Meta-I380 and Meta-II. It also lowers the enthalpy of Meta-I380 relative to Lumi and of Meta-II relative to Meta-I480. To help determine whether octanol affects the protein directly or indirectly through the lipid bilayer, similar experiments were conducted using rhodopsin solubilized in 0.13% dodecyl maltoside with and without octanol. Spectral shifts in the presence of octanol suggest that a direct protein interaction exists in addition to previously reported effects dependent on membrane free volume.

  8. Local peptide movement in the photoreaction intermediate of rhodopsin

    PubMed Central

    Nakamichi, Hitoshi; Okada, Tetsuji

    2006-01-01

    Photoactivation of the visual rhodopsin, a prototypical G protein-coupled receptor (GPCR), involves efficient conversion of the intrinsic inverse-agonist 11-cis-retinal to the all-trans agonist. This event leads to the rearrangement of the heptahelical transmembrane bundle, which is thought to be shared by hundreds of GPCRs. To examine this activation mechanism, we determined the x-ray crystallographic model of the photoreaction intermediate of rhodopsin, lumirhodopsin, which represents the conformational state having the nearly complete all-trans agonist form of the retinal. A difference electron density map clearly indicated that the distorted all-trans-retinal in the precedent intermediate bathorhodopsin relaxes by dislocation of the β-ionone ring in lumirhodopsin, along with significant peptide displacement in the middle of helix III, including approximately two helical turns. This local movement results in the breaking of the electrostatic interhelical restraints mediated by many of the conserved residues among rhodopsin-like GPCRs, with consequent acquisition of full activity. PMID:16908857

  9. Local peptide movement in the photoreaction intermediate of rhodopsin.

    PubMed

    Nakamichi, Hitoshi; Okada, Tetsuji

    2006-08-22

    Photoactivation of the visual rhodopsin, a prototypical G protein-coupled receptor (GPCR), involves efficient conversion of the intrinsic inverse-agonist 11-cis-retinal to the all-trans agonist. This event leads to the rearrangement of the heptahelical transmembrane bundle, which is thought to be shared by hundreds of GPCRs. To examine this activation mechanism, we determined the x-ray crystallographic model of the photoreaction intermediate of rhodopsin, lumirhodopsin, which represents the conformational state having the nearly complete all-trans agonist form of the retinal. A difference electron density map clearly indicated that the distorted all-trans-retinal in the precedent intermediate bathorhodopsin relaxes by dislocation of the beta-ionone ring in lumirhodopsin, along with significant peptide displacement in the middle of helix III, including approximately two helical turns. This local movement results in the breaking of the electrostatic interhelical restraints mediated by many of the conserved residues among rhodopsin-like GPCRs, with consequent acquisition of full activity.

  10. Functional characterization of flavobacteria rhodopsins reveals a unique class of light-driven chloride pump in bacteria.

    PubMed

    Yoshizawa, Susumu; Kumagai, Yohei; Kim, Hana; Ogura, Yoshitoshi; Hayashi, Tetsuya; Iwasaki, Wataru; DeLong, Edward F; Kogure, Kazuhiro

    2014-05-01

    Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton- or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08(T) encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H(+) and Na(+) pumps, respectively. Functional analyses further revealed that the light-activated NM-R3 rhodopsin pumps Cl(-) ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl(-)-pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins.

  11. Rhodopsin topography and rod-mediated function in cats with the retinal degeneration of taurine deficiency.

    PubMed

    Jacobson, S G; Kemp, C M; Borruat, F X; Chaitin, M H; Faulkner, D J

    1987-10-01

    Cats on a taurine-deficient diet were studied with imaging fundus reflectometry and full-field electroretinography. The pattern of rhodopsin loss and the natural history of the disease were determined from maps of the rhodopsin distribution in the central and nasal retina of cats with different degrees of severity of the retinopathy. Rhodopsin loss is first detectable in a focal region of the central retina. Subsequently, there are decreases in rhodopsin in the paracentral and nasal midperipheral retina. The horizontal streak of high rhodopsin levels is preferentially reduced in this retinopathy. The b-wave amplitude of the rod-dominated ERG is markedly reduced in cats with only mildly decreased levels of rhodopsin in the peripheral retina. In an affected cat with moderate rhodopsin loss in the central retina but minimal loss nasally, a light-microscopic study of the retina showed that there was disorganization and shortening of rod outer segments and loss of rod photoreceptor cells in the areas of reduced rhodopsin levels.

  12. Complete genome sequence of Anabaena variabilis ATCC 29413

    SciTech Connect

    Thiel, Teresa; Pratte, Brenda S.; Zhong, Jinshun; Goodwin, Lynne A.; Copeland, A; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2013-01-01

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Ana-baena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40 C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence.

  13. Complete genome sequence of Anabaena variabilis ATCC 29413.

    PubMed

    Thiel, Teresa; Pratte, Brenda S; Zhong, Jinshun; Goodwin, Lynne; Copeland, Alex; Lucas, Susan; Han, Cliff; Pitluck, Sam; Land, Miriam L; Kyrpides, Nikos C; Woyke, Tanja

    2014-06-15

    Anabaena variabilis ATCC 29413 is a filamentous, heterocyst-forming cyanobacterium that has served as a model organism, with an extensive literature extending over 40 years. The strain has three distinct nitrogenases that function under different environmental conditions and is capable of photoautotrophic growth in the light and true heterotrophic growth in the dark using fructose as both carbon and energy source. While this strain was first isolated in 1964 in Mississippi and named Anabaena flos-aquae MSU A-37, it clusters phylogenetically with cyanobacteria of the genus Nostoc. The strain is a moderate thermophile, growing well at approximately 40(°) C. Here we provide some additional characteristics of the strain, and an analysis of the complete genome sequence. PMID:25197444

  14. Monogalactosyldiacylglycerol biosynthesis by direct acyl transfer in Anabaena variabilis. [Anabaena variabilis

    SciTech Connect

    Chen, H.H.; Wickrema, A.; Jaworski, J.

    1987-05-01

    The authors previously reported the direct acylation of monogalactosyldiacylglycerol (MGDG) by an enzyme in the membranes of the cyanobacterium (Anabaena variabilis. The enzyme requires acyl-acyl carrier protein (acyl-ACP) as substrate, but had no other additional cofactor requirements. Palmitoyl-, stearoyl- and oleoyl-ACP were all effective substrates. The A. variabilis membranes also had a hydrolase activity which metabolized the acyl-ACP to yield free fatty acid and ACP. Possible mechanisms for the acylation reaction include either acyl exchange with existing MGDG or direct acyl transfer to a lyso-MGDG, with concomitant release of free ACP. The mechanism of this reaction has been resolved using a double labelled (/sup 14/C)acyl-(/sup 14/C)ACP substrate prepared with E. coli acyl-ACP synthetase. Following incubation with the enzyme, the unreacted (/sup 14/C)acyl-(/sup 14/C)ACP was isolated and the (/sup 14/C)acyl/(/sup 14/C)ACP ratio determined. Comparison of this ratio to that of the original substrate indicated no change and eliminated acyl exchange as a possible mechanism. Therefore, the direct acylation of lyso-MGDG is the proposed mechanism for this enzyme. The reaction is apparently specific for MGDG synthesis, as other glycolipids and phospholipids were not labelled during incubations.

  15. Comparative Mutagenesis Studies of Retinal Release in Light-Activated Zebrafish Rhodopsin Using Fluorescence Spectroscopy.

    PubMed

    Morrow, J M; Chang, B S W

    2015-07-28

    Rhodopsin is the visual pigment responsible for initiating scotopic (dim-light) vision in vetebrates. Once activated by light, release of all-trans-retinal from rhodopsin involves hydrolysis of the Schiff base linkage, followed by dissociation of retinal from the protein moiety. This kinetic process has been well studied in model systems such as bovine rhodopsin, but not in rhodopsins from cold-blooded animals, where physiological temperatures can vary considerably. Here, we characterize the rate of retinal release from light-activated rhodopsin in an ectotherm, zebrafish (Danio rerio), demonstrating in a fluorescence assay that this process occurs more than twice as fast as bovine rhodopsin at similar temperatures in 0.1% dodecyl maltoside. Using site-directed mutagenesis, we found that differences in retinal release rates can be attributed to a series of variable residues lining the retinal channel in three key structural motifs: an opening in metarhodopsin II between transmembrane helix 5 (TM5) and TM6, in TM3 near E122, and in the "retinal plug" formed by extracellular loop 2 (EL2). The majority of these sites are more proximal to the β-ionone ring of retinal than the Schiff base, indicating their influence on retinal release is more likely due to steric effects during retinal dissociation, rather than alterations to Schiff base stability. An Arrhenius plot of zebrafish rhodopsin was consistent with this model, inferring that the activation energy for Schiff base hydrolysis is similar to that of bovine rhodopsin. Functional variation at key sites identified in this study is consistent with the idea that retinal release might be an adaptive property of rhodopsin in vertebrates. Our study is one of the few investigating a nonmammalian rhodopsin, which will help establish a better understanding of the molecular mechanisms contributing to vision in cold-blooded vertebrates.

  16. Comparative Mutagenesis Studies of Retinal Release in Light-Activated Zebrafish Rhodopsin Using Fluorescence Spectroscopy.

    PubMed

    Morrow, J M; Chang, B S W

    2015-07-28

    Rhodopsin is the visual pigment responsible for initiating scotopic (dim-light) vision in vetebrates. Once activated by light, release of all-trans-retinal from rhodopsin involves hydrolysis of the Schiff base linkage, followed by dissociation of retinal from the protein moiety. This kinetic process has been well studied in model systems such as bovine rhodopsin, but not in rhodopsins from cold-blooded animals, where physiological temperatures can vary considerably. Here, we characterize the rate of retinal release from light-activated rhodopsin in an ectotherm, zebrafish (Danio rerio), demonstrating in a fluorescence assay that this process occurs more than twice as fast as bovine rhodopsin at similar temperatures in 0.1% dodecyl maltoside. Using site-directed mutagenesis, we found that differences in retinal release rates can be attributed to a series of variable residues lining the retinal channel in three key structural motifs: an opening in metarhodopsin II between transmembrane helix 5 (TM5) and TM6, in TM3 near E122, and in the "retinal plug" formed by extracellular loop 2 (EL2). The majority of these sites are more proximal to the β-ionone ring of retinal than the Schiff base, indicating their influence on retinal release is more likely due to steric effects during retinal dissociation, rather than alterations to Schiff base stability. An Arrhenius plot of zebrafish rhodopsin was consistent with this model, inferring that the activation energy for Schiff base hydrolysis is similar to that of bovine rhodopsin. Functional variation at key sites identified in this study is consistent with the idea that retinal release might be an adaptive property of rhodopsin in vertebrates. Our study is one of the few investigating a nonmammalian rhodopsin, which will help establish a better understanding of the molecular mechanisms contributing to vision in cold-blooded vertebrates. PMID:26098991

  17. Sensory analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sensory evaluation can answer questions about a product that instruments cannot. The human subject is the instrument, and data can provide a wealth of information for a product developer, or results can be very variable and erroneous if all the precautions to minimize bias and external noise are no...

  18. Sensory Dysfunction

    MedlinePlus

    ... to Web version Sensory Dysfunction Overview Why are smell and taste important? Your senses of smell and taste let you fully enjoy the scents ... bitter and sour. Flavor involves both taste and smell. For example, because a person is able to ...

  19. Optical control of a rhodopsin-based switch

    NASA Astrophysics Data System (ADS)

    Ovryn, Ben; Li, Xiang; Chiel, Hillel; Herlitze, Stefan

    2004-07-01

    A preliminary result supports the feasibility of using visible light to modulate the membrane potential of a cell. Human embryonic kidney cells (HEK293) were transfected with vertebrate rhodopsin and a gradient inward rectifying potassium (GIRK) channel. Whole cell patch clamp recordings of HEK293 cells exposed to 9-cis retinal showed that illumination increases the potassium current compared with recordings obtained in the dark. When combined with a rapid scanning device, this approach has the potential to control the activity of many neurons.

  20. Regulation of Development and Nitrogen Fixation in Anabaena

    SciTech Connect

    James W Golden

    2004-08-05

    The nitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 is being used as a simple model of microbial development and pattern formation in a multicellular prokaryotic organism. Anabaena reduces atmospheric nitrogen to ammonia in highly specialized, terminally differentiated cells called heterocysts. Anabaena is an important model system because of the multicellular growth pattern, the suspected antiquity of heterocyst development, and the contribution of fixed nitrogen to the environment. We are especially interested in understanding the molecular signaling pathways and genetic regulation that control heterocyst development. In the presence of an external source of reduced nitrogen, the differentiation of heterocysts is inhibited. When Anabaena is grown on dinitrogen, a one-dimensional developmental pattern of single heterocysts separated by approximately ten vegetative cells is established to form a multicellular organism composed of two interdependent cell types. The goal of this project is to understand the signaling and regulatory pathways that commit a vegetative cell to terminally differentiate into a nitrogen-fixing heterocyst. Several genes identified by us and by others were chosen as entry points into the regulatory network. Our research, which was initially focused on transcriptional regulation by group 2 sigma factors, was expanded to include group 3 sigma factors and their regulators after the complete Anabaena genome sequence became available. Surprisingly, no individual sigma factor is essential for heterocyst development. We have used the isolation of extragenic suppressors to study genetic interactions between key regulatory genes such as patS, hetR, and hetC in signaling and developmental pathways. We identified a hetR R223W mutation as a bypass suppressor of patS overexpression. Strains containing the hetR R223W allele fail to respond to pattern formation signals and overexpression of this allele results in a lethal phenotype

  1. Biodegradation of polychlorinated biphenyls (PCBs) by the novel identified cyanobacterium Anabaena PD-1

    PubMed Central

    Zhang, Hangjun; Jiang, Xiaojun; Lu, Liping; Xiao, Wenfeng

    2015-01-01

    Polychlorinated biphenyls (PCBs), a class of hazardous pollutants, are difficult to dissipate in the natural environment. In this study, a cyanobacterial strain Anabaena PD-1 showed good resistance against PCB congeners. Compared to a control group, chlorophyll a content decreased 3.7% and 11.7% when Anabaena PD-1 was exposed to 2 and 5 mg/L PCBs for 7 d. This cyanobacterial strain was capable of decomposing PCB congeners which was conclusively proved by determination of chloride ion concentrations in chlorine-free medium. After 7 d, the chloride ion concentrations in PCB-treated groups (1, 2, 5 mg/L) were 3.55, 3.05, and 2.25 mg/L, respectively. The genetic information of strain PD-1 was obtained through 16S rRNA sequencing analysis. The GenBank accession number of 16S rRNA of Anabaena PD-1 was KF201693.1. Phylogenetic tree analysis clearly indicated that Anabaena PD-1 belonged to the genus Anabaena. The degradation half-life of Aroclor 1254 by Anabaena PD-1 was 11.36 d; the total degradation rate for Aroclor 1254 was 84.4% after 25 d. Less chlorinated PCB congeners were more likely to be degraded by Anabaena PD-1 in comparison with highly chlorinated congeners. Meta- and para-chlorines in trichlorodiphenyls and tetrachlorobiphenyls were more susceptible to dechlorination than ortho-chlorines during the PCB-degradation process by Anabaena PD-1. Furthermore, Anabaena PD-1 can decompose dioxin-like PCBs. The percent biodegradation of 12 dioxin-like PCBs by strain PD-1 ranged from 37.4% to 68.4% after 25 days. Results above demonstrate that Anabaena PD-1 is a PCB-degrader with great potential for the in situ bioremediation of PCB-contaminated paddy soils. PMID:26177203

  2. Biodegradation of polychlorinated biphenyls (PCBs) by the novel identified cyanobacterium Anabaena PD-1.

    PubMed

    Zhang, Hangjun; Jiang, Xiaojun; Lu, Liping; Xiao, Wenfeng

    2015-01-01

    Polychlorinated biphenyls (PCBs), a class of hazardous pollutants, are difficult to dissipate in the natural environment. In this study, a cyanobacterial strain Anabaena PD-1 showed good resistance against PCB congeners. Compared to a control group, chlorophyll a content decreased 3.7% and 11.7% when Anabaena PD-1 was exposed to 2 and 5 mg/L PCBs for 7 d. This cyanobacterial strain was capable of decomposing PCB congeners which was conclusively proved by determination of chloride ion concentrations in chlorine-free medium. After 7 d, the chloride ion concentrations in PCB-treated groups (1, 2, 5 mg/L) were 3.55, 3.05, and 2.25 mg/L, respectively. The genetic information of strain PD-1 was obtained through 16S rRNA sequencing analysis. The GenBank accession number of 16S rRNA of Anabaena PD-1 was KF201693.1. Phylogenetic tree analysis clearly indicated that Anabaena PD-1 belonged to the genus Anabaena. The degradation half-life of Aroclor 1254 by Anabaena PD-1 was 11.36 d; the total degradation rate for Aroclor 1254 was 84.4% after 25 d. Less chlorinated PCB congeners were more likely to be degraded by Anabaena PD-1 in comparison with highly chlorinated congeners. Meta- and para-chlorines in trichlorodiphenyls and tetrachlorobiphenyls were more susceptible to dechlorination than ortho-chlorines during the PCB-degradation process by Anabaena PD-1. Furthermore, Anabaena PD-1 can decompose dioxin-like PCBs. The percent biodegradation of 12 dioxin-like PCBs by strain PD-1 ranged from 37.4% to 68.4% after 25 days. Results above demonstrate that Anabaena PD-1 is a PCB-degrader with great potential for the in situ bioremediation of PCB-contaminated paddy soils.

  3. Mechanism of voltage-sensitive fluorescence in a microbial rhodopsin

    PubMed Central

    Maclaurin, Dougal; Venkatachalam, Veena; Lee, Hohjai; Cohen, Adam E.

    2013-01-01

    Microbial rhodopsins were recently introduced as genetically encoded fluorescent indicators of membrane voltage. An understanding of the mechanism underlying this function would aid in the design of improved voltage indicators. We asked, what states can the protein adopt, and which states are fluorescent? How does membrane voltage affect the photostationary distribution of states? Here, we present a detailed spectroscopic characterization of Archaerhodopsin 3 (Arch). We performed fluorescence spectroscopy on Arch and its photogenerated intermediates in Escherichia coli and in single HEK293 cells under voltage-clamp conditions. These experiments probed the effects of time-dependent illumination and membrane voltage on absorption, fluorescence, membrane current, and membrane capacitance. The fluorescence of Arch arises through a sequential three-photon process. Membrane voltage modulates protonation of the Schiff base in a 13-cis photocycle intermediate (M ⇌ N equilibrium), not in the ground state as previously hypothesized. We present experimental protocols for optimized voltage imaging with Arch, and we discuss strategies for engineering improved rhodopsin-based voltage indicators. PMID:23530193

  4. Nuclear Wavepacket Propogation Model for the Retinal Chromophore in Rhodopsin

    NASA Astrophysics Data System (ADS)

    Corn, Brittany; Malinovskaya, Svetlana

    2009-05-01

    Rhodopsin, consisting of a retinal chromophore and a protein opsin, is responsible for the first steps in the vision process through a cis to trans photoisomerization, which is completed within 200 fs[1]. Efforts to control the ultrafast dynamics of this molecule have been carried out experimentally[2] as well as through quantum mechanical modeling of nuclear wave packet propagation[3]. We propose a two state model in which the ground electronic Potential Energy Surface (PES) is made up of two adjacent harmonic potentials, representing the cis and trans retinal saddle points, as well as an excited PES, characterized by the Morse potential, which meets the ground PES at a conical intersection. We explore the achievement of a high quantum yield of the trans retinal configuration by varying parameters of the external field and choosing the most adequate shape. Another investigation is presented in which we compare the charge distribution of cis and trans retinal in order to reveal a charge transfer mechanism behind the isomerization of rhodopsin. The results of the Lowdin and Natural Population Analyses demonstrate a significant transfer of charge in and around the isomerization region. [1] RW Schoenlein, LA Peteanu, RA Mathies, CV Shank, Science 254, 412 (1991) [2] VI Prokhorenko, AM Nagy, SA Waschuk, LS Brown, RR Birge, RJD Miller, Science 313, 1257 (2006) [3] S Hahn, G Stock, Chem Phys 259, 297-312 (2000)

  5. Acetabularia rhodopsin I is a light-stimulated proton pump.

    PubMed

    Lee, Sang-Soo; Choi, Ah Reum; Kim, So Young; Kang, Ho-Won; Jung, Kwang-Hwan; Lee, Jung-Ha

    2011-05-01

    We cloned an intronless, nuclear-encoded opsin gene from an EST library of Acetabularia acetabulum. Acetabularia rhodopsin I (ARI) encodes a protein of 246 amino acids with molecular weight of 27 kDa. ARI was reconstituted in the Xenopus oocyte expression system to characterize its electrophysiological properties utilizing the two-electrode voltage-clamping technique. Oocytes where ARI cRNA was injected displayed outward directed currents in response to light. The maximum action spectrum of ARI was detected at 520 nm green light. Light-stimulated ARI current amplitude was altered by the protons, but not by the other ions in recording solutions, suggesting that the algal rhodopsin is a light-stimulated proton pump. Typical proton-mediated outward current elicited by 520 nm light was characterized with two phases of non-inactivating outward current following initial transient current. Taken together, we here reported cloning of a novel Acetabularia opsin gene which was characterized to be a proton-pump stimulated by light.

  6. Gloeobacter Rhodopsin, Limitation of Proton Pumping at High Electrochemical Load

    PubMed Central

    Vogt, Arend; Wietek, Jonas; Hegemann, Peter

    2013-01-01

    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. PMID:24209850

  7. Axial gradients of rhodopsin in light-exposed retinal rods of the toad

    PubMed Central

    1990-01-01

    Exposure of an intact vertebrate eye to light bleaches the rhodopsin in the photoreceptor outer segments in spatially nonuniform patterns. Some axial bleaching patterns produced in toad rods were determined using microspectrophotometric techniques. More rhodopsin was bleached at the base of the outer segment than at the distal tip. The shape of the bleaching gradient varied with the extent of bleach and with the spectral content of the illuminant. Monochromatic light at the lambda max of the rhodopsin gave rise to the steepest bleaching gradients and induced the greatest changes in the form of the gradient with increasing extent of bleach. These results were consistent with a mathematical model for pigment bleaching in an unstirred sample. The model did not fit bleaching patterns resulting from special lighting conditions that promoted the photoregeneration of rhodopsin from the intermediates of bleaching. Prolonged light adaptation of toads could also produce axial rhodopsin gradients that were not fit by the bleaching model. Under certain conditions the axial gradient of rhodopsin in a rod outer segment reversed with time in the light: the rhodopsin content became highest at the base. This result could be explained by an interaction between the pattern of bleaching and the intracellular topography of regeneration. PMID:2126801

  8. Thermal and spectroscopic characterization of a proton pumping rhodopsin from an extreme thermophile.

    PubMed

    Tsukamoto, Takashi; Inoue, Keiichi; Kandori, Hideki; Sudo, Yuki

    2013-07-26

    So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

  9. A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas.

    PubMed

    Foster, K W; Saranak, J; Patel, N; Zarilli, G; Okabe, M; Kline, T; Nakanishi, K

    Rhodopsin is a visual pigment ubiquitous in multicellular animals. If visual pigments have a common ancient origin, as is believed, then some unicellular organisms might also use a rhodopsin photoreceptor. We show here that the unicellular alga Chlamydomonas does indeed use a rhodopsin photoreceptor. We incorporated analogues of its retinal chromophore into a blind mutant; normal photobehaviour was restored and the colour of maximum sensitivity was shifted in a manner consistent with the nature of the retinal analogue added. The data suggest that 11-cis-retinal is the natural chromophore and that the protein environment of this retinal is similar to that found in bovine rhodopsin, suggesting homology with the rhodopsins of higher organisms. This is the first demonstration of a rhodopsin photoreceptor in an alga or eukaryotic protist and also the first report of behavioural spectral shifts caused by exogenous synthetic retinals in a eukaryote. A survey of the morphology and action spectra of other protists suggests that rhodopsins may be common photoreceptors of chlorophycean, prasinophycean and dinophycean algae. Thus, Chlamydomonas represents a useful new model for studying photoreceptor cells.

  10. MicRhoDE: a curated database for the analysis of microbial rhodopsin diversity and evolution

    PubMed Central

    Boeuf, Dominique; Audic, Stéphane; Brillet-Guéguen, Loraine; Caron, Christophe; Jeanthon, Christian

    2015-01-01

    Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life and in viruses. Today, microbial rhodopsin research is a flourishing research field in which new understandings of rhodopsin diversity, function and evolution are contributing to broader microbiological and molecular knowledge. Here, we describe MicRhoDE, a comprehensive, high-quality and freely accessible database that facilitates analysis of the diversity and evolution of microbial rhodopsins. Rhodopsin sequences isolated from a vast array of marine and terrestrial environments were manually collected and curated. To each rhodopsin sequence are associated related metadata, including predicted spectral tuning of the protein, putative activity and function, taxonomy for sequences that can be linked to a 16S rRNA gene, sampling date and location, and supporting literature. The database currently covers 7857 aligned sequences from more than 450 environmental samples or organisms. Based on a robust phylogenetic analysis, we introduce an operational classification system with multiple phylogenetic levels ranging from superclusters to species-level operational taxonomic units. An integrated pipeline for online sequence alignment and phylogenetic tree construction is also provided. With a user-friendly interface and integrated online bioinformatics tools, this unique resource should be highly valuable for upcoming studies of the biogeography, diversity, distribution and evolution of microbial rhodopsins. Database URL: http://micrhode.sb-roscoff.fr. PMID:26286928

  11. MicRhoDE: a curated database for the analysis of microbial rhodopsin diversity and evolution.

    PubMed

    Boeuf, Dominique; Audic, Stéphane; Brillet-Guéguen, Loraine; Caron, Christophe; Jeanthon, Christian

    2015-01-01

    Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life and in viruses. Today, microbial rhodopsin research is a flourishing research field in which new understandings of rhodopsin diversity, function and evolution are contributing to broader microbiological and molecular knowledge. Here, we describe MicRhoDE, a comprehensive, high-quality and freely accessible database that facilitates analysis of the diversity and evolution of microbial rhodopsins. Rhodopsin sequences isolated from a vast array of marine and terrestrial environments were manually collected and curated. To each rhodopsin sequence are associated related metadata, including predicted spectral tuning of the protein, putative activity and function, taxonomy for sequences that can be linked to a 16S rRNA gene, sampling date and location, and supporting literature. The database currently covers 7857 aligned sequences from more than 450 environmental samples or organisms. Based on a robust phylogenetic analysis, we introduce an operational classification system with multiple phylogenetic levels ranging from superclusters to species-level operational taxonomic units. An integrated pipeline for online sequence alignment and phylogenetic tree construction is also provided. With a user-friendly interface and integrated online bioinformatics tools, this unique resource should be highly valuable for upcoming studies of the biogeography, diversity, distribution and evolution of microbial rhodopsins. Database URL: http://micrhode.sb-roscoff.fr. PMID:26286928

  12. Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile*

    PubMed Central

    Tsukamoto, Takashi; Inoue, Keiichi; Kandori, Hideki; Sudo, Yuki

    2013-01-01

    So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding. PMID:23740255

  13. Coexpression of Spectrally Distinct Rhodopsins in Aedes aegypti R7 Photoreceptors

    PubMed Central

    Hu, Xiaobang; Whaley, Michelle A.; Stein, Michelle M.; Mitchell, Bronwen E.; O'Tousa, Joseph E.

    2011-01-01

    The retina of the mosquito Aedes aegypti can be divided into four regions based on the non-overlapping expression of a UV sensitive Aaop8 rhodopsin and a long wavelength sensitive Aaop2 type rhodopsin in the R7 photoreceptors. We show here that another rhodopsin, Aaop9, is expressed in all R7 photoreceptors and a subset of R8 photoreceptors. In the dorsal region, Aaop9 is expressed in both the cell body and rhabdomere of R7 and R8 cells. In other retinal regions Aaop9 is expressed only in R7 cells, being localized to the R7 rhabdomere in the central and ventral regions and in both the cell body and rhabdomere within the ventral stripe. Within the dorsal-central transition area ommatidia do not show a strict pairing of R7–R8 cell types. Thus, Aaop9 is coexpressed in the two classes of R7 photoreceptors previously distinguished by the non-overlapping expression of Aaop8 and Aaop2 rhodopsins. Electroretinogram analysis of transgenic Drosophila shows that Aaop9 is a short wavelength rhodopsin with an optimal response to 400–450 nm light. The coexpressed Aaop2 rhodopsin has dual wavelength sensitivity of 500–550 nm and near 350 nm in the UV region. As predicted by the spectral properties of each rhodopsin, Drosophila photoreceptors expressing both Aaop9 and Aaop2 rhodopsins exhibit a uniform sensitivity across the broad 350–550 nm light range. We propose that rhodopsin coexpression is an adaptation within the R7 cells to improve visual function in the low-light environments in which Ae. aegypti is active. PMID:21858005

  14. Regulation of Development and Nitrogen Fixation in Anabaena

    SciTech Connect

    James W. Golden

    2008-10-17

    The regulation of development and cellular differentiation is important for all multicellular organisms. The nitrogen-fixing filamentous cyanobacterium Anabaena (also Nostoc) sp. PCC 7120 (hereafter Anabaena) provides a model of multicellular microbial development and pattern formation. Anabaena reduces N2 to ammonia in specialized terminally differentiated cells called heterocysts. A one-dimensional developmental pattern of single heterocysts regularly spaced along filaments of photosynthetic vegetative cells is established to form a multicellular organism composed of these two interdependent cell types. This multicellular growth pattern, the distinct phylogeny of cyanobacteria, and the suspected antiquity of heterocyst development make this an important model system. Our long-term goal is to understand the regulatory network required for heterocyst development and nitrogen fixation. This project is focused on two key aspects of heterocyst regulation: one, the mechanism by which HetR controls the initiation of differentiation, and two, the cis and trans acting factors required for expression of the nitrogen-fixation (nif) genes. HetR is thought to be a central regulator of heterocyst development but the partners and mechanisms involved in this regulation are unknown. Our recent results indicate that PatS and other signals that regulate heterocyst pattern cannot interact, directly or indirectly, with a R223W mutant of HetR. We plan to use biochemical and genetic approaches to identify proteins that interact with the HetR protein, which will help reveal the mechanisms underlying its regulation of development. Our second goal is to determine how the nif genes are expressed. It is important to understand the mechanisms controlling nif genes since they represent the culmination of the differentiation process and the essence of heterocyst function. The Anabaena genome lacks the genes required for expression of nif genes present in other organisms such as rpoN (sigma 54

  15. New Anabaena and Nostoc cyanophages from sewage settling ponds

    SciTech Connect

    Hu, N.; Thiel, T.; Giddings, T.H., Jr.; Wolk, C.P.

    1981-10-15

    We have isolated, from sewage settling ponds, 16 cyanophages for heterocyst forming, filamentous cyanobacteria of the genera Anabaena and Nostoc. These phages fall into three groups based on morphology, host range, one-step growth curves, and restriction digests. On the basis of these criteria they can be distinguished from cyanophages A-1(L), A-4(L), N-1, and AN-10 which we received from other laboratories. Certain of the newly described phages are similar in morphology to the short-tailed LPP cyanophages, and others to the long-tailed AS cyanophages.

  16. Sensitivity of Anabaena spiroides to zinc and cobalt

    SciTech Connect

    Kostyayev, V. Ya.; Yagodka, S.N.; Sokolov, V.A.

    1980-01-01

    The influence of zinc and cobalt chlorous salts on destruction, nitrogen fixation photosynthesis, and Na/sup +/ and K/sup +/-ion concentrations in the cells was studied for the blue-green alga Anabaena spiroides. Stability of A. spiroides depends on the developmental stage. The algae are most sensitive to cobalt at the beginning of the growth lag-phase, and they are least sensitive to it at the end of this phase. Cobalt ions are more toxic for A. spiroides than zinc ions. Salts of heavy metals inhibit active ion transport, which results in a sharp decrease of K and Na ions concentration in the algae cells.

  17. Fluorapatite as Inorganic Phosphate Source for the Cyanobacterium Anabaena PCC 7120

    NASA Astrophysics Data System (ADS)

    Schaperdoth, I.; Brantley, S.

    2003-12-01

    We investigated the hypothesis that the cyanobacterium Anabaena PCC 7120 is able to use fluorapatite (FAP) as sole phosphate source for growth. In the experimental setup the dissolution of FAP was tested in a phosphate free growth medium in the presence and absence of the Anabaena, as well as the cell free supernatant of an Anabaena culture. The results were compared with that of an Anabaena culture grown without fluorapatite. Parameters measured were pH, dissolved P and Ca, as well as cell density. The FAP grains were analyzed using SEM and XPS. Additionally, the differential expression of secreted proteins in cultures with and without dissolved phosphate was examined. P-limited Anabaena cultures tend to aggregate and in the presence of FAP the cells attached themselves to the mineral grains. The cultures benefit from the presence of FAP. The cells have a very effective P-uptake system that is able to take up dissolved phosphate very efficiently and draw the concentrations down to very low levels. Furthermore, the SEM analysis of FAP showed an etching of the mineral grains in the samples from the Anabaena cultures. The mechanism of apatite dissolution with and without Anabaena will be discussed in terms of these experimental observations.

  18. Characterization and chromosomal localization of the gene for human rhodopsin kinase

    SciTech Connect

    Khani, S.C.; Yamamoto, S.; Dryja, T.P.

    1996-08-01

    G-protein-dependent receptor kinases (GRKs) play a key role in the adapatation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid densensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of FRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently clone human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family. 39 refs., 3 figs.

  19. Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors

    PubMed Central

    1977-01-01

    Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr). PMID:591915

  20. Sequence and intramolecular distance scoring analyses of microbial rhodopsins

    PubMed Central

    Asano, Miki; Ide, Shunta; Kamata, Atsushi; Takahasi, Kiyohiro; Okada, Tetsuji

    2016-01-01

    Recent accumulation of sequence and structural data, in conjunction with systematical classification into a set of families, has significantly advanced our understanding of diverse and specific protein functions. Analysis and interpretation of protein family data requires comprehensive sequence and structural alignments. Here, we present a simple scheme for analyzing a set of experimental structures of a given protein or family of proteins, using microbial rhodopsins as an example. For a data set comprised of around a dozen highly similar structures to each other (overall pairwise root-mean-squared deviation < 2.3 Å), intramolecular distance scoring analysis yielded valuable information with respect to structural properties, such as differences in the relative variability of transmembrane helices. Furthermore, a comparison with recent results for G protein-coupled receptors demonstrates how the results of the present analysis can be interpreted and effectively utilized for structural characterization of diverse protein families in general. PMID:26998236

  1. Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis.

    PubMed Central

    Martin, G; Haehnel, W; Böger, P

    1997-01-01

    In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH. GS inactivation was completed within 30 min. Both the inactivated GS and the active enzyme were isolated. No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide. Mass spectrometry revealed that the molecular masses of active and inactive GS are equal. While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract. This result indicates that the active site was affected. From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration. GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen. In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2. The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen. Accordingly, the inactivating fraction was inhibited by catalase and EDTA. This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli. We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation. PMID:9006027

  2. Study of the schiff base mode in bovine rhodopsin and bathorhodopsin

    SciTech Connect

    Deng, H.; Callender, R.H.

    1987-11-17

    The authors have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC=NH/sup +/-, -HC=ND/sup +/-. -H/sup 13/C=NH/sup +/-, and -H/sup 13/C=ND/sup +/-. Apart from the doubly labeled retinal, they find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. They develop a force field that accurately fits the observed ethylenic (C=C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, they investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, they find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. They discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process.

  3. Rhodopsin expression in the zebrafish pineal gland from larval to adult stage.

    PubMed

    Magnoli, Domenico; Zichichi, Rosalia; Laurà, Rosaria; Guerrera, Maria Cristina; Campo, Salvatore; de Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

    2012-03-01

    The zebrafish pineal gland plays an important role in different physiological functions including the regulation of the circadian clock. In the fish pineal gland the pinealocytes are made up of different segments: outer segment, inner segment and basal pole. Particularly, in the outer segment the rhodopsin participates in the external environment light reception that represents the first biochemical step in the melatonin production. It is well known that the rhodopsin in the adult zebrafish is well expressed in the pineal gland but both the expression and the cellular localization of this protein during development remain still unclear. In this study using qRT-PCR, sequencing and immunohistochemistry the expression as well as the protein localization of the rhodopsin in the zebrafish from larval (10 dpf) to adult stage (90 dpf) were demonstrated. The rhodopsin mRNA expression presents a peak of expression at 10 dpf, a further reduction to 50 dpf before increasing again in the adult stage. Moreover, the cellular localization of the rhodopsin-like protein was always localized in the pinealocyte at all ages examined. Our results demonstrated the involvement of the rhodopsin in the zebrafish pineal gland physiology particularly in the light capture during the zebrafish lifespan.

  4. pH-dependent interaction of rhodopsin with cyanidin-3-glucoside. 1. Structural aspects.

    PubMed

    Yanamala, Naveena; Tirupula, Kalyan C; Balem, Fernanda; Klein-Seetharaman, Judith

    2009-01-01

    Anthocyanins are a class of natural compounds common in flowers and vegetables. Because of the increasing preference of consumers for food containing natural colorants and the demonstrated beneficial effects of anthocyanins on human health, it is important to decipher the molecular mechanisms of their action. Previous studies indicated that the anthocyanin cyanidin-3-glucoside (C3G) modulates the function of the photoreceptor rhodopsin. In this paper, we show using selective excitation (1)H NMR spectroscopy that C3G binds to rhodopsin. Ligand resonances broaden upon rhodopsin addition and rhodopsin resonances exhibit chemical shift changes as well as broadening effects in specific resonances, in an activation state-dependent manner. Furthermore, dark-adapted and light-activated states of rhodopsin show preferences for different C3G species. Molecular docking studies of the flavylium cation, quinoidal base, carbinol pseudobase and chalcone forms of C3G to models of the dark, light-activated and opsin structures of rhodopsin also support this conclusion. The results provide new insights into anthocyanin-protein interactions and may have relevance for the enhancement of night vision by this class of compounds. This work is also the first report of the study of ligand binding to a full-length membrane receptor in detergent micelles by (1)H NMR spectroscopy. Such studies were previously hampered by the presence of detergent micelle resonances, a problem overcome by the selective excitation approach. PMID:19192199

  5. Dynamics of voltage profile in enzymatic ion transporters, demonstrated in electrokinetics of proton pumping rhodopsin.

    PubMed

    Hagedorn, Rolf; Gradmann, Dietrich; Hegemann, Peter

    2008-12-01

    H(+)-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H(+)-pumping rhodopsin. The electrical properties of H(+)-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H(+)-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H(+) transfer to an H(+) acceptor, reprotonation of retinal from the intracellular face via an H(+) donor, and proton release to the extracellular space via an H(+) release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby--as in an Ohmic voltage divider--the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H(+)-binding sites involved.

  6. Azolla-Anabaena relationship. XIII. Fixation of (/sup 13/N)N/sub 2/. [Azolla caroliniana; Anabaena azollae

    SciTech Connect

    Meeks, J.C.; Steinberg, N.A.; Enderlin, C.S.; Joseph, C.M.; Peters, G.A.

    1987-07-01

    The major radioactive products of the fixation of (/sup 13/N)N/sub 2/ by Azolla caroliniana willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of (/sup 13/N)N/sub 2/-derived /sup 13/NH/sub 4//sup +/ after long incubation periods was attributed to the spatial separation between the site of N/sub 2/-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from (/sup 13/N)N/sub 2/, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and (/sup 13/N)N/sub 2/-derived /sup 13/NH/sub 4//sup +/, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Azabaena.

  7. Novel surface associated polyphosphate bodies sequester uranium in the filamentous, marine cyanobacterium, Anabaena torulosa.

    PubMed

    Acharya, Celin; Apte, Shree Kumar

    2013-12-01

    A filamentous, heterocystous, nitrogen-fixing marine cyanobacterium, Anabaena torulosa, has been shown to harbour surface associated, acid soluble polyphosphate bodies. Uranium immobilization by such polyphosphate bodies, reported in cyanobacteria for the first time, demonstrates a novel uranium sequestration phenomenon.

  8. Chemical analysis of the phenol-water-extractable materials from Anabaena flos-aquae.

    PubMed

    Wang, A W; Hill, A

    1977-04-01

    The high molecular-weight carbohydrate substances extracted in the aqueous and phenol phases by phenol-water extraction of Anabaena flos-aquae A-37 were found to be polysaccharides without lipid attached.

  9. [Effect of light and temperature on growth kinetics of Anabaena flosaquae under phosphorus limitation].

    PubMed

    Yin, Zhi-Kun; Li, Zhe; Wang, Sheng; Guo, Jin-Song; Xiao, Yan; Liu, Jing; Zhang, Ping

    2015-03-01

    Phosphorus, light and temperature are the key environmental factors leading to algae growth. But the effects of interaction between light and temperature on the growth of Anabaena flosaquae under phosphorus limitation were not well documented in literature. Anabaena flosaquae was selected for the study and lab-scale experiment and simulation were carried out. The results showed that the optimal temperature of Anabaena flosaquae was 20 degrees C under phosphorus limitation when the light intensity was constant, and the optimal light intensity (illuminance) of Anabaena flosaquae was 3 000 lx under phosphorus limitation when the temperature was constant. Based on model fitting and parameter calibration, the optimal temperature and light intensity of Anabaena flosaquae were 21.03 degress C ± 1.55 degrees C and 2 675.12 lx ± 262.93 lx, respectively. These data were close to the actual water environmental condition at the end of spring. Results of this study will provide important foundation for prediction of Anabaena blooms. PMID:25929064

  10. [Effect of light and temperature on growth kinetics of Anabaena flosaquae under phosphorus limitation].

    PubMed

    Yin, Zhi-Kun; Li, Zhe; Wang, Sheng; Guo, Jin-Song; Xiao, Yan; Liu, Jing; Zhang, Ping

    2015-03-01

    Phosphorus, light and temperature are the key environmental factors leading to algae growth. But the effects of interaction between light and temperature on the growth of Anabaena flosaquae under phosphorus limitation were not well documented in literature. Anabaena flosaquae was selected for the study and lab-scale experiment and simulation were carried out. The results showed that the optimal temperature of Anabaena flosaquae was 20 degrees C under phosphorus limitation when the light intensity was constant, and the optimal light intensity (illuminance) of Anabaena flosaquae was 3 000 lx under phosphorus limitation when the temperature was constant. Based on model fitting and parameter calibration, the optimal temperature and light intensity of Anabaena flosaquae were 21.03 degress C ± 1.55 degrees C and 2 675.12 lx ± 262.93 lx, respectively. These data were close to the actual water environmental condition at the end of spring. Results of this study will provide important foundation for prediction of Anabaena blooms.

  11. Rhodopsins carrying modified chromophores--the 'making of', structural modelling and their light-induced reactivity.

    PubMed

    Ockenfels, Andreas; Schapiro, Igor; Gärtner, Wolfgang

    2016-02-01

    A series of vitamin-A aldehydes (retinals) with modified alkyl group substituents (9-demethyl-, 9-ethyl-, 9-isopropyl-, 10-methyl, 10-methyl-13-demethyl-, and 13-demethyl retinal) was synthesized and their 11-cis isomers were used as chromophores to reconstitute the visual pigment rhodopsin. Structural changes were selectively introduced around the photoisomerizing C11=C12 bond. The effect of these structural changes on rhodopsin formation and bleaching was determined. Global fit of assembly kinetics yielded lifetimes and spectral features of the assembly intermediates. Rhodopsin formation proceeds stepwise with prolonged lifetimes especially for 9-demethyl retinal (longest lifetime τ3 = 7500 s, cf., 3500 s for retinal), and for 10-methyl retinal (τ3 = 7850 s). These slowed-down processes are interpreted as either a loss of fixation (9dm) or an increased steric hindrance (10me) during the conformational adjustment within the protein. Combined quantum mechanics and molecular mechanics (QM/MM) simulations provided structural insight into the retinal analogues-assembled, full-length rhodopsins. Extinction coefficients, quantum yields and kinetics of the bleaching process (μs-to-ms time range) were determined. Global fit analysis yielded lifetimes and spectral features of bleaching intermediates, revealing remarkably altered kinetics: whereas the slowest process of wild-type rhodopsin and of bleached and 11-cis retinal assembled rhodopsin takes place with lifetimes of 7 and 3.8 s, respectively, this process for 10-methyl-13-demethyl retinal was nearly 10 h (34670 s), coming to completion only after ca. 50 h. The structural changes in retinal derivatives clearly identify the precise interactions between chromophore and protein during the light-induced changes that yield the outstanding efficiency of rhodopsin.

  12. Four of the six Drosophila rhodopsin-expressing photoreceptors can mediate circadian entrainment in low light.

    PubMed

    Saint-Charles, Alexandra; Michard-Vanhée, Christine; Alejevski, Faredin; Chélot, Elisabeth; Boivin, Antoine; Rouyer, François

    2016-10-01

    Light is the major stimulus for the synchronization of circadian clocks with day-night cycles. The light-driven entrainment of the clock that controls rest-activity rhythms in Drosophila relies on different photoreceptive molecules. Cryptochrome (CRY) is expressed in most brain clock neurons, whereas six different rhodopsins (RH) are present in the light-sensing organs. The compound eye includes outer photoreceptors that express RH1 and inner photoreceptors that each express one of the four rhodopsins RH3-RH6. RH6 is also expressed in the extraretinal Hofbauer-Buchner eyelet, whereas RH2 is only found in the ocelli. In low light, the synchronization of behavioral rhythms relies on either CRY or the canonical rhodopsin phototransduction pathway, which requires the phospholipase C-β encoded by norpA (no receptor potential A). We used norpA(P24) cry(02) double mutants that are circadianly blind in low light and restored NORPA function in each of the six types of photoreceptors, defined as expressing a particular rhodopsin. We first show that the NORPA pathway is less efficient than CRY for synchronizing rest-activity rhythms with delayed light-dark cycles but is important for proper phasing, whereas the two light-sensing pathways can mediate efficient adjustments to phase advances. Four of the six rhodopsin-expressing photoreceptors can mediate circadian entrainment, and all are more efficient for advancing than for delaying the behavioral clock. In contrast, neither RH5-expressing retinal photoreceptors nor RH2-expressing ocellar photoreceptors are sufficient to mediate synchronization through the NORPA pathway. Our results thus reveal different contributions of rhodopsin-expressing photoreceptors and suggest the existence of several circuits for rhodopsin-dependent circadian entrainment. J. Comp. Neurol. 524:2828-2844, 2016. © 2016 Wiley Periodicals, Inc.

  13. Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

    PubMed

    Keffer, J L; Sabanayagam, C R; Lee, M E; DeLong, E F; Hahn, M W; Maresca, J A

    2015-05-15

    Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples. PMID:25769822

  14. Four of the six Drosophila rhodopsin-expressing photoreceptors can mediate circadian entrainment in low light.

    PubMed

    Saint-Charles, Alexandra; Michard-Vanhée, Christine; Alejevski, Faredin; Chélot, Elisabeth; Boivin, Antoine; Rouyer, François

    2016-10-01

    Light is the major stimulus for the synchronization of circadian clocks with day-night cycles. The light-driven entrainment of the clock that controls rest-activity rhythms in Drosophila relies on different photoreceptive molecules. Cryptochrome (CRY) is expressed in most brain clock neurons, whereas six different rhodopsins (RH) are present in the light-sensing organs. The compound eye includes outer photoreceptors that express RH1 and inner photoreceptors that each express one of the four rhodopsins RH3-RH6. RH6 is also expressed in the extraretinal Hofbauer-Buchner eyelet, whereas RH2 is only found in the ocelli. In low light, the synchronization of behavioral rhythms relies on either CRY or the canonical rhodopsin phototransduction pathway, which requires the phospholipase C-β encoded by norpA (no receptor potential A). We used norpA(P24) cry(02) double mutants that are circadianly blind in low light and restored NORPA function in each of the six types of photoreceptors, defined as expressing a particular rhodopsin. We first show that the NORPA pathway is less efficient than CRY for synchronizing rest-activity rhythms with delayed light-dark cycles but is important for proper phasing, whereas the two light-sensing pathways can mediate efficient adjustments to phase advances. Four of the six rhodopsin-expressing photoreceptors can mediate circadian entrainment, and all are more efficient for advancing than for delaying the behavioral clock. In contrast, neither RH5-expressing retinal photoreceptors nor RH2-expressing ocellar photoreceptors are sufficient to mediate synchronization through the NORPA pathway. Our results thus reveal different contributions of rhodopsin-expressing photoreceptors and suggest the existence of several circuits for rhodopsin-dependent circadian entrainment. J. Comp. Neurol. 524:2828-2844, 2016. © 2016 Wiley Periodicals, Inc. PMID:26972685

  15. Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

    PubMed

    Keffer, J L; Sabanayagam, C R; Lee, M E; DeLong, E F; Hahn, M W; Maresca, J A

    2015-05-15

    Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low-comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls-these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples.

  16. Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

    PubMed

    Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

    2016-03-01

    Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications.

  17. Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis

    SciTech Connect

    Thiel, T.

    1988-03-01

    Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells.Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of P/sub i/. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent K/sub i/ values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.

  18. Outdoor biophotolytic system using the cyanobacterium anabaena cylindrica B629

    SciTech Connect

    Smith, G.D.; Lambert, G.R.

    1981-01-01

    The cyanobacterium Anabaena cylindrica B629 was suspended in small glass beads and incubated in a gas-tight glass vessel outdoors under a gas atmosphere comprising carbon monoxide (0.2%), acetylene (5%), oxygen (6.5%), and nitrogen. The solution phase initially contained sodium bicarbonate (10mM) at pH 7. Under these conditions the organism continuously produced hydrogen gas for over three weeks. The temperature of the culture was maintained below 30 /degree/C and the minimum night temperatures were recorded. The vessel was covered by a shadecloth, which reduced the natural illumination by approximately 70%. The system is an alternative to those requiring the strict absence of oxygen and little nitrogen, and requires virtually no attention during the incubation period. 18 refs.

  19. UV-inducible DNA repair in the cyanobacteria Anabaena spp

    SciTech Connect

    Levine, E.; Thiel, T.

    1987-09-01

    Strains of the filamentous cyanobacteria Anabaena spp. were capable of very efficient photoreactivation of UV irradiation-induced damage to DNA. Cells were resistant to several hundred joules of UV irradiation per square meter under conditions that allowed photoreactivation, and they also photoreactivated UV-damaged cyanophage efficiently. Reactivation of UV-irradiated cyanophage (Weigle reactivation) also occurred; UV irradiation of host cells greatly enhanced the plaque-forming ability of irradiated phage under nonphotoreactivating conditions. Postirradiation incubation of the host cells under conditions that allowed photoreactivation abolished the ability of the cells to perform Weigle reactivation of cyanophage N-1. Mitomycin C also induced Weigle reactivation of cyanophage N-1, but nalidixic acid did not. The inducible repair system (defined as the ability to perform Weigle reactivation of cyanophages) was relatively slow and inefficient compared with photoreactivation.

  20. Structure of the retinal chromophore in 7,9-dicis-rhodopsin

    SciTech Connect

    Loppnow, G.R.; Miley, M.E.; Mathies, R.A. ); Liu, R.S.H. ); Kandori, H.; Shichida, Y.; Fukada, Y.; Yoshizawa, T. )

    1990-09-25

    Bovine rhodopsin was bleached and regenerated with 7,9-dicis-retinal to form 7,9-dicis-rhodopsin, which was purified on a concanavalin A affinity column. The absorption maximum of the 7,9-dicis pigment is 453 nm, giving an opsin shift of 1600 cm{sup {minus}1} compared to 2500 cm{sup {minus}1} for 11-cis-rhodopsin and 2400 cm{sup {minus}1} for 9-cis-rhodopsin. Rapid-flow resonance Raman spectra have been obtained of 7,9-dicis-rhodopsin in H{sub 2}O and D{sub 2}O at room temperature. The shift of the 1654-cm{sup {minus}1} C{double bond}N stretch to 1627 cm{sup {minus}1} in D{sub 2}O demonstrates that the Schiff base nitrogen is protonated. The absence of any shift in the 1201-cm{sup {minus}1} mode, which is assigned as the C{sub 14}-C{sub 15} stretch, or of any other C-C stretching modes in D{sub 2}O indicates that the Schiff base C{double bond}N configuration is trans (anti). Assuming that the cyclohexenyl ring binds with the same orientation in 7,9-dicis-, 9-cis-, and 11-cis-rhodopsins, the presence of two cis bonds requires that the N-H bond of the 7,9-dicis chromophore points in the opposite direction from that in the 9-cis or 11-cis pigment. However, the Schiff base C{double bond}NH{sup +} stretching frequency and its D{sub 2}O shift in 7,9-dicis-rhodopsin are very similar to those in 11-cis- and 9-cis-rhodopsin, indicating that the Schiff base electrostatic/hydrogen-bonding environments are effectively the same. The C{double bond}N trans (anti) Schiff base geometry of 7,9-dicis-rhodopsin and the insensitivity of its Schiff base vibrational properties to orientation are rationalized by examining the binding site specificity with molecular modeling.

  1. Investigation of evolution-related aspects of bacterial rhodopsins

    NASA Technical Reports Server (NTRS)

    1994-01-01

    We have investigated evolution-related aspects of bacterial rhodopsins, the unique retinal-based energy transducing systems of halophilic archae. The approach was to describe both structural and functional aspects: the structure by sequencing genes to explore which regions are conserved, and the function by comparing proton and chloride transport in the closely related systems, bacteriorhodopsin and halorhodopsin, respectively. In the latter, we have made a good start toward the ultimate goal of separating the attributes of the general principles of retinal-based ionic pumps from those of the specific ion specificities, by determining the thermodynamics of the internal steps of the protein-mediated active transport process, as well as some of the intraprotein ion-transfer steps. Our present emphasis is on continuing to acquire the tools for studying what distinguishes proton transport from chloride transport. We consider it important, therefore, that we have been able to provide firm mathematical grounds for the kinetics analyses which underlies these studies. Our molecular biological studies have received a great boost from the expression vector for the bop gene based on a halobacterial plasmid, that we recently developed.

  2. Focal cone ERGs of rhodopsin Pro347Leu transgenic rabbits.

    PubMed

    Ueno, Shinji; Koyasu, Toshiyuki; Kominami, Taro; Sakai, Takao; Kondo, Mineo; Yasuda, Shunsuke; Terasaki, Hiroko

    2013-10-18

    A rhodopsin P347L transgenic (Tg) rabbit, a model of retinitis pigmentosa, has been generated in our laboratory. The purpose of this study was to determine the properties of focal areas of the retina in this rabbit model during the course of retinal degeneration. To accomplish this, we recorded focal ERGs from wild-type (WT) and Tg rabbits at ages 3, 6, and 12 months. A 15° stimulus spot was used to elicit the focal ERGs from the center of the visual streak and from four surrounding areas. We found that the amplitudes of the focal cone ERG b-waves and oscillatory potentials (OPs) of the Tg rabbits in the five areas decreased progressively with increasing age and became almost non-recordable at 12 months. There were no significant regional differences in the b-waves of Tg rabbits recorded from the 5 areas. The amplitudes of the OPs were better preserved than the b-waves and the OPs/b-wave ratio was higher than that in WT rabbits at every recording area. The summed OPs amplitudes, which most likely originate from the amacrine and/or ganglion cells, recorded from the area superior to the optic disc was significantly larger than that from other areas at 3- and 6-months-old. This indicated that the inner retinal neurons were not altered equally after photoreceptor degeneration in this rabbit model.

  3. Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity.

    PubMed

    Gaub, Benjamin M; Berry, Michael H; Holt, Amy E; Isacoff, Ehud Y; Flannery, John G

    2015-10-01

    Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination. PMID:26137852

  4. Optogenetic Vision Restoration Using Rhodopsin for Enhanced Sensitivity

    PubMed Central

    Gaub, Benjamin M; Berry, Michael H; Holt, Amy E; Isacoff, Ehud Y; Flannery, John G

    2015-01-01

    Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination. PMID:26137852

  5. Rhodopsin targeted transcriptional silencing by DNA-binding

    PubMed Central

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-01-01

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations. DOI: http://dx.doi.org/10.7554/eLife.12242.001 PMID:26974343

  6. A Photochromic Histidine Kinase Rhodopsin (HKR1) That Is Bimodally Switched by Ultraviolet and Blue Light*

    PubMed Central

    Luck, Meike; Mathes, Tilo; Bruun, Sara; Fudim, Roman; Hagedorn, Rolf; Tran Nguyen, Tra My; Kateriya, Suneel; Kennis, John T. M.; Hildebrandt, Peter; Hegemann, Peter

    2012-01-01

    Rhodopsins are light-activated chromoproteins that mediate signaling processes via transducer proteins or promote active or passive ion transport as ion pumps or directly light-activated channels. Here, we provide spectroscopic characterization of a rhodopsin from the Chlamydomonas eyespot. It belongs to a recently discovered but so far uncharacterized family of histidine kinase rhodopsins (HKRs). These are modular proteins consisting of rhodopsin, a histidine kinase, a response regulator, and in some cases an effector domain such as an adenylyl or guanylyl cyclase, all encoded in a single protein as a two-component system. The recombinant rhodopsin fragment, Rh, of HKR1 is a UVA receptor (λmax = 380 nm) that is photoconverted by UV light into a stable blue light-absorbing meta state Rh-Bl (λmax = 490 nm). Rh-Bl is converted back to Rh-UV by blue light. Raman spectroscopy revealed that the Rh-UV chromophore is in an unusual 13-cis,15-anti configuration, which explains why the chromophore is deprotonated. The excited state lifetime of Rh-UV is exceptionally stable, probably caused by a relatively unpolar retinal binding pocket, converting into the photoproduct within about 100 ps, whereas the blue form reacts 100 times faster. We propose that the photochromic HKR1 plays a role in the adaptation of behavioral responses in the presence of UVA light. PMID:23027869

  7. Expression and light-triggered movement of rhodopsins in the larval visual system of mosquitoes

    PubMed Central

    Rocha, Manuel; Kimler, Kyle J.; Leming, Matthew T.; Hu, Xiaobang; Whaley, Michelle A.; O'Tousa, Joseph E.

    2015-01-01

    ABSTRACT During the larval stages, the visual system of the mosquito Aedes aegypti contains five stemmata, often referred to as larval ocelli, positioned laterally on each side of the larval head. Here we show that stemmata contain two photoreceptor types, distinguished by the expression of different rhodopsins. The rhodopsin Aaop3 (GPROP3) is expressed in the majority of the larval photoreceptors. There are two small clusters of photoreceptors located within the satellite and central stemmata that express the rhodopsin Aaop7 (GPROP7) instead of Aaop3. Electroretinogram analysis of transgenic Aaop7 Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectral properties. Light triggers an extensive translocation of Aaop3 from the photosensitive rhabdoms to the cytoplasmic compartment, whereas light-driven translocation of Aaop7 is limited. The results suggest that these photoreceptor cell types play distinct roles in larval vision. An additional component of the larval visual system is the adult compound eye, which starts to develop at the anterior face of the larval stemmata during the 1st instar stage. The photoreceptors of the developing compound eye show rhodopsin expression during the 4th larval instar stage, consistent with indications from previous reports that the adult compound eye contributes to larval and pupal visual capabilities. PMID:25750414

  8. Study of the orientation of retinal in bovine rhodopsin: the use of a photoactivatable retinal analog

    SciTech Connect

    Nakayama, T.

    1987-05-01

    Rhodopsin is the major transmembrane protein in the photoreceptor cells of vertebrate and invertebrate retina. Bovine rhodopsin consists of a polypeptide chain of 348 amino acids of known sequence in which the chromophore, 11-cis-retinal, is linked to Lys-296 as a Schiff base. To investigate the orientation of retinal in the protein and to study the interactions between retinal and the protein, the authors have developed a crosslinking approach using a /sup 3/H-labeled photoactivatable analog of retinal. Bleached rhodopsin in rod outer segments was reconstituted with the analog to give a pigment with lambda/sub max/ at 460nm. Reduction of the Schiff base with borane dimenthylamine, followed by degradation with CNBr and sequencing of the radioactive fragment showed that the analog is attached to Lys-296, as in the native rhodopsin. Further, the reconstitute protein after photolysis was phosphorylated by rhodopsin kinase. Photolysis of the reconstituted pigment at -15/sup 0/C resulted in crosslinking of the analog to the opsin to the extent of 30% as analyzed by SDS electrophoresis. The site(s) of crosslinking in the protein are under investigation.

  9. The Retromer Complex Is Required for Rhodopsin Recycling and Its Loss Leads to Photoreceptor Degeneration

    PubMed Central

    Wang, Shiuan; Tan, Kai Li; Agosto, Melina A.; Xiong, Bo; Yamamoto, Shinya; Sandoval, Hector; Jaiswal, Manish; Bayat, Vafa; Zhang, Ke; Charng, Wu-Lin; David, Gabriela; Duraine, Lita; Venkatachalam, Kartik; Wensel, Theodore G.; Bellen, Hugo J.

    2014-01-01

    Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity. PMID:24781186

  10. Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation.

    PubMed

    Tian, Yao; Li, Tao; Sun, Mingkuan; Wan, Didi; Li, Qian; Li, Peipei; Zhang, Zi Chao; Han, Junhai; Xie, Wei

    2013-01-23

    Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. Here, we show that the Drosophila homolog of α-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. We show that the decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, we identify that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, we demonstrate that Neurexin is essential for the apolipoproteins level. Our results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function.

  11. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

    PubMed Central

    Sung, C H; Schneider, B G; Agarwal, N; Papermaster, D S; Nathans, J

    1991-01-01

    Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP. Images PMID:1924344

  12. Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors

    PubMed Central

    Satoh, Akiko K.; Xia, Hongai; Yan, Limin; Liu, Che-Hsiung; Hardie, Roger C.; Ready, Donald F.

    2010-01-01

    Upon illumination visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments - vertebrate outer segments or invertebrate rhabdomeres - where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s), and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq and the light-sensitive Ca2+ permeable TRP channels, slows translocation by 10-100 fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx. PMID:20869596

  13. Rhodopsin regeneration in the normal and in the detached/replaced retina of the skate.

    PubMed

    Sun, Y; Ripps, H

    1992-11-01

    The bleaching and regeneration of rhodopsin in the skate retina was studied by means of fundus reflectometry, both in the normal eyecup preparation and after the retina had been detached and then replaced on the surface of the pigment epithelium (RPE). After bleaching virtually all the rhodopsin in the retinal test area of the normal eyecup, more than 90% of the photopigment was reformed after about 2 hr in darkness; over most of this time course, rhodopsin density rose linearly at a rate of 0.875% min-1 with a half-time of 55 min. Detaching the retina from its pigment epithelium resulted in a number of abnormalities, both structural and functional. Histological examination of the detached/replaced (D/R) retina showed striking alterations in the structural integrity of the RPE cells at their interface with the neural retina. The cells appeared vacuolated and misshapen, and the apical processes of the RPE, which normally ensheath the receptor outer segments, were shredded and free of their association with the visual cells. These morphological changes, as well as dilution of the IRBP content of the subretinal space caused by separation of the tissues, appear to be the main factors contributing to the functional abnormalities in rhodopsin kinetics. But despite these abnormalities and the persistent detachment, the rate of regeneration and the amount of rhodopsin reformed after bleaching were reduced by less than 50% of their normal values. The fact that a significant fraction of the bleached rhodopsin was regenerated under these conditions indicates that 11-cis retinal formed in the RPE was able to traverse a much greater than normal subretinal space to reach the opsin-bearing photoreceptor membranes. PMID:1478278

  14. Missense mutation (E150K) of rhodopsin in autosomal recessive retinitis pigmentosa

    SciTech Connect

    Orth, U.; Oehlmann, R.; Gal, A.

    1994-09-01

    Missense or nonsense mutations of the rhodopsin gene have been implied in the pathogenesis of at least 3 different traits; autosomal dominant retinitis pigmentosa (adRP), congenital stationary night blindness (CSNB), and autosomal recessive retinitis pigmentosa (arRP). For the latter, a single patient has been reported with a nonsense mutation at codon 249 on both alleles. Heterozygous carriers of missense mutations of rhodopsin develop either adRP or CSNB depending on the particular amino acid substitution. Four of the 9 siblings from a consanguineous marriage in southern India were reported the have arRP. Mutational screening and sequencing of the rhodopsin gene revealed a G-to-A transition of the first nucleotide at codon 150 in exon II, which alters glutamate to lysine. The E150K mutation was present in the 4 patients in homozygous form, whereas the parents and 2 of the siblings were heterozygotes. Two-point analysis produced a Zmax=3.46 at theta=0.00. Two unaffected siblings who are heterozygotes for the E150K mutation underwent a thorough ophthalmological and psychophysical examination. No clinical abnormalities were found although these individuals were over forty, whereas the onset of RP in their affected siblings was in the second decade. Collectively, both the genetic and clinical findings strongly suggest that the E150K mutation of rhodopsin is recessive in this family. Glu150 forms part of the CD cytoplasmic loop of rhodopsin, which has been implicated in the binding and activation of transducin. We speculate that E150K leads to RP because the mutant protein may be incapable of activating transducin. It is tempting to speculate that, in addition to mutations in the genes for rhodopsin and the {beta}-subunit of PDE, mutations in the genes for transducin may also result in arRP.

  15. A single gene all3940 (Dps) overexpression in Anabaena sp. PCC 7120 confers multiple abiotic stress tolerance via proteomic alterations.

    PubMed

    Narayan, Om Prakash; Kumari, Nidhi; Bhargava, Poonam; Rajaram, Hema; Rai, Lal Chand

    2016-01-01

    DNA-binding proteins (Dps) induced during starvation play an important role in gene regulation and maintaining homeostasis in bacteria. The nitrogen-fixing cyanobacterium, Anabaena PCC7120, has four genes annotated as coding for Dps; however, the information on their physiological roles is limiting. One of the genes coding for Dps, 'all3940' was found to be induced under different abiotic stresses in Anabaena and upon overexpression enhanced the tolerance of Anabaena to a multitude of stresses, which included salinity, heat, heavy metals, pesticide, and nutrient starvation. On the other hand, mutation in the gene resulted in decreased growth of Anabaena. The modulation in the levels of All3940 in Anabaena, achieved either by overexpression of the protein or mutation of the gene, resulted in changes in the proteome, which correlated well with the physiological changes observed. Proteins required for varied physiological activities, such as photosynthesis, carbon-metabolism, oxidative stress alleviation, exhibited change in protein profile upon modulation of All3940 levels in Anabaena. This suggested a direct or an indirect effect of All3940 on the expression of the above stress-responsive proteins, thereby enhancing tolerance in Anabaena PCC7120. Thus, All3940, though categorized as a Dps, is possibly a general stress protein having a global role in regulating tolerance to multitude of stresses in Anabaena.

  16. Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant

    PubMed Central

    Sandberg, Megan N.; Greco, Jordan A.; Wagner, Nicole L.; Amora, Tabitha L.; Ramos, Lavoisier A.; Chen, Min-Hsuan; Knox, Barry E.; Birge, Robert R.

    2015-01-01

    Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λmax = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λmax = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λmax = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early

  17. CIS-TRANS ISOMERS OF VITAMIN A AND RETINENE IN THE RHODOPSIN SYSTEM

    PubMed Central

    Hubbard, Ruth; Wald, George

    1952-01-01

    Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (λmax· 500 mµ). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (λmax· 487 mµ). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the

  18. Proteinase-treated photoreceptor discs. Photoelectric activity of the partially-digested rhodopsin and membrane orientation.

    PubMed

    Bayramashvili, D I; Drachev, A L; Drachev, L A; Kaulen, A D; Kudelin, A B; Martynov, V I; Skulachev, V P

    1984-08-01

    Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively. In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid-impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light-induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one. Incubation of papain-treated or thermolysin-treated discs at pH 6.0 induced formation of inside-out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase-sensitive site, localized between 104 and 105 residues, has been discovered in the inside-out vesicles treated with thermolysin. This fact consistent with the scheme of the 'seven column' arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179-191]. Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer. The main specific feature inherent in the photoelectric response of the papain-treated or thermolysin-treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion

  19. A single mutation converts bacterial Na(+) -transporting rhodopsin into an H(+) transporter.

    PubMed

    Mamedov, Mahir D; Mamedov, Adalyat M; Bertsova, Yulia V; Bogachev, Alexander V

    2016-09-01

    Na(+) -rhodopsins are light-driven pumps used by marine bacteria to extrude Na(+) ions from the cytoplasm. We show here that replacement of Gln123 on the cytoplasmic side of the ion-conductance channel with aspartate or glutamate confers H(+) transport activity to the Na(+) -rhodopsin from Dokdonia sp. PRO95. The Q123E variant could transport H(+) out of Escherichia coli cells in a medium containing 100 mm Na(+) and SCN(-) as the penetrating anion. The rates of the photocycle steps of this variant were only marginally dependent on Na(+) , and the major electrogenic steps were the decays of the K and O intermediates. PMID:27447358

  20. [Effects of sediment on the growth of Microcystis and Anabaena in Yanghe reservoir].

    PubMed

    Chu, Zhao-Sheng; Zhang, Yu-Bao; Jin, Xiang-Can; Xu, Ying; Yang, Hong-Jun

    2012-03-01

    Batch culture experiments were used to study the effect of leachate from sediment of Yanghe reservoir on the growth of Microcystis and Anabaena isolated from Yanghe reservoir. The results showed that the growth of Microcystis was significantly inhibited when the addition of anaerobic leachate from sediment in M11 culture medium was high (> or = 20% V/V). The maximum biomass of Microcystis was lower than that grown in pure M11 culture medium. But there was an obvious promotion on the growth of Anabaena when anaerobic leachate from sediment was added. The growth rate of Anabaena increased 36.6%, 47.2% and 36.0%, respectively, compared to M11 culture medium when adding 2%, 20% and 50% (V/V) anaerobic leachate. Compared to adding anaerobic leachate, aerobic leachate had no influence on the growth of Microcystis, while the growth rate of Anabaena was promoted by aerobic leachate. The growth rate of Anabaena increased 37.2% compared with M11 when adding 20% (V/V) of aerobic leachate. But after addition of Fe-citrate to the mixed culture medium (50% M11 culture medium +50% anaerobic leachate), the maximum biomass of Microcystis significantly increased. The results suggest that high organic matter concentration decreases iron availability for Microcystis.

  1. UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species.

    PubMed

    Shrivastava, Alok Kumar; Chatterjee, Antra; Yadav, Shivam; Singh, Prashant Kumar; Singh, Shilpi; Rai, L C

    2015-09-01

    Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

  2. Modulation of fungicidal potential of Anabaena strains by light and temperature.

    PubMed

    Chaudhary, Vidhi; Prasanna, Radha; Bhatnagar, A K

    2012-05-01

    The regulation of fungicidal and hydrolytic enzyme activity was investigated in a set of cyanobacterial strains belonging to the genus Anabaena (Anabaena laxa RPAN8, Anabaena iyengarii RPAN9, Anabaena variabilis RPAN59 and Anabaena oscillarioides RPAN69), with A. variabilis RPAN16 serving as negative control. Time course studies undertaken with cultures incubated under different light and temperature conditions revealed enhancement in growth and fungicidal activity under continuous light (CL) and light dark (LD, 16:8) conditions and temperature of 30 °C and 40 °C. A significant increase of 3-18 % in chitosanase activity was recorded in all the 4-week-old cultures under CL condition and at 40 °C. Endoglucanase activity of RPAN8 and 9 was twofolds higher than the other strains under all light/dark conditions and temperature in the 4-week-old cultures, while continuous dark (CD) enhanced CMCase activity in RPAN69. This study provided useful information regarding the most suitable conditions of light and temperature for maximizing hydrolytic enzyme activity and fungicidal activity, as a prelude to their effective use as biocontrol agents.

  3. Genetic studies on a nitrogen-fixing cyanobacterium. [Anabaena; Escherichi coli

    SciTech Connect

    Wolk, C.P.; Cardemil, L.; Elhai, J.; Flores, E.; Murry, M.; Schmetterer, G.; Schrautemeier, B.

    1987-04-01

    Mutants of Anabaena PCC7120 capable of aerobic growth with NO/sub 3//sup -/ but not N/sub 2/, and capable of microaerobic reduction of C/sub 2/H/sub 2/, were isolated by penicillin enrichment after UV irradiation. Heterocysts of two mutants lack the principal envelope glycolipid, those of EF116 have a non-cohesive envelope polysaccharide, and those of other strains have other defects. A Nm/sup r/ cosmid library of DNA from wild type Anabaena PCC7120 was established in Escherichia coli bearing the Ap helper plasmid pDS4101. A conjugative plasmid was introduced, and the bacteria replicated to lawns of individual mutant strains of Anabaena. After one day of non-selective growth, selection was applied for Nm/sup r/ and nitrogen fixation. Overlapping cosmids complementing EF116 and one complementing another mutant have been mapped. The complementing genes are thought to act early in differentiation. Inclusion, in an E. coli donor of an appropriate methylase gene enhanced, by a factor of 10/sup 2/ to 10/sup 3/, transfer to Anabaena PCC7120 of a plasmid containing numerous sites for the Anabaena restriction endonuclease, AvaII.

  4. Dark rearing rescues P23H rhodopsin-induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: a chromophore-dependent mechanism characterized by production of N-terminally truncated mutant rhodopsin.

    PubMed

    Tam, Beatrice M; Moritz, Orson L

    2007-08-22

    To elucidate the molecular mechanisms underlying the light-sensitive retinal degeneration caused by the rhodopsin mutation P23H, which causes retinitis pigmentosa (RP) in humans, we expressed Xenopus laevis, bovine, human, and murine forms of P23H rhodopsin in transgenic X. laevis rod photoreceptors. All P23H rhodopsins caused aggressive retinal degeneration associated with low expression levels and retention of P23H rhodopsin in the endoplasmic reticulum (ER), suggesting involvement of protein misfolding and ER stress. However, light sensitivity varied dramatically between these RP models, with complete or partial rescue by dark rearing in the case of bovine and human P23H rhodopsin, and no rescue for X. laevis P23H rhodopsin. Rescue by dark rearing required an intact 11-cis-retinal chromophore binding site within the mutant protein and was associated with truncation of the P23H rhodopsin N terminus. This yielded an abundant nontoxic approximately 27 kDa form that escaped the ER and was transported to the rod outer segment. The truncated protein was produced in the greatest quantities in dark-reared retinas expressing bovine P23H rhodopsin and was not observed with X. laevis P23H rhodopsin. These results are consistent with a mechanism involving enhanced protein folding in the presence of 11-cis-retinal chromophore, with ER exit assisted by proteolytic truncation of the N terminus. This study provides a molecular mechanism for light sensitivity observed in other transgenic models of RP and for phenotypic variation among RP patients.

  5. Utilization of the cyanobacteria Anabaena sp. CH1 in biological carbon dioxide mitigation processes.

    PubMed

    Chiang, Chang-Ling; Lee, Chi-Mei; Chen, Pei-Chung

    2011-05-01

    Before switching totally to alternative fuel stage, CO(2) mitigation process has considered a transitional strategy for combustion of fossil fuels inevitably. In comparison to other CO(2) mitigation options, such as oceanic or geologic injection, the biological photosynthetic process would present a far superior and sustainable solution under both environmental and social considerations. The utilization of the cyanobacteria Anabaena sp. CH1 in carbon dioxide mitigation processes is analyzed in our research. It was found that an original developed photobioreactor with internal light source exhibits high light utilization. Anabaena sp. CH1 demonstrates excellent CO(2) tolerance even at 15% CO(2) level. This enables flue gas from power plant to be directly introduced to Anabaena sp. CH1 culture. Double light intensity and increased 47% CO(2) bubble retention time could enhance CO(2) removal efficiencies by 79% and 67%, respectively. A maximum CO(2) fixation rate of 1.01 g CO(2)L(-1)day(-1) was measured experimentally.

  6. Enhanced resistance to UV-B radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by repeated exposure.

    PubMed

    Qin, Hongjie; Li, Dunhai

    2014-07-01

    In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N2-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats.

  7. Methyl viologen responsive proteome dynamics of Anabaena sp. strain PCC7120.

    PubMed

    Panda, Bandita; Basu, Bhakti; Rajaram, Hema; Kumar Apte, Shree

    2014-08-01

    A proteomic approach was employed to elucidate the response of an agriculturally important microbe, Anabaena sp. strain PCC7120, to methyl viologen (MV). Exposure to 2 μM MV caused 50% lethality (LD50 ) within 6 h and modified the cellular levels of several proteins. About 31 proteins increased in abundance and 24 proteins decreased in abundance, while 55 proteins showed only a minor change in abundance. Of these, 103 proteins were identified by MS. Levels of proteins involved in ROS detoxification and chaperoning activities were enhanced but that of crucial proteins involved in light and dark reactions of photosynthesis declined or constitutive. The abundance of proteins involved in carbon and energy biogenesis were altered. The study elaborated the oxidative stress defense mechanism deployed by Anabaena, identified carbon metabolism and energy biogenesis as possible major targets of MV sensitivity, and suggested potential biotechnological interventions for improved stress tolerance in Anabaena 7120.

  8. The effects of SO sub 2 on Azolla - Anabaena symbiosis

    SciTech Connect

    Jaeseoun Hur; Wellburn, A.R. )

    1991-05-01

    Cultures of Azolla pinnata containing Anabaena were investigated as a sensitive and reproducible bioindicator of air pollution. Three equal doses of SO{sub 2} (week*ppb: 1*100, 2*50, 4*25) were applied to Azolla cultures growing in nitrogen-free medium in a specially-designed exposure system. Exposure to high concentrations of SO{sub 2} showed highly significant reductions in growth of the fern, while nitrogen fixation and heterocyst development were severely damaged. This was associated with a reduction of protein content in the SO{sub 2}-exposed ferns and again more significant at higher SO{sub 2} levels. There was a variation in the absolute amount of the individual pigments between SO{sub 2} doses and/or treatments which was related to the physiological development of the ferns throughout the fumigations. Moreover, the ratio of violaxanthin to antheraxanthin in the 100 ppb SO{sub 2}-treated ferns was significantly higher than that in the clean air-grown ferns. The results clearly demonstrate that SO{sub 2} has adverse effects on the symbiosis and suggest that this fern is a promising bioindicator of air pollution and a very good model to investigate the inter-relationships between photosynthesis, nitrogen fixation and air pollution stress.

  9. Regeneration of filaments (colonies) from Anabaena variabilis spheroplasts

    SciTech Connect

    Lem, N.W.; Perrin, C.G.; Nemeth, M.A.

    1986-04-01

    A simple method for regeneration of filaments (clones) from spheroplasts of the cyanobacterium (blue-green alga), Anabaena variabilis, was developed and used to quantify cell growth in the presence of two antibiotics. Cells from exponential phase cultures of ATCC 29413 and M3 were harvested and incubated with lysozyme (0.12% in 0.03M K-phosphate, pH 6.8, 0.55M sorbitol; 37 C) to produce spheroplasts. The spheroplasts were washed with buffer, plated onto soft agar and incubated (18 h light: 6 hr dark, 27 C). Colonies became visible at 7 - 9 days and were monitored for times up to 21 days. The concentration of chloramphenicol which inhibited cell growth by 50% was approximately 1.8 mg ml/sup -1/ medium and the concentrations of ampicillin which inhibited cell growth by 50% were approximately 4 and 15 pg ml/sup -1/ medium for ATCC 29413 and M3, respectively. This method may be useful for genetic manipulation of cells from these and other filamentous, N/sub 2/-fixing cyanobacteria.

  10. Radiation characteristics and optical properties of filamentous cyanobacterium Anabaena cylindrica.

    PubMed

    Heng, Ri-Liang; Lee, Euntaek; Pilon, Laurent

    2014-04-01

    This study presents experimental measurements of the absorption and scattering cross sections and the spectral complex index of refraction of filamentous cyanobacteria. Filamentous heterocystous cyanobacterium Anabaena cylindrica was chosen as a model organism. Its filaments consisted of long chains of polydisperse cells. Their average mass scattering and absorption cross sections were measured from 400 to 750 nm at four different times during their batch growth in medium BG-11(-N) under 3000 lux of white fluorescent light. The effective real (or refraction index) and imaginary (or absorption index) parts of the complex index of refraction were retrieved using an inverse method based on a genetic algorithm. The microorganisms were modeled as infinitely long and randomly oriented volume-equivalent cylinders. The absorption index featured peaks corresponding to chlorophyll a (Chl a) at 436 and 676 nm and phycocyanin (PCCN) at 630 nm and a shoulder around 480 nm, corresponding to photoprotective carotenoids. The absorption peaks of Chl a and PCCN concentrations increased and the shoulder due to carotenoids decreased in response to photolimitation caused by biomass growth. Subsequent nitrogen limitation caused the PCCN absorption peak to decrease significantly due to degradation of PCCN as an endogenous source of nitrogen for nitrogenase maintenance and synthesis, as confirmed by increasing heterocyst differentiation. The results can be used for predicting and optimizing light transfer in photobioreactors for wastewater treatment and ammonia or biofuel production. PMID:24695147

  11. Hydrogen production by nitrogen-starved cultures of Anabaena cylindrica.

    PubMed Central

    Weissman, J C; Benemann, J R

    1977-01-01

    Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen continuously for 7 to 19 days. Hydrogen production attained a maximum level after 1 to 2 days of starvation and was followed by a slow decline. The maximum rates were 30 ml of H2 evolved per liter of culture per h or 32 mul of H2 per mg of dry weight per h. In 5 to 7 days the rate of H2 evolution by the more productive cultures fell to one-half its maximum value. The addition of 10(-4) to 5 X 10(-4) M ammonium increased the rate of oxygen evolution and the total hydrogen production of the cultures. H2-O2 ratios were 4:1 under conditions of complete nitrogen starvation and about 1.7:1 after the addition of ammonium. Thus, oxygen evolution was affected by the extent of the nitrogen starvation. Thermodynamic efficiencies of converting incident light energy to free energy of hydrogen via algal photosynthesis were 0.4%. Possible factors limiting hydrogen production were decline of reductant supply and filament breakage. Hydrogen production by filamentous, heterocystous blue-green algae could be used for development of a biophotolysis system. PMID:402109

  12. Physiological Reactions of the Reversible Hydrogenase from Anabaena 7120 1

    PubMed Central

    Houchins, Jeffrey P.; Burris, Robert H.

    1981-01-01

    The reversible hydrogenase from Anabaena 7120 appeared when O2 was continuously removed from a growing culture. Activity increased further when cells were incubated under argon in the dark or in the light plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Hydrogenase existed in an inactive state during periods of O2 evolution. It could be reductively activated by exposure to reduced methyl viologen or by dark, anaerobic incubation. Hydrogenase-containing cells evolved H2 slowly during dark anaerobic incubations, and the rate of H2 evolution was increased by illumination with low intensity light. Light enhancement of H2 evolution was of short duration and was eliminated by the ferredoxin antagonist disalicylidene diaminopropane. Physiological acceptors that supported H2 uptake included NO3−, NO2−, and HSO3−, and light had a slight influence on the rate of H2 uptake with these acceptors. Low levels of O2 supported H2 uptake, but higher concentrations of O2 inactivated the hydrogenase. Hydrogen uptake with HCO3− as acceptor was the most rapid reaction measured, and it was strictly light-dependent. It occurred only at low light intensities, and higher light intensities restored normal O2-evolving photosynthesis. It is suggested that hydrogenase is present to capture exogenous H2 as a source of reducing equivalents during growth in anaerobic environments. PMID:16661986

  13. Mercury-induced dark-state instability and photobleaching alterations of the visual g-protein coupled receptor rhodopsin.

    PubMed

    Morillo, Margarita; Toledo, Darwin; Pérez, Juan Jesús; Ramon, Eva; Garriga, Pere

    2014-07-21

    Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin immunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light. PMID:24911398

  14. Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Isu, Atsuko; Fukaya, Yuki; Hisabori, Toru

    2016-02-01

    In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena. PMID:26684202

  15. Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Isu, Atsuko; Fukaya, Yuki; Hisabori, Toru

    2016-02-01

    In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena.

  16. Biotransformation of 2,4,6-trinitrotoluene in Anabaena sp. cultures

    SciTech Connect

    Pavlostathis, S.G.; Jackson, G.H.

    1999-03-01

    The transformation of 2,4,6-trinitrotoluene (TNT) was investigated in cultures of the cyanobacterium Anabaena sp. by conducting a series of batch assays. 2,4,6-Trinitrotoluene was added to Anabaena sp. cultures in single and consecutive additions, at various initial concentrations, to determine its transformation kinetics, to identify products formed, to evaluate potential toxicity, and to determine the effect of light deprivation on the TNT transformation process. 2,4,6-Trinitrotoluene disappearance occurred only in the presence of Anabaena sp. cultures maintained under a normal 16-h photoperiod. Toxicity leading to culture chlorosis and death was observed in batch systems with an initial TNT concentration greater than 10 mg/L. A low rate and extent of TNT disappearance was observed in light-deprived cultures, which were inhibited even at low TNT concentrations. At pH values between 7.5 and 8.5, azoxy-tetranitrotoluene isomers were detected in both the culture medium and solvent extracts of biomass and accounted for only 20 and 4.4% of the initially added TNT moles, respectively. At a culture pH range between 5.6 and 5.9, achieved by aeration with a 5% CO{sub 2}/air mixture, hydroxylaminodinitrotoluene equimolar to the TNT addition was produced and then depleted from the culture medium with prolonged incubation. Although TNT reduction in Anabaena sp. cultures occurred, yielding low levels of azoxy-tetranitrotoluene isomers or hydroxylaminodinitrotoluene, uptake and other transformation reactions of TNT and/or its transformation products by Anabaena sp. may have taken place. Based on a less than 15% observed increase of biomass concentration over the relatively short incubation periods and by considering the mean biomass concentration constant, the TNT disappearance rate followed pseudo-first-order kinetics. The biomass carbon-normalized TNT disappearance rates in Anabaena sp. cultures were about three orders of magnitude higher than previously reported TNT

  17. [Influence of nutrient sources on Anabaena spiroides growth and odorous compounds production characteristics].

    PubMed

    Yu, Jian-Wei; Chen, Ke-Yun; Su, Ming; Yang, Min; Liu, Dai-Cheng

    2011-08-01

    The occurrence of taste and odors, produced by secondary metabolites of cyanobacteria, has been one of the major water quality problems in drinking water. However, the odorous compounds produced by cyanobacteria usually differ significantly with different species. One cyanobacterium isolated from Yanghe reservoir was identified as Anabaena sp., which can produce high level of geosmin consistently during laboratory culture. By culture expanding experiments, the algal growth and geosmin production characteristics of the Anabaena sp. were studied on different conditions of nitrogen and phosphorus sources. The results indicated that geosmin mainly remained in the intracellular algal cells regardless of the nutrient sources, and the extracellular content was only in th range of 0.2% - 9.6%. Compared with ammonia nitrogen conditions, the growth of Anabaena sp. in nitrate nitrogen conditions was much higher, with a 1.4-fold variation in geosmin production. While ammonia nitrogen concentration was 0.5 mg/L, the algal biomass and geosmin production achieved the highest level of 3.8 x 10(4) cells, mL(-1) and 1.1 x 10(4) ngL(-1), respectively. When the nitrate nitrogen concentration was 2.0 mg/L, the algal biomass and geosmin production achieved the highest level of 6.6 x 10(4) cells x mL(-1) and 1.3 x 10(4) ng x L(-1), respectively. Compared with nitrogen sources, the growth of Anabaena sp. could be promoted significantly until phosphorus level attained 0.12 mg/L, indicating that phosphorus is the main limiting nutrient source for Anabaena sp.. For Yanghe reservoir, the nutrient level has already been enough for the growth of Anabaena sp. Therefore, the nutrient source content, especially phosphorus, should be reduced effectively to control the cyanobacterium bloom and taste and odor problems.

  18. Transcription control of ribulose bisphosphate carboxylase/oxygenase activase and adjacent genes in Anabaena species.

    PubMed Central

    Li, L A; Tabita, F R

    1994-01-01

    The gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activase (rca) was uniformly localized downstream from the genes encoding the large and small subunits of RubisCO (rbcL and rbcS) in three strains of Anabaena species. However, two open reading frames (ORF1 and ORF2), situated between rbcS and rca in Anabaena sp. strain CA, were not found in the intergenic region of Anabaena variabilis and Anabaena sp. strain PCC 7120. During autotrophic growth of Anabaena cells, rca and rbc transcripts accumulated in the light and diminished in the dark; light-dependent expression of these genes was not affected by the nitrogen source and the concentration of exogenous CO2 supplied to the cells. When grown on fructose, rca- and rbc-specific transcripts accumulated in A. variabilis regardless of whether the cells were illuminated. Transcript levels, however, were much lower in dark-grown heterotrophic cultures than in photoheterotrophic cultures. In photoheterotrophic cultures, the expression of the rca and rbc genes was similar to that in cultures grown with CO2 as the sole source of carbon. Although the rbcL-rbcS and rca genes are linked and are in the same transcriptional orientation in Anabaena strains, hybridization of rbc and rca to distinct transcripts suggested that these genes are not cotranscribed, consistent with the results of primer extension and secondary structure analysis of the nucleotide sequence. Transcription from ORF1 and ORF2 was not detected under the conditions examined, and the function of these putative genes remains unknown. Images PMID:7961423

  19. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

    PubMed

    Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2015-07-30

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

  20. Salt Effects on the Conformational Stability of the Visual G-Protein-Coupled Receptor Rhodopsin

    PubMed Central

    Reyes-Alcaraz, Arfaxad; Martínez-Archundia, Marlet; Ramon, Eva; Garriga, Pere

    2011-01-01

    Membrane protein stability is a key parameter with important physiological and practical implications. Inorganic salts affect protein stability, but the mechanisms of their interactions with membrane proteins are not completely understood. We have undertaken the study of a prototypical G-protein-coupled receptor, the α-helical membrane protein rhodopsin from vertebrate retina, and explored the effects of inorganic salts on the thermal decay properties of both its inactive and photoactivated states. Under high salt concentrations, rhodopsin significantly increased its activation enthalpy change for thermal bleaching, whereas acid denaturation affected the formation of a denatured loose-bundle state for both the active and inactive conformations. This behavior seems to correlate with changes in protonated Schiff-base hydrolysis. However, chromophore regeneration with the 11-cis-retinal chromophore and MetarhodopsinII decay kinetics were slower only in the presence of sodium chloride, suggesting that in this case, the underlying phenomenon may be linked to the activation of rhodopsin and the retinal release processes. Furthermore, the melting temperature, determined by means of circular dichroism and differential scanning calorimetry measurements, was increased in the presence of high salt concentrations. The observed effects on rhodopsin could indicate that salts favor electrostatic interactions in the retinal binding pocket and indirectly favor hydrophobic interactions at the membrane protein receptor core. These effects can be exploited in applications where the stability of membrane proteins in solution is highly desirable. PMID:22261069

  1. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    SciTech Connect

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Tan, M. H. Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce D.; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E.; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S.; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

  2. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    DOE PAGESBeta

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotationmore » between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.« less

  3. Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

    SciTech Connect

    Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; Kick, Leonhard M.; Gati, Cornelius; Nelson, Garrett; Deupi, Xavier; Standfuss, Jörg; Schertler, Gebhard; Panneels, Valérie

    2015-06-27

    A new batch preparation method is presented for high-density micrometre-sized crystals of the G protein-coupled receptor rhodopsin for use in time-resolved serial femtosecond crystallography at an X-ray free-electron laser using a liquid jet. Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

  4. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    PubMed Central

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Eileen Tan, M. H.; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-01-01

    G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  5. Recombination reaction of rhodopsin in situ studied by photoconversion of "indicator yellow".

    PubMed

    Kolesnikov, A V; Shukolyukov, S A; Cornwall, M C; Govardovskii, V I

    2006-05-01

    We measured the kinetics of recombination of 11-cis-retinal with opsin in intact frog rod outer segment (ROS). The rhodopsin in ROS was bleached and allowed to decay to "indicator yellow," a photoproduct where all-trans-retinal is partly free, and partly bound to non-specific amino groups of disk membranes. By briefly illuminating the "indicator yellow" by an intense 465 or 380-nm flash, we then photoconverted all-trans-retinal to (mostly) the 11-cis- form thus introducing into ROS a certain amount of cis-chromophore. The recombination of cis-retinal with opsin and the formation of rhodopsin were followed by fast single-cell microspectrophotometry. Regeneration proceeded with a time constant of approximately 3.5 min; up to 27% of bleached visual pigment was restored. The regenerated pigment consisted of 91% rhodopsin (11-cis-chromophore) and 9% of presumably isorhodopsin (9-cis-chromophore). The recombination of 11-cis-retinal with opsin inside the ROS proceeds substantially faster than rhodopsin regeneration in the intact eye and, hence, is not the rate-limiting step in the visual cycle.

  6. The density and photosensitivity of human rhodopsin in the living retina

    PubMed Central

    Alpern, M.; Pugh, E. N.

    1974-01-01

    1. The visual pigment in a 5° circular patch of the living human retina 18° temporal from the fovea was studied with the Rushton retinal densitometer. The measuring light (570 nm) was selected to obviate artifacts from colour photoproducts. 2. The action spectrum of a 10% bleach agrees well with the action spectrum at absolute threshold for the same patch of retina. The quantized C.I.E. scotopic spectral sensitivity curve is a good description of both spectra. Therefore, the visual pigment studied must be human rhodopsin. 3. Its density has been estimated in five different ways. The results are in reasonable agreement. The optical density of human rhodopsin in vivo is about 0·35 (common logarithmic units) at its γmax. 4. The photosensitivity of human rhodopsin in vivo was determined by studying its rate of bleaching in response to steps of monochromatic light exposed to the dark adapted eye, by measuring the amount bleached in the steady state by monochromatic lights as well as the amount bleached by 10 sec flashes of white light. 5. The results obtained by the different methods are in good agreement with each other and with previous estimates made by others using white light. 6. The photosensitivity of human rhodopsin in vivo [εγmax = 62,000 to 120,000 l./cm mole] is much higher than expected from in vitro measurements. PMID:4825455

  7. Functional role of positively selected amino acid substitutions in mammalian rhodopsin evolution

    PubMed Central

    Fernández-Sampedro, Miguel A.; Invergo, Brandon M.; Ramon, Eva; Bertranpetit, Jaume; Garriga, Pere

    2016-01-01

    Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution. PMID:26865329

  8. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

    PubMed

    Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2015-07-30

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  9. Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors

    PubMed Central

    2014-01-01

    Background In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. Results We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. Conclusions We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription. PMID:24499263

  10. Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin.

    PubMed

    Ames, James B; Levay, Konstantin; Wingard, Jennifer N; Lusin, Jacqueline D; Slepak, Vladlen Z

    2006-12-01

    Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).

  11. Screening for mutations in rhodopsin and peripherin/RDS in patients with autosomal dominant retinitis pigmentosa

    SciTech Connect

    Rodriguez, J.A.; Gannon, A.M.; Daiger, S.P.

    1994-09-01

    Mutations in rhodopsin account for approximately 30% of all cases of autosomal dominant retinits pigmentosa (adRP) and mutations in peripherin/RDS account for an additional 5% of cases. Also, mutations in rhodopsin can cause autosomal recessive retinitis pigmentosa and mutations in peripherin/RDS can cause dominant macular degeneration. Most disease-causing mutations in rhodopsin and peripherin/RDS are unique to one family or, at most, to a few families within a limited geographic region, though a few mutations are found in multiple, unrelated families. To further determine the spectrum of genetic variation in these genes, we screened DNA samples from 134 unrelated patients with retinitis pigmentosa for mutations in both rhodopsin and peripherin/RDS using SSCP followed by genomic sequencing. Of the 134 patients, 86 were from families with apparent adRP and 48 were either isolated cases or were from families with an equivocal mode of inheritance. Among these patients we found 14 distinct rhodopsin mutations which are likely to cause retinal disease. Eleven of these mutations were found in one individual or one family only, whereas the Pro23His mutation was found in 14 {open_quotes}unrelated{close_quotes}individuals. The splice-site mutation produces dominant disease though with highly variable expression. Among the remaining patients were found 6 distinct peripherin/RDS mutations which are likely to cause retinal disease. These mutations were also found in one patient or family only, except the Gly266Asp mutation which was found in two unrelated patients. These results confirm the expected frequency and broad spectrum of mutations causing adRP.

  12. An ecophysiological study of the Azolla filiculoides- Anabaena azollae association

    NASA Astrophysics Data System (ADS)

    van Kempen, Monique; Smolders, Fons; Speelman, Eveline; Reichart, Gert Jan; Barke, Judith; Brinkhuis, Henk; Lotter, Andy; Roelofs, Jan

    2010-05-01

    The long term effects of salinity stress on the growth, nutrient content and amino acid composition of the Azolla filiculoides - Anabaena azollae association was studied in a laboratory experiment. It was demonstrated that the symbiosis could tolerate salt stress up to 90 mM NaCl, even after a 100 day period of preconditioning at salt concentrations that were 30 mM NaCl lower. In the 120 mM NaCl treatment the Azolla filiculoides survived, but hardly any new biomass was produced. It was shown that during the experiment, A. filiculoides became increasingly efficient in excluding salt ions from the plant tissue and was thus able to increase its salt tolerance. The amino acid analysis revealed that the naturally occurring high glutamine concentration in the plants was strongly reduced at salt concentrations of 120 mM NaCl and higher. This was the result of the reduced nitrogenase activity at these salt concentrations, as was demonstrated in an acetylene reduction assay. We suggest that the high glutamine concentration in the plants might play a role in the osmoregulatory response against salt stress, enabling growth of the A. filiculoides -Anabaena azollae association up to 90 mM NaCl. In a mesocosm experiment it furthermore was demonstrated that Azolla might manipulate its own microenvironment when grown at elevated salt concentration (up to ~50 mmol•L-1) by promoting salinity stratification, especially when it has formed a dense cover at the water surface. Beside salt stress, we also studied the growth of Azolla filiculoides in response to elevated atmospheric carbon dioxide concentration, in combination with different light intensities and different pH of the nutrient solution. The results demonstrated that as compared to the control (ambient pCO2 concentrations), Azolla filiculoides was able to produce twice as much biomass at carbon dioxide concentrations that were five times as high as the ambient pCO2 concentration. However, it was also shown that this

  13. Complete Genome Sequence of a Novel Strain of Cyanobacterium, Anabaena sp. 4-3.

    PubMed

    Pfeffer, Sarah; Sowa, Steven; Brown, R Malcolm

    2016-01-01

    We report the complete nucleotide sequence of Anabaena sp. 4-3, an efficient producer of sucrose. It was isolated from salt flats near the University of Texas Marine Science Institute in Port Aransas, Texas. The genome may provide insight into the utilization of cyanobacteria as a source for biofuels. PMID:27540066

  14. Multiple modes of iron uptake by the filamentous, siderophore-producing cyanobacterium, Anabaena sp. PCC 7120.

    PubMed

    Rudolf, Mareike; Kranzler, Chana; Lis, Hagar; Margulis, Ketty; Stevanovic, Mara; Keren, Nir; Schleiff, Enrico

    2015-08-01

    Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore- and non-siderophore-producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore-producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self-secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe') via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore- and non-siderophore-mediated iron uptake. While assimilation of Fe' and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe' reduction and uptake is advantageous for low-density cultures, while at higher densities siderophore uptake is preferred. PMID:25943160

  15. Effect of butachlor on growth and nitrogen fixation by Anabaena sphaerica.

    PubMed

    Suseela, M R

    2001-07-01

    Present study was carried out to examine the effect of Butachlor on growth and nitrogen fixation by Anabaena sphaerica. The increased concentration of the pesticide did not have any adverse effect on the alga. Rather it accelerated the algal contribution in terms of biomass and nitrogen fixation.

  16. Multiple modes of iron uptake by the filamentous, siderophore-producing cyanobacterium, Anabaena sp. PCC 7120.

    PubMed

    Rudolf, Mareike; Kranzler, Chana; Lis, Hagar; Margulis, Ketty; Stevanovic, Mara; Keren, Nir; Schleiff, Enrico

    2015-08-01

    Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore- and non-siderophore-producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore-producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self-secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe') via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore- and non-siderophore-mediated iron uptake. While assimilation of Fe' and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe' reduction and uptake is advantageous for low-density cultures, while at higher densities siderophore uptake is preferred.

  17. Complete Genome Sequence of a Novel Strain of Cyanobacterium, Anabaena sp. 4-3

    PubMed Central

    Sowa, Steven

    2016-01-01

    We report the complete nucleotide sequence of Anabaena sp. 4-3, an efficient producer of sucrose. It was isolated from salt flats near the University of Texas Marine Science Institute in Port Aransas, Texas. The genome may provide insight into the utilization of cyanobacteria as a source for biofuels. PMID:27540066

  18. Role of manganese in protection against oxidative stress under iron starvation in cyanobacterium Anabaena 7120.

    PubMed

    Kaushik, Manish Singh; Srivastava, Meenakshi; Verma, Ekta; Mishra, Arun Kumar

    2015-06-01

    The cyanobacterium Anabaena sp. PCC 7120 was grown in presence and absence of iron to decipher the role of manganese in protection against the oxidative stress under iron starvation and growth, manganese uptake kinetics, antioxidative enzymes, lipid peroxidation, electrolyte leakage, thiol content, total peroxide, proline and NADH content was investigated. Manganese supported the growth of cyanobacterium Anabaena 7120 under iron deprived conditions where maximum uptake rate of manganese was observed with lower K(m) and higher V(max) values. Antioxidative enzymes were also found to be elevated in iron-starved conditions. Estimation of lipid peroxidation and electrolyte leakage depicted the role of manganese in stabilizing the integrity of the membrane which was considered as the prime target of oxygen free radicals in oxidative stress. The levels of total peroxide, thiol, proline and NADH content, which are the representative of oxidative stress response in Anabaena 7120, were also showed increasing trends in iron starvation. Hence, the results discerned, clearly suggested the role of manganese in protection against the oxidative stress in cyanobacterium Anabaena 7120 under iron starvation either due to its antioxidative properties or involvement as cofactor in a number of antioxidative enzymes.

  19. Effect of eight polynuclear hydrocarbons on growth of Anabaena flos-aquae

    SciTech Connect

    Bastian, M.V.; Toetz, D.W.

    1982-11-01

    Unialgal cultures of Anabaena flos-aquae were exposed to toxicants and assayed for growth, as measured by the maximum standing crop. Toxicant degradation was measured by spectrofluorometry. Data show that the hydrophilic compounds tested (acenaphthene, naphthalene) stimulated growth in marked contrast to the growth-inhibiting hydrophobic polynuclear aromatic hydrocarbons.

  20. Scanning Laser Ophthalmoscope Measurement of Local Fundus Reflectance and Autofluorescence Changes Arising from Rhodopsin Bleaching and Regeneration

    PubMed Central

    Morgan, Jessica I. W.; Pugh, Edward N.

    2013-01-01

    Purpose. We measured the bleaching and regeneration kinetics of rhodopsin in the living human eye with two-wavelength, wide-field scanning laser ophthalmoscopy (SLO), and investigated the effect of rhodopsin bleaching on autofluorescence intensity. Methods. The retina was imaged with an Optos P200C SLO by its reflectance of 532 and 633 nm light, and its autofluorescence excited by 532 nm light, before and after exposure to lights calibrated to bleach rhodopsin substantially. Bleaching was confined to circular retinal regions of 4.8° visual angle located approximately 16° superotemporal and superonasal to fixation. Images were captured as 12-bit tiff files and postprocessed to extract changes in reflectance and autofluorescence. Results. At the locus of bleaching transient increases in reflectance of the 532 nm, but not the 633 nm beam were observed readily and quantified. A transient increase in autofluorescence also occurred. The action spectrum, absolute sensitivity, and recovery of the 532 nm reflectance increase were consistent with previous measurements of human rhodopsin's spectral sensitivity, photosensitivity, and regeneration kinetics. The autofluorescence changes closely tracked the changes in rhodopsin density. Conclusions. The bleaching and regeneration kinetics of rhodopsin can be measured locally in the human retina with a widely available SLO. The increased autofluorescence excited by 532 nm light upon bleaching appears primarily due to transient elimination of rhodopsin's screening of autofluorescent fluorochromes in the RPE. The spatially localized measurement with a widely available SLO of rhodopsin, the most abundant protein in the retina, could be a valuable adjunct to retinal health assessment. PMID:23412087

  1. The severe autosomal dominant retinitis pigmentosa rhodopsin mutant Ter349Glu mislocalizes and induces rapid rod cell death.

    PubMed

    Hollingsworth, T J; Gross, Alecia K

    2013-10-01

    Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype. PMID:23940033

  2. Volume and enthalpy changes after photoexcitation of bovine rhodopsin: laser-induced optoacoustic studies.

    PubMed Central

    Strassburger, J M; Gärtner, W; Braslavsky, S E

    1997-01-01

    Laser-induced optoacoustic measurements were performed with bovine rhodopsin in the temperature range 5-32 degrees C in its natural environment (i.e., in washed membranes) as well as solubilized in dodecyl-beta-D-maltoside. A signal deconvolution procedure using a simple sequential kinetic scheme for the photobaric time evolution revealed, in the case of the washed membranes, the presence of an intermediate with a 14-ns lifetime at 25 degrees C, of the same order as that reported for the BSI intermediate in solubilized rhodopsin (Hug, S. J., W. J. Lewis, C. M. Einterz, T. E. Thorgeirsson, and D. S. Kliger. 1990. Nanosecond photolysis of rhodopsin: evidence for a new, blue-shifted intermediate. Biochemistry. 29:1475-1485), with an energy content of (85 +/- 20) kJ/mol, and accompanied by an expansion of 26 +/- 3 ml/mol. The difference in energy content between BSI and the next transient lumi was estimated in only -1 +/- 5 kJ/mol, concomitant with an expansion of 9 +/- 3 ml/mol. Thus, this transition, which according to literature involves an equilibrium, should be controlled by an entropic change, rather than by an enthalpic difference. This is supported by the fact that both activation parameters for the decay of batho and BSI decrease upon solubilization. For detergent-solubilized rhodopsin, two time constants were enough to fit the sample signal. A short lifetime ascribable to BSI was not detected in this case. For the first intermediate (probably batho in equilibrium with BSI), an energy content of 50 +/- 20 kJ/mol and an expansion of 20 +/- 1 ml/mol, and for lumi an energy content of 11 +/- 20 kJ/mol and a further expansion of 11 +/- 2 ml/mol were determined. Thus, the intermediates of the membrane-embedded form of rhodopsin (in contrast to solubilized samples) are kept in a higher energy level, although the total expansion from rhodopsin to lumi is similar for both conditions (35 +/- 6 and 31 +/- 3 ml/mol). The expansions are interpreted as protein

  3. Genomic makeup of the marine flavobacterium Nonlabens (Donghaeana) dokdonensis and identification of a novel class of rhodopsins.

    PubMed

    Kwon, Soon-Kyeong; Kim, Byung Kwon; Song, Ju Yeon; Kwak, Min-Jung; Lee, Choong Hoon; Yoon, Jung-Hoon; Oh, Tae Kwang; Kim, Jihyun F

    2013-01-01

    Rhodopsin-containing marine microbes such as those in the class Flavobacteriia play a pivotal role in the biogeochemical cycle of the euphotic zone (Fuhrman JA, Schwalbach MS, Stingl U. 2008. Proteorhodopsins: an array of physiological roles? Nat Rev Microbiol. 6:488-494). Deciphering the genome information of flavobacteria and accessing the diversity and ecological impact of microbial rhodopsins are important in understanding and preserving the global ecosystems. The genome sequence of the orange-pigmented marine flavobacterium Nonlabens dokdonensis (basonym: Donghaeana dokdonensis) DSW-6 was determined. As a marine photoheterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The sequence analysis also uncovered a gene encoding an unexpected type of microbial rhodopsin containing a unique motif in addition to a proteorhodopsin gene and a number of photolyase or cryptochrome genes. Homologs of the novel rhodopsin gene were found in other flavobacteria, alphaproteobacteria, a species of Cytophagia, a deinococcus, and even a eukaryote diatom. They all contain the characteristic NQ motif and form a phylogenetically distinct group. Expression analysis of this rhodopsin gene in DSW-6 indicated that it is induced at high NaCl concentrations, as well as in the presence of light and the absence of nutrients. Genomic and metagenomic surveys demonstrate the diversity of the NQ rhodopsins in nature and the prevalent occurrence of the encoding genes among microbial communities inhabiting hypersaline niches, suggesting its involvement in sodium metabolism and the sodium-adapted lifestyle.

  4. Genomic Makeup of the Marine Flavobacterium Nonlabens (Donghaeana) dokdonensis and Identification of a Novel Class of Rhodopsins

    PubMed Central

    Kwon, Soon-Kyeong; Kim, Byung Kwon; Song, Ju Yeon; Kwak, Min-Jung; Lee, Choong Hoon; Yoon, Jung-Hoon; Oh, Tae Kwang; Kim, Jihyun F.

    2013-01-01

    Rhodopsin-containing marine microbes such as those in the class Flavobacteriia play a pivotal role in the biogeochemical cycle of the euphotic zone (Fuhrman JA, Schwalbach MS, Stingl U. 2008. Proteorhodopsins: an array of physiological roles? Nat Rev Microbiol. 6:488–494). Deciphering the genome information of flavobacteria and accessing the diversity and ecological impact of microbial rhodopsins are important in understanding and preserving the global ecosystems. The genome sequence of the orange-pigmented marine flavobacterium Nonlabens dokdonensis (basonym: Donghaeana dokdonensis) DSW-6 was determined. As a marine photoheterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The sequence analysis also uncovered a gene encoding an unexpected type of microbial rhodopsin containing a unique motif in addition to a proteorhodopsin gene and a number of photolyase or cryptochrome genes. Homologs of the novel rhodopsin gene were found in other flavobacteria, alphaproteobacteria, a species of Cytophagia, a deinococcus, and even a eukaryote diatom. They all contain the characteristic NQ motif and form a phylogenetically distinct group. Expression analysis of this rhodopsin gene in DSW-6 indicated that it is induced at high NaCl concentrations, as well as in the presence of light and the absence of nutrients. Genomic and metagenomic surveys demonstrate the diversity of the NQ rhodopsins in nature and the prevalent occurrence of the encoding genes among microbial communities inhabiting hypersaline niches, suggesting its involvement in sodium metabolism and the sodium-adapted lifestyle. PMID:23292138

  5. Genomic makeup of the marine flavobacterium Nonlabens (Donghaeana) dokdonensis and identification of a novel class of rhodopsins.

    PubMed

    Kwon, Soon-Kyeong; Kim, Byung Kwon; Song, Ju Yeon; Kwak, Min-Jung; Lee, Choong Hoon; Yoon, Jung-Hoon; Oh, Tae Kwang; Kim, Jihyun F

    2013-01-01

    Rhodopsin-containing marine microbes such as those in the class Flavobacteriia play a pivotal role in the biogeochemical cycle of the euphotic zone (Fuhrman JA, Schwalbach MS, Stingl U. 2008. Proteorhodopsins: an array of physiological roles? Nat Rev Microbiol. 6:488-494). Deciphering the genome information of flavobacteria and accessing the diversity and ecological impact of microbial rhodopsins are important in understanding and preserving the global ecosystems. The genome sequence of the orange-pigmented marine flavobacterium Nonlabens dokdonensis (basonym: Donghaeana dokdonensis) DSW-6 was determined. As a marine photoheterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The sequence analysis also uncovered a gene encoding an unexpected type of microbial rhodopsin containing a unique motif in addition to a proteorhodopsin gene and a number of photolyase or cryptochrome genes. Homologs of the novel rhodopsin gene were found in other flavobacteria, alphaproteobacteria, a species of Cytophagia, a deinococcus, and even a eukaryote diatom. They all contain the characteristic NQ motif and form a phylogenetically distinct group. Expression analysis of this rhodopsin gene in DSW-6 indicated that it is induced at high NaCl concentrations, as well as in the presence of light and the absence of nutrients. Genomic and metagenomic surveys demonstrate the diversity of the NQ rhodopsins in nature and the prevalent occurrence of the encoding genes among microbial communities inhabiting hypersaline niches, suggesting its involvement in sodium metabolism and the sodium-adapted lifestyle. PMID:23292138

  6. Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment.

    PubMed

    Earl, James B; Britt, Steven G

    2006-10-01

    The R7 and R8 photoreceptor cells of the Drosophila retina are thought to mediate color discrimination and polarized light detection. This is based on the patterned expression of different visual pigments, rhodopsins, in different photoreceptor cells. In this report, we examined the developmental timing of retinal patterning. There is genetic evidence that over the majority of the eye, patterned expression of opsin genes is regulated by a signal from one subtype of R7 cells to adjacent R8 cells. We examined the onset of expression of the rhodopsin genes to determine the latest time point by which photoreceptor subtype commitment must have occurred. We found that the onset of rhodopsin expression in all photoreceptors of the compound eye occurs during a narrow window from 79% to 84% of pupal development (approximately 8 h), pupal stages P12-P14. Rhodopsin 1 has the earliest onset, followed by Rhodopsins 3, 4, and 5 at approximately the same time, and finally Rhodopsin 6. This sequence mimics the model for how R7 and R8 photoreceptor cells are specified, and defines the timing of photoreceptor cell fate decisions with respect to other events in eye development. PMID:16495161

  7. Effects of acid stress on Scenedesmus quadricauda (chlorophyta) and Anabaena sp. (cyanophyta)

    SciTech Connect

    Hadden-Carter, P.J.

    1984-01-01

    The effects of pH in conjunction with light and temperature on growth of Scenedesmus quadricauda (Chlorophyta) and Anabaena sp. (Cyanophyta) were examined in culture. Decreasing pH from 7 to 3 inhibited growth, more so in the blue-green alga. Effects were greatly influenced by light and temperature. Above a critical level (pH4 with the blue-green, pH 3 with the green) both algae recovered when acid stress was removed; post-acidification growth rates varied inversely with pH for the green alga and directly for the blue-green. Two sheathed blue-green algae (Lyngbya and Gleocapsa) grew below pH 6, while two unsheathed blue-green algae (Anabaena and Oscillatoria) did not. Cell dimensions of both S. quadricaude and Anabaena sp. generally increased as pH declined; the green alga was the more plastic of the two. Acid stress significantly decreased photosynthetic rate in S. quadricauda but did not for Anabaena sp. Respiratory rates were not significantly related to pH for either alga. Chlorophyll a per cell was higher than controls (pH 7) at pH 5 and 6 in Anabaena sp. and at pH 4 through 6 for S. quadricauda. Both cell division and total culture biomass declined with pH. When grown in mixed culture, the green alga usually predominated at pH 4 and often at pH 5; the blue-green was favored at lower light intensities and higher temperatures. In no instance did one alga stimulate growth of the other, although mutual inhibition occurred in several instances.

  8. Classification and phylogeny of the cyanobiont Anabaena azollae Strasburger: an answered question?

    PubMed

    Pereira, Ana L; Vasconcelos, Vitor

    2014-06-01

    The symbiosis Azolla-Anabaena azollae, with a worldwide distribution in pantropical and temperate regions, is one of the most studied, because of its potential application as a biofertilizer, especially in rice fields, but also as an animal food and in phytoremediation. The cyanobiont is a filamentous, heterocystic cyanobacterium that inhabits the foliar cavities of the pteridophyte and the indusium on the megasporocarp (female reproductive structure). The classification and phylogeny of the cyanobiont is very controversial: from its morphology, it has been named Nostoc azollae, Anabaena azollae, Anabaena variabilis status azollae and recently Trichormus azollae, but, from its 16S rRNA gene sequence, it has been assigned to Nostoc and/or Anabaena, and from its phycocyanin gene sequence, it has been assigned as non-Nostoc and non-Anabaena. The literature also points to a possible co-evolution between the cyanobiont and the Azolla host, since dendrograms and phylogenetic trees of fatty acids, short tandemly repeated repetitive (STRR) analysis and restriction fragment length polymorphism (RFLP) analysis of nif genes and the 16S rRNA gene give a two-cluster association that matches the two-section ranking of the host (Azolla). Another controversy surrounds the possible existence of more than one genus or more than one species strain. The use of freshly isolated or cultured cyanobionts is an additional problem, since their morphology and protein profiles are different. This review gives an overview of how morphological, chemical and genetic analyses influence the classification and phylogeny of the cyanobiont and future research.

  9. Signaling by Sensory Receptors

    PubMed Central

    Julius, David; Nathans, Jeremy

    2012-01-01

    Sensory systems detect small molecules, mechanical perturbations, or radiation via the activation of receptor proteins and downstream signaling cascades in specialized sensory cells. In vertebrates, the two principal categories of sensory receptors are ion channels, which mediate mechanosensation, thermosensation, and acid and salt taste; and G-protein-coupled receptors (GPCRs), which mediate vision, olfaction, and sweet, bitter, and umami tastes. GPCR-based signaling in rods and cones illustrates the fundamental principles of rapid activation and inactivation, signal amplification, and gain control. Channel-based sensory systems illustrate the integration of diverse modulatory signals at the receptor, as seen in the thermosensory/pain system, and the rapid response kinetics that are possible with direct mechanical gating of a channel. Comparisons of sensory receptor gene sequences reveal numerous examples in which gene duplication and sequence divergence have created novel sensory specificities. This is the evolutionary basis for the observed diversity in temperature- and ligand-dependent gating among thermosensory channels, spectral tuning among visual pigments, and odorant binding among olfactory receptors. The coding of complex external stimuli by a limited number of sensory receptor types has led to the evolution of modality-specific and species-specific patterns of retention or loss of sensory information, a filtering operation that selectively emphasizes features in the stimulus that enhance survival in a particular ecological niche. The many specialized anatomic structures, such as the eye and ear, that house primary sensory neurons further enhance the detection of relevant stimuli. PMID:22110046

  10. Noninvasive optical inhibition with a red-shifted microbial rhodopsin

    PubMed Central

    Chuong, Amy S; Miri, Mitra L; Busskamp, Volker; Matthews, Gillian A C; Acker, Leah C; Sørensen, Andreas T; Young, Andrew; Klapoetke, Nathan C; Henninger, Mike A; Kodandaramaiah, Suhasa B; Ogawa, Masaaki; Ramanlal, Shreshtha B; Bandler, Rachel C; Allen, Brian D; Forest, Craig R; Chow, Brian Y; Han, Xue; Lin, Yingxi; Tye, Kay M; Roska, Botond; Cardin, Jessica A; Boyden, Edward S

    2014-01-01

    Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience. PMID:24997763

  11. NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

    PubMed

    Govorunova, Elena G; Sineshchekov, Oleg A; Janz, Roger; Liu, Xiaoqin; Spudich, John L

    2015-08-01

    Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

  12. Feeding and the rhodopsin family g-protein coupled receptors in nematodes and arthropods.

    PubMed

    Cardoso, João C R; Félix, Rute C; Fonseca, Vera G; Power, Deborah M

    2012-01-01

    In vertebrates, receptors of the rhodopsin G-protein coupled superfamily (GPCRs) play an important role in the regulation of feeding and energy homeostasis and are activated by peptide hormones produced in the brain-gut axis. These peptides regulate appetite and energy expenditure by promoting or inhibiting food intake. Sequence and function homologs of human GPCRs involved in feeding exist in the nematode roundworm, Caenorhabditis elegans (C. elegans), and the arthropod fruit fly, Drosophila melanogaster (D. melanogaster), suggesting that the mechanisms that regulate food intake emerged early and have been conserved during metazoan radiation. Nematodes and arthropods are the most diverse and successful animal phyla on Earth. They can survive in a vast diversity of environments and have acquired distinct life styles and feeding strategies. The aim of the present review is to investigate if this diversity has affected the evolution of invertebrate GPCRs. Homologs of the C. elegans and D. melanogaster rhodopsin receptors were characterized in the genome of other nematodes and arthropods and receptor evolution compared. With the exception of bombesin receptors (BBR) that are absent from nematodes, a similar gene complement was found. In arthropods, rhodopsin GPCR evolution is characterized by species-specific gene duplications and deletions and in nematodes by gene expansions in species with a free-living stage and gene deletions in representatives of obligate parasitic taxa. Based upon variation in GPCR gene number and potentially divergent functions within phyla we hypothesize that life style and feeding diversity practiced by nematodes and arthropods was one factor that contributed to rhodopsin GPCR gene evolution. Understanding how the regulation of food intake has evolved in invertebrates will contribute to the development of novel drugs to control nematodes and arthropods and the pests and diseases that use them as vectors. PMID:23264768

  13. Interphotoreceptor retinoid binding protein (IRBP) enhances rhodopsin regeneration in the experimentally detached retina.

    PubMed

    Duffy, M; Sun, Y; Wiggert, B; Duncan, T; Chader, G J; Ripps, H

    1993-12-01

    Results obtained in a previous study showed that, compared with a normal eyecup preparation, the amount of rhodopsin regenerated and the rate at which it was resynthesized after bleaching were reduced by about 50% when the skate retina was detached from its pigment epithelium (RPE) and replaced immediately on the apical surface of the RPE (Sun and Ripps, 1992). In the present study, these observations have been extended to preparations in which the detachment procedure was performed under fluid in order to dilute the IRBP content of the interphotoreceptor matrix. The goal initially was to determine whether lowering the IRBP concentration of the subretinal space affected the regenerative process. Using fundus reflectometry, it was found that allowing fluid to enter the subretinal space exposed by the detachment procedure caused profound deficits in both the rate and amount of rhodopsin that regenerated after bleaching. Results obtained with SDS-PAGE and immunohistochemistry showed that the molecular weight of the IRBP extracted from the skate retina is similar to that of many other vertebrate species, and that antibodies prepared against mammalian IRBP react with epitopes on skate IRBP within the interphotoreceptor matrix. Accordingly, it was investigated whether it is possible to reverse the detachment-induced anomalies in rhodopsin kinetics by introducing ligand-free IRBP purified from bovine retina to the subretinal space. Again using fundus reflectometry, it was found that instilling 5 microM of a 130 microM IRBP solution between the neural retina and the RPE increased significantly the rate of regeneration, and more than doubled the amount of rhodopsin reformed in darkness. PMID:8150029

  14. Role of RDS and Rhodopsin in Cngb1-Related Retinal Degeneration

    PubMed Central

    Chakraborty, Dibyendu; Conley, Shannon M.; Pittler, Steven J.; Naash, Muna I.

    2016-01-01

    Purpose Rod photoreceptor outer segment (OS) morphogenesis, structural integrity, and proper signal transduction rely on critical proteins found in the different OS membrane domains (e.g., plasma, disc, and disc rim membrane). Among these key elements are retinal degeneration slow (RDS, also known as peripherin-2), rhodopsin, and the beta subunit of the cyclic nucleotide gated channel (CNGB1a), which have been found to interact in a complex. The purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of CNGB1a, rhodopsin, and RDS. Methods Outer segment trafficking, retinal function, and photoreceptor structure were evaluated using knockout mouse lines. Results Eliminating Cngb1 and reducing RDS leads to additive defects in RDS expression levels and rod electroretinogram (ERG) function, (e.g., Cngb1−/−/rds+/− versus rds+/− or Cngb1−/−) but not to additive defects in rod ultrastructure. These additive effects also manifested in cone function: Photopic ERG responses were significantly lower in the Cngb1−/−/rds+/− versus rds+/− or Cngb1−/−, suggesting that eliminating Cngb1 can accelerate the cone degeneration that usually presents later in the rds+/−. This was not the case with rhodopsin; reducing rhodopsin levels in concert with eliminating CNGB1a did not lead to phenotypes more severe than those observed in the Cngb1 knockout alone. Conclusions These data support a role for RDS as the core component of a multiprotein plasma membrane-rim-disc complex that has both a structural role in photoreceptor OS formation and maintenance and a functional role in orienting proteins for optimal signal transduction. PMID:26934134

  15. Transmembrane Helices Tilt, Bend, Slide, Torque, and Unwind between Functional States of Rhodopsin

    PubMed Central

    Ren, Zhong; Ren, Peter X.; Balusu, Rohith; Yang, Xiaojing

    2016-01-01

    The seven-helical bundle of rhodopsin and other G-protein coupled receptors undergoes structural rearrangements as the transmembrane receptor protein is activated. These structural changes are known to involve tilting and bending of various transmembrane helices. However, the cause and effect relationship among structural events leading to a cytoplasmic crevasse for G-protein binding is less well defined. Here we present a mathematical model of the protein helix and a simple procedure to determine multiple parameters that offer precise depiction of a helical conformation. A comprehensive survey of bovine rhodopsin structures shows that the helical rearrangements during the activation of rhodopsin involve a variety of angular and linear motions such as torsion, unwinding, and sliding in addition to the previously reported tilting and bending. These hitherto undefined motion components unify the results obtained from different experimental approaches, and demonstrate conformational similarity between the active opsin structure and the photoactivated structures in crystallo near the retinal anchor despite their marked differences. PMID:27658480

  16. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

  17. Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum.

    PubMed

    Kikukawa, Takashi; Shimono, Kazumi; Tamogami, Jun; Miyauchi, Seiji; Kim, So Young; Kimura-Someya, Tomomi; Shirouzu, Mikako; Jung, Kwang-Hwan; Yokoyama, Shigeyuki; Kamo, Naoki

    2011-10-18

    Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.

  18. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation. PMID:26375013

  19. Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser.

    PubMed

    Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; Kick, Leonhard M; Gati, Cornelius; Nelson, Garrett; Deupi, Xavier; Standfuss, Jörg; Schertler, Gebhard; Panneels, Valérie

    2015-07-01

    Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup. PMID:26144230

  20. Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser.

    PubMed

    Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; Kick, Leonhard M; Gati, Cornelius; Nelson, Garrett; Deupi, Xavier; Standfuss, Jörg; Schertler, Gebhard; Panneels, Valérie

    2015-07-01

    Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

  1. Relationship between the excited state relaxation paths of rhodopsin and isorhodopsin.

    PubMed

    Strambi, Angela; Coto, Pedro B; Frutos, Luis Manuel; Ferré, Nicolas; Olivucci, Massimo

    2008-03-19

    The pigment Isorhodopsin, an analogue of the visual pigment Rhodopsin, is investigated via quantum-mechanics/molecular-mechanics computations based on an ab initio multiconfigurational quantum chemical treatment. The limited <5 kcal mol(-1) error found for the spectral parameters allows for a nearly quantitative analysis of the excited-state structure and reactivity of its 9-cis-retinal chromophore. We demonstrate that, similar to Rhodopsin, Isorhodopsin features a shallow photoisomerization path. However, the structure of the reaction coordinate appears to be reversed. In fact, while the coordinate still corresponds to an asynchronous crankshaft motion, the dominant isomerization component involves a counterclockwise, rather than clockwise, twisting of the 9-cis bond. Similarly, the minor component involves a clockwise, rather than counterclockwise, twisting of the 11-trans bond. Ultimately, these results indicate that Rhodopsin and Isorhodopsin relax along a common excited-state potential energy valley starting from opposite ends. The fact that the central and lowest energy region of such valley runs along a segment of the intersection space between the ground and excited states of the protein explains why the pigments decay at distinctive conical intersection structures. PMID:18302369

  2. NEUROPHYSIOLOGICAL EVALUATION OF SENSORY SYSTEMS'

    EPA Science Inventory

    Exposure to many neurotoxic compounds has been shown to produce a sensory system dysfunction. Neurophysiological assessment of sensory function in humans and animal models often uses techniques known as sensory evoked potentials. Because both humans and animals show analogous res...

  3. Physiological and proteomic analysis of salinity tolerance of the halotolerant cyanobacterium Anabaena sp.

    PubMed

    Yadav, Ravindra Kumar; Thagela, Preeti; Tripathi, Keshawanand; Abraham, G

    2016-09-01

    The halotolerant cyanobacterium Anabaena sp was grown under NaCl concentration of 0, 170 and 515 mM and physiological and proteomic analysis was performed. At 515 mM NaCl the cyanobacterium showed reduced photosynthetic activities and significant increase in soluble sugar content, proline and SOD activity. On the other hand Anabaena sp grown at 170 mM NaCl showed optimal growth, photosynthetic activities and comparatively low soluble sugar content, proline accumulation and SOD activity. The intracellular Na(+) content of the cells increased both at 170 and 515 mM NaCl. In contrast, the K(+) content of the cyanobacterium Anabaena sp remained stable in response to growth at identical concentration of NaCl. While cells grown at 170 mM NaCl showed highest intracellular K(+)/Na(+) ratio, salinity level of 515 mM NaCl resulted in reduced ratio of K(+)/Na(+). Proteomic analysis revealed 50 salt-responsive proteins in the cyanobacterium Anabaena sp under salt treatment compared with control. Ten protein spots were subjected to MALDI-TOF-MS/MS analysis and the identified proteins are involved in photosynthesis, protein folding, cell organization and energy metabolism. Differential expression of proteins related to photosynthesis, energy metabolism was observed in Anabaena sp grown at 170 mM NaCl. At 170 mM NaCl increased expression of photosynthesis related proteins and effective osmotic adjustment through increased antioxidant enzymes and modulation of intracellular ions contributed to better salinity tolerance and optimal growth. On the contrary, increased intracellular Na(+) content coupled with down regulation of photosynthetic and energy related proteins resulted in reduced growth at 515 mM NaCl. Therefore reduced growth at 515 mM NaCl could be due to accumulation of Na(+) ions and requirement to maintain higher organic osmolytes and antioxidants which is energy intensive. The results thus show that the basis of salt tolerance is different when the

  4. Selection on synonymous codons in mammalian rhodopsins: a possible role in optimizing translational processes

    PubMed Central

    2014-01-01

    Background Synonymous codon usage can affect many cellular processes, particularly those associated with translation such as polypeptide elongation and folding, mRNA degradation/stability, and splicing. Highly expressed genes are thought to experience stronger selection pressures on synonymous codons. This should result in codon usage bias even in species with relatively low effective population sizes, like mammals, where synonymous site selection is thought to be weak. Here we use phylogenetic codon-based likelihood models to explore patterns of codon usage bias in a dataset of 18 mammalian rhodopsin sequences, the protein mediating the first step in vision in the eye, and one of the most highly expressed genes in vertebrates. We use these patterns to infer selection pressures on key translational mechanisms including polypeptide elongation, protein folding, mRNA stability, and splicing. Results Overall, patterns of selection in mammalian rhodopsin appear to be correlated with post-transcriptional and translational processes. We found significant evidence for selection at synonymous sites using phylogenetic mutation-selection likelihood models, with C-ending codons found to have the highest relative fitness, and to be significantly more abundant at conserved sites. In general, these codons corresponded with the most abundant tRNAs in mammals. We found significant differences in codon usage bias between rhodopsin loops versus helices, though there was no significant difference in mean synonymous substitution rate between these motifs. We also found a significantly higher proportion of GC-ending codons at paired sites in rhodopsin mRNA secondary structure, and significantly lower synonymous mutation rates in putative exonic splicing enhancer (ESE) regions than in non-ESE regions. Conclusions By focusing on a single highly expressed gene we both distinguish synonymous codon selection from mutational effects and analytically explore underlying functional mechanisms

  5. Hereditary sensory neuropathies.

    PubMed

    Auer-Grumbach, Michaela

    2004-05-01

    Hereditary sensory neuropathies (HSNs) are a group of genetically determined peripheral neuropathies with prominent disturbance of the peripheral sensory neurons. They are characterized by sensory loss, insensitivity to pain, a variable degree of muscle weakness and wasting, as well as autonomic features. Frequent complications are foot ulcerations and infections that may lead to osteomyelitis, followed by necrosis and amputations. Consequently, the hereditary sensory neuropathies have also been termed ulceromutilating neuropathies. On the other hand, in the presence of additional motor weakness, they have been subclassified among the group of Charcot-Marie-Tooth (CMT) disorders. Sporadic and familial cases with different modes of inheritance are known to affect both children and adults. The most prevalent forms of the autosomal dominantly inherited hereditary sensory neuropathies are HSN I and CMT 2b. HSN I is associated with mutations in the SPTLC1 gene, whereas mutations in the RAB7 gene have been identified in CMT 2b. However, at least one more hitherto unknown gene responsible for autosomal-dominant hereditary sensory neuropathies must exist. Autosomal-recessive hereditary sensory neuropathies types III and IV, and probably also type V, result from mutations in the IKBKAP and NTRK1 genes. Very recently, the gene in HSN II (HSN2) has been identified. A spontaneous autosomal-recessive mutation in the Cct4 gene has been reported in the Sprague-Dawley rat strain with early onset sensory neuropathy. Although no curative treatment is available so far, and current therapy is limited to symptom relief, these molecular genetic advances in knowledge about the hereditary sensory neuropathies can be translated into clinical practice by improving diagnosis and genetic counseling. They will also be the basis for functional studies in the future. PMID:15319794

  6. Novel heterocyst-specific flavodiiron proteins in Anabaena sp. PCC 7120.

    PubMed

    Ermakova, Maria; Battchikova, Natalia; Allahverdiyeva, Yagut; Aro, Eva-Mari

    2013-01-01

    Flavodiiron proteins present in many prokaryotic and some eukaryotic organisms have a capacity to protect cells against nitrosative or oxidative stress. In Anabaena sp. PCC 7120, Flv1 and Flv3 proteins are encoded by families of two genes. We demonstrate here that flv1A and flv3A genes are up-regulated in vegetative cells in low CO₂ and high light conditions. In contrast, flv1B and flv3B genes are expressed in N₂-fixing conditions and corresponding proteins are located exclusively in heterocysts. It is suggested that Flv1B and Flv3B protect enzymes of N₂-fixation in heterocysts of Anabaena 7120 by reducing molecular oxygen directly to water.

  7. AAV delivery of wild-type rhodopsin preserves retinal function in a mouse model of autosomal dominant retinitis pigmentosa.

    PubMed

    Mao, Haoyu; James, Thomas; Schwein, Alison; Shabashvili, Arseniy E; Hauswirth, William W; Gorbatyuk, Marina S; Lewin, Alfred S

    2011-05-01

    Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / +  or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.

  8. Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

    PubMed Central

    Thiel, T

    1993-01-01

    Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis. Images PMID:8407800

  9. Assessment of salinity-induced photorespiratory glycolate metabolism in Anabaena sp. PCC 7120.

    PubMed

    Srivastava, Ashish Kumar; Alexova, Ralitza; Jeon, Young Jae; Kohli, Gurjeet S; Neilan, Brett A

    2011-03-01

    This paper reports an investigation of salinity-induced glycolate metabolism in the cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena PCC 7120). Quantitative analysis of transcripts for the photosynthesis-associated genes encoding ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), phosphoribulokinase and transketolase, as well as those involved in glycolate metabolism (phosphoglycolate phosphatase, glycolate oxidase, alanine-glyoxylate aminotransferase and serine hydroxymethyltransferase) was performed. The expression of all investigated photosynthesis-associated genes except Rubisco was downregulated after 24 h NaCl treatment. However, under the same conditions, the transcripts encoding enzymes involved in glycolate metabolism were overexpressed. This was further confirmed by the quantitative analysis of the intermediates involved in glycolate metabolism. The intracellular levels of organic acids (glyceric, glycolic and glyoxylic acids) and amino acids (glycine and serine) were elevated in salt-treated cells as compared to those in the control cells. Transcriptional inhibition of photosynthesis-associated genes, and upregulation of genes and enhanced synthesis of intermediates associated with glycolate metabolism, indicate the occurrence of this photorespiratory metabolic pathway metabolism in Anabaena PCC 7120 under salt stress. PMID:21163840

  10. All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120.

    PubMed

    Klemke, Friederike; Beyer, Gabriele; Sawade, Linda; Saitov, Ali; Korte, Thomas; Maldener, Iris; Lockau, Wolfgang; Nürnberg, Dennis J; Volkmer, Thomas

    2014-12-01

    The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis-Menten kinetics, with kcat = 48.2 s(-1) at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the 'bi-bi ping-pong mechanism'. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

  11. Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity

    SciTech Connect

    Clarke, S.E.; Stuart, J.; Sanders-Loehr, J.

    1987-05-01

    Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 ..mu..M copper and was abolished by copper concentrations of 10 /sup +/M or higher. When the copper was chelated to schizokinen, the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 ..mu..M added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.

  12. Assessment of salinity-induced photorespiratory glycolate metabolism in Anabaena sp. PCC 7120.

    PubMed

    Srivastava, Ashish Kumar; Alexova, Ralitza; Jeon, Young Jae; Kohli, Gurjeet S; Neilan, Brett A

    2011-03-01

    This paper reports an investigation of salinity-induced glycolate metabolism in the cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena PCC 7120). Quantitative analysis of transcripts for the photosynthesis-associated genes encoding ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), phosphoribulokinase and transketolase, as well as those involved in glycolate metabolism (phosphoglycolate phosphatase, glycolate oxidase, alanine-glyoxylate aminotransferase and serine hydroxymethyltransferase) was performed. The expression of all investigated photosynthesis-associated genes except Rubisco was downregulated after 24 h NaCl treatment. However, under the same conditions, the transcripts encoding enzymes involved in glycolate metabolism were overexpressed. This was further confirmed by the quantitative analysis of the intermediates involved in glycolate metabolism. The intracellular levels of organic acids (glyceric, glycolic and glyoxylic acids) and amino acids (glycine and serine) were elevated in salt-treated cells as compared to those in the control cells. Transcriptional inhibition of photosynthesis-associated genes, and upregulation of genes and enhanced synthesis of intermediates associated with glycolate metabolism, indicate the occurrence of this photorespiratory metabolic pathway metabolism in Anabaena PCC 7120 under salt stress.

  13. Functional properties of LptA and LptD in Anabaena sp. PCC 7120.

    PubMed

    Hsueh, Yi-Ching; Brouwer, Eva-M; Marzi, Julian; Mirus, Oliver; Schleiff, Enrico

    2015-09-01

    Lipopolysaccharides (LPS) are central components of the outer membrane and consist of Lipid A, the core polysaccharide, and the O-antigen. The synthesis of LPS is initiated at the cytosolic face of the cytoplasmic membrane. The subsequent transport to and across the outer membrane involves multiple lipopolysaccharide transport (Lpt) proteins. Among those proteins, the periplasmic-localized LptA and the outer membrane-embedded LptD participate in the last steps of transfer and insertion of LPS into the outer membrane. While the process is described for proteobacterial model systems, not much is known about the machinery in cyanobacteria. We demonstrate that anaLptD (alr1278) of Anabaena sp. PCC 7120 is important for cell wall function and its pore domain shows a Lipid A sensitive cation-selective gating behavior. The N-terminal domain of anaLptD recognizes anaLptA (alr4067), but not ecLptA. Furthermore, anaLptA specifically interacts with the Lipid A from Anabaena sp. PCC 7120 only, while anaLptD binds to Lipid A isolated from Escherichia coli as well. Based on the comparative analysis of proteins from E. coli and Anabaena sp. we discuss the properties of the cyanobacterial Lpt system.

  14. Influence of phosphorus and pH on the fungicidal potential of Anabaena strains.

    PubMed

    Chaudhary, Vidhi; Prasanna, Radha; Bhatnagar, Ashok Kumar

    2013-03-01

    The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains - A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.

  15. All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120.

    PubMed

    Klemke, Friederike; Beyer, Gabriele; Sawade, Linda; Saitov, Ali; Korte, Thomas; Maldener, Iris; Lockau, Wolfgang; Nürnberg, Dennis J; Volkmer, Thomas

    2014-12-01

    The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis-Menten kinetics, with kcat = 48.2 s(-1) at pH 7.5 and 28 °C and KM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the 'bi-bi ping-pong mechanism'. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions. PMID:25320362

  16. Enhanced resistance to UV-B radiation in Anabaena sp. PCC 7120 (Cyanophyceae) by repeated exposure.

    PubMed

    Qin, Hongjie; Li, Dunhai

    2014-07-01

    In natural habitats, organisms especially phytoplankton are not always continuously subjected to ultraviolet-B radiation (UVBR). By simulation of the natural situation, the N2-fixing cyanobacterium Anabaena sp. PCC 7120 was subjected to UV-B exposure and recovery cycles. A series of morphological and physiological changes were observed in Anabaena sp. PCC 7120 under repeated UVBR when compared with controls. Such as the breakage of filaments, intervals between heterocysts, heterocyst frequency, total carbohydrate, and carotenoids were increased, while the nitrogenase activity and photosynthetic activity were inhibited by repeated UVBR; however, these activities could recover when UV-B stress was removed. Unexpectedly, the over-compensatory growth was observed at the end of the second round of exposure and recovery cycle. Our results showed that discontinuous UVBR could increase the growth rate and the tolerance as well as repair capacity of Anabaena sp. PCC 7120. These results indicate that moderate UVBR may increase the growth of cyanobacteria in natural habitats. PMID:24562674

  17. Neurocontrol in sensory cortex

    NASA Astrophysics Data System (ADS)

    Ritt, Jason; Nandi, Anirban; Schroeder, Joseph; Ching, Shinung

    Technology to control neural ensembles is rapidly advancing, but many important challenges remain in applications, such as design of controls (e.g. stimulation patterns) with specificity comparable to natural sensory encoding. We use the rodent whisker tactile system as a model for active touch, in which sensory information is acquired in a closed loop between feedforward encoding of sensory information and feedback guidance of sensing motions. Motivated by this system, we present optimal control strategies that are tailored for underactuation (a large ratio of neurons or degrees of freedom to stimulation channels) and limited observability (absence of direct measurement of the system state), common in available stimulation technologies for freely behaving animals. Using a control framework, we have begun to elucidate the feedback effect of sensory cortex activity on sensing in behaving animals. For example, by optogenetically perturbing primary sensory cortex (SI) activity at varied timing relative to individual whisker motions, we find that SI modulates future sensing behavior within 15 msec, on a whisk by whisk basis, changing the flow of incoming sensory information based on past experience. J.T.R. and S.C. hold Career Awards at the Scientific Interface from the Burroughs Wellcome Fund.

  18. Examining Sensory Quadrants in Autism

    ERIC Educational Resources Information Center

    Kern, Janet K.; Garver, Carolyn R.; Carmody, Thomas; Andrews, Alonzo A.; Trivedi, Madhukar H.; Mehta, Jyutika A.

    2007-01-01

    The purpose of this study was to examine sensory quadrants in autism based on Dunn's Theory of Sensory Processing. The data for this study was collected as part of a cross-sectional study that examined sensory processing (using the Sensory Profile) in 103 persons with autism, 3-43 years of age, compared to 103 age- and gender-matched community…

  19. Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state.

    PubMed Central

    Bentrop, J; Schwab, K; Pak, W L; Paulsen, R

    1997-01-01

    The cytoplasmic surface of Drosophila melanogaster Rh1 rhodopsin (ninaE) harbours amino acids which are highly conserved among G-protein-coupled receptors. Site-directed mutations which cause Leu81Gln or Asn86Ile amino acid substitutions in the first cytoplasmic loop of the Rh1 opsin protein, are shown to block rhodopsin synthesis in the nascent, glycosylated state from which the mutant opsin is degraded rapidly. In mutants Leu81Gln and Asn86Ile, only 20-30% and <2% respectively, of functional rhodopsins are synthesized and transported to the photoreceptive membrane. Thus, conserved amino acids in opsin's cytoplasmic surface are a critical factor in the interaction of opsin with proteins of the rhodopsin processing machinery. Photoreceptor cells expressing mutant rhodopsins undergo age-dependent degeneration in a recessive manner. PMID:9130705

  20. Modeling Photo-Bleaching Kinetics to Create High Resolution Maps of Rod Rhodopsin in the Human Retina.

    PubMed

    Ehler, Martin; Dobrosotskaya, Julia; Cunningham, Denise; Wong, Wai T; Chew, Emily Y; Czaja, Wojtek; Bonner, Robert F

    2015-01-01

    We introduce and describe a novel non-invasive in-vivo method for mapping local rod rhodopsin distribution in the human retina over a 30-degree field. Our approach is based on analyzing the brightening of detected lipofuscin autofluorescence within small pixel clusters in registered imaging sequences taken with a commercial 488nm confocal scanning laser ophthalmoscope (cSLO) over a 1 minute period. We modeled the kinetics of rhodopsin bleaching by applying variational optimization techniques from applied mathematics. The physical model and the numerical analysis with its implementation are outlined in detail. This new technique enables the creation of spatial maps of the retinal rhodopsin and retinal pigment epithelium (RPE) bisretinoid distribution with an ≈ 50μm resolution. PMID:26196397

  1. Modeling Photo-Bleaching Kinetics to Create High Resolution Maps of Rod Rhodopsin in the Human Retina

    PubMed Central

    Ehler, Martin; Dobrosotskaya, Julia; Cunningham, Denise; Wong, Wai T.; Chew, Emily Y.; Czaja, Wojtek; Bonner, Robert F.

    2015-01-01

    We introduce and describe a novel non-invasive in-vivo method for mapping local rod rhodopsin distribution in the human retina over a 30-degree field. Our approach is based on analyzing the brightening of detected lipofuscin autofluorescence within small pixel clusters in registered imaging sequences taken with a commercial 488nm confocal scanning laser ophthalmoscope (cSLO) over a 1 minute period. We modeled the kinetics of rhodopsin bleaching by applying variational optimization techniques from applied mathematics. The physical model and the numerical analysis with its implementation are outlined in detail. This new technique enables the creation of spatial maps of the retinal rhodopsin and retinal pigment epithelium (RPE) bisretinoid distribution with an ≈ 50μm resolution. PMID:26196397

  2. Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent

    PubMed Central

    Sakata, Rie; Kabutomori, Ryo; Okano, Keiko; Mitsui, Hiromasa; Takemura, Akihiro; Miwa, Tetsuya; Yamamoto, Hiroyuki; Okano, Toshiyuki

    2015-01-01

    Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C. rhodomelas eyeball and subjected to spectroscopic analysis, which revealed the presence of a pigment characterized by an absorption maximum (λmax) at 480 nm. Immunoblot analysis of the ocular protein showed a rhodopsin-like immunoreactivity. We also isolated a retinal cDNA encoding the entire coding sequence of putative C. rhodomelas rhodopsin (CrRh). HEK293EBNA cells were transfected with the CrRh cDNA and the proteins extracted from the cells were subjected to spectroscopic analysis. The recombinant CrRh showed the absorption maximum at 480 nm in the presence of 11-cis retinal. Comparison of the results from the eyeball extract and the recombinant CrRh strongly suggests that CrRh has an A1-based 11-cis-retinal chromophore and works as a photoreceptor in the C. rhodomelas retina, and hence that C. rhodomelas responds to dim blue light much the same as other deep-sea fishes. Because hydrothermal vent is a huge supply of viable food, C. rhodomelas likely do not need to participate diel vertical migration and may recognize the bioluminescence produced by aquatic animals living near the hydrothermal vents. PMID:26275172

  3. Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent.

    PubMed

    Sakata, Rie; Kabutomori, Ryo; Okano, Keiko; Mitsui, Hiromasa; Takemura, Akihiro; Miwa, Tetsuya; Yamamoto, Hiroyuki; Okano, Toshiyuki

    2015-01-01

    Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C. rhodomelas eyeball and subjected to spectroscopic analysis, which revealed the presence of a pigment characterized by an absorption maximum (λmax) at 480 nm. Immunoblot analysis of the ocular protein showed a rhodopsin-like immunoreactivity. We also isolated a retinal cDNA encoding the entire coding sequence of putative C. rhodomelas rhodopsin (CrRh). HEK293EBNA cells were transfected with the CrRh cDNA and the proteins extracted from the cells were subjected to spectroscopic analysis. The recombinant CrRh showed the absorption maximum at 480 nm in the presence of 11-cis retinal. Comparison of the results from the eyeball extract and the recombinant CrRh strongly suggests that CrRh has an A1-based 11-cis-retinal chromophore and works as a photoreceptor in the C. rhodomelas retina, and hence that C. rhodomelas responds to dim blue light much the same as other deep-sea fishes. Because hydrothermal vent is a huge supply of viable food, C. rhodomelas likely do not need to participate diel vertical migration and may recognize the bioluminescence produced by aquatic animals living near the hydrothermal vents.

  4. Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish, Careproctus rhodomelas, Living near the Deep-Sea Hydrothermal Vent.

    PubMed

    Sakata, Rie; Kabutomori, Ryo; Okano, Keiko; Mitsui, Hiromasa; Takemura, Akihiro; Miwa, Tetsuya; Yamamoto, Hiroyuki; Okano, Toshiyuki

    2015-01-01

    Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C. rhodomelas eyeball and subjected to spectroscopic analysis, which revealed the presence of a pigment characterized by an absorption maximum (λmax) at 480 nm. Immunoblot analysis of the ocular protein showed a rhodopsin-like immunoreactivity. We also isolated a retinal cDNA encoding the entire coding sequence of putative C. rhodomelas rhodopsin (CrRh). HEK293EBNA cells were transfected with the CrRh cDNA and the proteins extracted from the cells were subjected to spectroscopic analysis. The recombinant CrRh showed the absorption maximum at 480 nm in the presence of 11-cis retinal. Comparison of the results from the eyeball extract and the recombinant CrRh strongly suggests that CrRh has an A1-based 11-cis-retinal chromophore and works as a photoreceptor in the C. rhodomelas retina, and hence that C. rhodomelas responds to dim blue light much the same as other deep-sea fishes. Because hydrothermal vent is a huge supply of viable food, C. rhodomelas likely do not need to participate diel vertical migration and may recognize the bioluminescence produced by aquatic animals living near the hydrothermal vents. PMID:26275172

  5. Chemoheterotrophic Growth of the Cyanobacterium Anabaena sp. Strain PCC 7120 Dependent on a Functional Cytochrome c Oxidase

    PubMed Central

    Stebegg, Ronald; Wurzinger, Bernhard; Mikulic, Markus

    2012-01-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth. PMID:22730128

  6. Assessment of Anabaena sp. Strain PCC 7120 as a Heterologous Expression Host for Cyanobacterial Natural Products: Production of Lyngbyatoxin A.

    PubMed

    Videau, Patrick; Wells, Kaitlyn N; Singh, Arun J; Gerwick, William H; Philmus, Benjamin

    2016-09-16

    Cyanobacteria are well-known producers of natural products of highly varied structure and biological properties. However, the long doubling times, difficulty in establishing genetic methods for marine cyanobacteria, and low compound titers have hindered research into the biosynthesis of their secondary metabolites. While a few attempts to heterologously express cyanobacterial natural products have occurred, the results have been of varied success. Here, we report the first steps in developing the model freshwater cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that Anabaena 7120 can heterologously synthesize lyngbyatoxin A in yields comparable to those of the native producer, Moorea producens, and detail the design and use of replicative plasmids for compound production. We also demonstrate that Anabaena 7120 recognizes promoters from various biosynthetic gene clusters from both free-living and obligate symbiotic marine cyanobacteria. Through simple genetic manipulations, the titer of lyngbyatoxin A can be improved up to 13-fold. The development of Anabaena 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways.

  7. Posttranscriptional regulation of glutamine synthetase in the filamentous Cyanobacterium Anabaena sp. PCC 7120: differential expression between vegetative cells and heterocysts.

    PubMed

    Galmozzi, Carla V; Saelices, Lorena; Florencio, Francisco J; Muro-Pastor, M Isabel

    2010-09-01

    Genes homologous to those implicated in glutamine synthetase (GS) regulation by protein-protein interaction in the cyanobacterium Synechocystis sp. strain PCC 6803 are conserved in several cyanobacterial sequenced genomes. We investigated this GS regulatory mechanism in Anabaena sp. strain PCC 7120. In this strain the system operates with only one GS inactivation factor (inactivation factor 7A [IF7A]), encoded by open reading frame (ORF) asl2329 (gifA). Following addition of ammonium, expression of gifA is derepressed, leading to the synthesis of IF7A, and consequently, GS is inactivated. Upon ammonium removal, the GS activity returns to the initial level and IF7A becomes undetectable. The global nitrogen control protein NtcA binds to the gifA promoter. Constitutive high expression levels of gifA were found in an Anabaena ntcA mutant (CSE2), indicating a repressor role for NtcA. In vitro studies demonstrate that Anabaena GS is not inactivated by Synechocystis IFs (IF7 and IF17), indicating the specificity of the system. We constructed an Anabaena strain expressing a second inactivating factor, containing the amino-terminal part of IF17 from Synechocystis fused to IF7A. GS inactivation in this strain is more effective than that in the wild type (WT) and resembles that observed in Synechocystis. Finally we found differential expression of the IF system between heterocysts and vegetative cells of Anabaena.

  8. Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt.

    PubMed

    Waditee-Sirisattha, Rungaroon; Singh, Meenakshi; Kageyama, Hakuto; Sittipol, Daungjai; Rai, Ashwani K; Takabe, Teruhiro

    2012-11-01

    Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine-synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.

  9. Chemoheterotrophic growth of the Cyanobacterium Anabaena sp. strain PCC 7120 dependent on a functional cytochrome c oxidase.

    PubMed

    Stebegg, Ronald; Wurzinger, Bernhard; Mikulic, Markus; Schmetterer, Georg

    2012-09-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth.

  10. Time-resolved photointermediate changes in rhodopsin glutamic acid 181 mutants.

    PubMed

    Lewis, James W; Szundi, Istvan; Kazmi, Manija A; Sakmar, Thomas P; Kliger, David S

    2004-10-01

    The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.

  11. Phosphoinositides, Ezrin/Moesin, and rac1 Regulate Fusion of Rhodopsin Transport Carriers in Retinal Photoreceptors

    PubMed Central

    Deretic, Dusanka; Traverso, Valerie; Parkins, Nilda; Jackson, Fannie; de Turco, Elena B. Rodriguez; Ransom, Nancy

    2004-01-01

    The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate–binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle. PMID:13679519

  12. Reconstitution of Gloeobacter violaceus Rhodopsin with a Light-Harvesting Carotenoid Antenna†

    PubMed Central

    Imasheva, Eleonora S.; Balashov, Sergei P.; Choi, Ah Reum; Jung, Kwang-Hwan; Lanyi, Janos K.

    2009-01-01

    We show that salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin, can be reconstituted into the retinal protein from Gloeobacter violaceus expressed in E. 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 ca. 50°, similar to that in xanthorhodopsin. The results indicate that gloeobacter rhodopsin binds salinixanthin in a similar way as 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. PMID:19842712

  13. Reconstitution of Gloeobacter violaceus rhodopsin with a light-harvesting carotenoid antenna.

    PubMed

    Imasheva, Eleonora S; Balashov, Sergei P; Choi, Ah Reum; Jung, Kwang-Hwan; Lanyi, Janos K

    2009-11-24

    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.

  14. Cryptogenic sensory polyneuropathy.

    PubMed

    Pasnoor, Mamatha; Dimachkie, Mazen M; Barohn, Richard J

    2013-05-01

    Chronic sensory or sensorimotor polyneuropathy is a common cause for referral to neurologists. Despite extensive diagnostic testing, up to one-third of these patients remain without a known cause, and are referred to as having cryptogenic sensory peripheral neuropathy. Symptoms progress slowly. On examination, there may be additional mild toe flexion and extension weakness. Electrophysiologic testing and histology reveals axonal neuropathy. Prognosis is usually favorable, as most patients maintain independent ambulation. Besides patient education and reassurance, management is focused on pharmacotherapy for neuropathic pain and physical therapy for balance training, and, occasionally, assistive devices.

  15. Anatoxin-a synthetase gene cluster of the cyanobacterium Anabaena sp. strain 37 and molecular methods to detect potential producers.

    PubMed

    Rantala-Ylinen, Anne; Känä, Suvi; Wang, Hao; Rouhiainen, Leo; Wahlsten, Matti; Rizzi, Ermanno; Berg, Katri; Gugger, Muriel; Sivonen, Kaarina

    2011-10-01

    Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Méjean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.

  16. Voltage imaging in vivo with a new class of rhodopsin-based indicators

    NASA Astrophysics Data System (ADS)

    Douglass, Adam

    2013-03-01

    Reliable, optical detection of single action potentials in an intact brain is one of the longest-standing challenges in neuroscience. We have recently shown that a number of microbial rhodopsins exhibit intrinsic fluorescence that is sensitive to transmembrane potential. One class of indicator, derived from Archaerhodopsin-3 (Arch), responds to voltage transients with a speed and sensitivity that enable near-perfect identification of single action potentials in cultured neurons [Nat Methods. (2011). 9:90-5]. We have extended the use of these indicators to an in vivo context through the application of advanced imaging techniques to the larval zebrafish. Using planar-illumination, spinning-disk confocal, and epifluorescence imaging modalities, we have successfully recorded electrical activity in a variety of fish structures, including the brain and heart, in a completely noninvasive manner. Transgenic lines expressing Arch variants in defined cells enable comprehensive measurements to be made from specific target populations. In parallel, we have also extended the capabilities of our indicators by improving their multiphoton excitability and overall brightness. Microbial rhodopsin-based voltage indicators now enable optical interrogation of complex neural circuits, and electrophysiology in systems for which electrode-based techniques are challenging.

  17. Dephosphorylation of the beta 2-adrenergic receptor and rhodopsin by latent phosphatase 2

    SciTech Connect

    Yang, S.D.; Fong, Y.L.; Benovic, J.L.; Sibley, D.R.; Caron, M.G.; Lefkowitz, R.J.

    1988-06-25

    Recent evidence suggests that the function of receptors coupled to guanine nucleotide regulatory proteins may be controlled by highly specific protein kinases, e.g. rhodopsin kinase and the beta-adrenergic receptor kinase. In order to investigate the nature of the phosphatases which might be involved in controlling the state of receptor phosphorylation we studied the ability of four highly purified well characterized protein phosphatases to dephosphorylate preparations of rhodopsin or beta 2-adrenergic receptor which had been highly phosphorylated by beta-adrenergic receptor kinase. These included: type 1 phosphatase, calcineurin phosphatase, type 2A phosphatase, and the high molecular weight latent phosphatase 2. Under conditions in which all the phosphatases could dephosphorylate such common substrates as (/sup 32/P)phosphorylase a and (/sup 32/P)myelin basic protein at similar rates only the latent phosphatase 2 was active on the phosphorylated receptors. Moreover, a latent phosphatase activity was found predominantly in a sequestered membrane fraction of frog erythrocytes. This parallels the distribution of a beta-adrenergic receptor phosphatase activity recently described in these cells. These data suggest a potential role for the latent phosphatase 2 as a specific receptor phosphatase.

  18. Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

    NASA Astrophysics Data System (ADS)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2016-02-01

    This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

  19. Small-angle neutron and X-ray scattering reveal conformational changes in rhodopsin activation

    NASA Astrophysics Data System (ADS)

    Shrestha, Utsab R.; Bhowmik, Debsindhu; Perera, Suchitrhanga M. C. D.; Chawla, Udeep; Struts, Andrey V.; Graziono, Vito; Pingali, Sai Venkatesh; Heller, William T.; Qian, Shuo; Brown, Michael F.; Chu, Xiang-Qiang

    2015-03-01

    Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photo-activation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from 11-cis to all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how protein organizational structure changes with detergent environment in solution. We use both small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) techniques to answer the above questions. For the first time we observe the change in protein conformational ensemble upon photo-activation by SANS with contrast variation, which enables the separate study of the protein structure within the detergent assembly. In addition, SAXS study of protein structure within detergent assembly suggests that the detergent molecules form a belt of monolayer (micelle) around protein with different geometrical shapes to keep the protein in folded state.

  20. Efficient femtosecond energy transfer from carotenoid to retinal in gloeobacter rhodopsin-salinixanthin complex.

    PubMed

    Iyer, E Siva Subramaniam; Gdor, Itay; Eliash, Tamar; Sheves, Mordechai; Ruhman, Sanford

    2015-02-12

    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.

  1. X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

    PubMed

    Tsukamoto, Takashi; Mizutani, Kenji; Hasegawa, Taisuke; Takahashi, Megumi; Honda, Naoya; Hashimoto, Naoki; Shimono, Kazumi; Yamashita, Keitaro; Yamamoto, Masaki; Miyauchi, Seiji; Takagi, Shin; Hayashi, Shigehiko; Murata, Takeshi; Sudo, Yuki

    2016-06-01

    Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance. PMID:27129243

  2. CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    PubMed Central

    Xu, Ying; Wang, Tao

    2016-01-01

    ABSTRACT Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity. PMID:26598556

  3. Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase

    PubMed Central

    Grigoriev, Ilya I.; Senin, Ivan I.; Tikhomirova, Natalya K.; Komolov, Konstantin E.; Permyakov, Sergei E.; Zernii, Evgeni Yu.; Koch, Karl-Wilhelm; Philippov, Pavel P.

    2012-01-01

    Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK. PMID:22408603

  4. Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation

    PubMed Central

    Kimata, Naoki; Pope, Andreyah; Eilers, Markus; Opefi, Chikwado A.; Ziliox, Martine; Hirshfeld, Amiram; Zaitseva, Ekaterina; Vogel, Reiner; Sheves, Mordechai; Reeves, Philip J.; Smith, Steven O.

    2016-01-01

    The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation. PMID:27585742

  5. Ocular findings in a form of retinitis pigmentosa with a rhodopsin gene defect.

    PubMed Central

    Berson, E L

    1990-01-01

    Ocular findings are presented in 17 unrelated patients with a form of autosomal dominant retinitis pigmentosa and the same C to A transversion in codon 23 of the rhodopsin gene. These patients (mean age, 36.6 years) had, on average, significantly better visual acuity and larger ERG amplitudes than 131 unrelated patients (mean age, 32.1 years) with autosomal dominant retinitis pigmentosa without this mutation. These 17 patients from separate families as well as 11 relatives with the mutation from 4 of these families showed interfamilial and intrafamilial variability with respect to severity of their ocular disease. This clinical heterogeneity among patients with the same mutation, with older patients sometimes showing less loss of visual function and less intraretinal bone spicule pigment than younger patients, suggests that some factor other than the gene defect itself is involved in the expression of this condition. This form of retinitis pigmentosa can now be detected by testing leukocyte DNA from peripheral blood. Patients so identified should have an ocular examination to determine the extent of their disease in view of the clinical heterogeneity that exists among patients with this mutation. Some mechanisms by which this mutation in the rhodopsin gene could lead to photoreceptor cell death are discussed. Opportunities for future clinical and laboratory research in search of possible treatments are considered. Images FIGURE 2 FIGURE 5 A FIGURE 5 B FIGURE 5 C FIGURE 5 D PMID:2095030

  6. Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation.

    PubMed

    Kimata, Naoki; Pope, Andreyah; Eilers, Markus; Opefi, Chikwado A; Ziliox, Martine; Hirshfeld, Amiram; Zaitseva, Ekaterina; Vogel, Reiner; Sheves, Mordechai; Reeves, Philip J; Smith, Steven O

    2016-01-01

    The 11-cis retinal chromophore is tightly packed within the interior of the visual receptor rhodopsin and isomerizes to the all-trans configuration following absorption of light. The mechanism by which this isomerization event drives the outward rotation of transmembrane helix H6, a hallmark of activated G protein-coupled receptors, is not well established. To address this question, we use solid-state NMR and FTIR spectroscopy to define the orientation and interactions of the retinal chromophore in the active metarhodopsin II intermediate. Here we show that isomerization of the 11-cis retinal chromophore generates strong steric interactions between its β-ionone ring and transmembrane helices H5 and H6, while deprotonation of its protonated Schiff's base triggers the rearrangement of the hydrogen-bonding network involving residues on H6 and within the second extracellular loop. We integrate these observations with previous structural and functional studies to propose a two-stage mechanism for rhodopsin activation. PMID:27585742

  7. Three-dimensional model for meta-II rhodopsin, an activated G-protein-coupled receptor.

    PubMed

    Nikiforovich, Gregory V; Marshall, Garland R

    2003-08-01

    A novel approach that iteratively combined the results of energy calculations and experimental data was used to generate a three-dimensional (3D) model of the photoactivated state (R*) of bovine rhodopsin (Rh). The approach started with simplified energy calculations in an effort to find a set of sterically and energetically reasonable options for transmembrane (TM) helix arrangements with all-trans-retinal. Various 3D models of TM helix packing found by computations were then compared to limited site-directed spin-label experimental data regarding the transition of the TM helices of Rh in the inactive state (R) to those in the R* state to identify the most plausible model of the TM helical bundle. At the next step, all non-TM structural elements, such as the non-TM helix 8, the N- and C-terminal fragments, and the loops connecting TM helices, were reconstructed, and after the entire R* structure had been relaxed, all other currently available additional experimental data, both mutational and spectroscopic, on the structure of the meta-II state of rhodopsin were used to validate the resulting 3D model.

  8. Initial excited-state dynamics of an N-alkylated indanylidene-pyrroline (NAIP) rhodopsin analog.

    PubMed

    Schapiro, Igor; Fusi, Stefania; Olivucci, Massimo; Andruniów, Tadeusz; Sasidharanpillai, Swaroop; Loppnow, Glen R

    2014-10-23

    N-Alkylated indanylidene-pyrroline-based molecular switches mimic different aspects of the light-induced retinal chromophore isomerization in rhodopsin: the vertebrate dim-light visual pigment. In particular, they display a similar ultrashort excited-state lifetime, subpicosecond photoproduct appearance time, and photoproduct vibrational coherence. To better understand the early light-induced dynamics of such systems, we measured and modeled the resonance Raman spectra of the Z-isomer of the N-methyl-4-(5'-methoxy-2',2'-dimethyl-indan-1'-ylidene)-5-methyl-2,3-dihydro-2H-pyrrolium (NAIP) switch in methanol solution. It is shown that the data, complemented with a <70 fs excited-state trajectory computation, demonstrate initial excited-state structural dynamics dominated by double-bond expansion and single-bond contraction stretches. This mode subsequently couples with the five-membered ring inversion and double-bond torsion. These results are discussed in the context of the mechanism of the excited-state photoisomerization of NAIP switches in solution and the 11-cis retinal in rhodopsin. PMID:25255466

  9. Rhodopsin and retinochrome in the retina of a marine gastropod, Conomulex luhuanus.

    PubMed

    Ozaki, K; Terakita, A; Hara, R; Hara, T

    1986-01-01

    Photopigments in the conch retina were examined with special attention given to the photic vesicles characteristic of gastropod photoreceptors. Three different fractions of visual cell fragments were prepared from the retina: the MV-fraction containing the rhabdomal microvilli, and the PVH- and PVL-fractions containing the photic vesicles located in the visual cell body. Rhodopsin was found in the MV-fraction (lambda max = 474 nm), and yielded a photoequilibrium mixture with metarhodopsin (lambda max = 512 nm) on irradiation with blue light. Retinochrome was found in both of the PVH- and PVL-fractions (lambda max = approximately 510 nm), and was bleached into metaretinochrome by exposure to orange light, showing no marked shift of the absorption peak. Unlike the PVH-fraction, the PVL-fraction contains much aporetinochrome in addition to retinochrome, suggesting that the large mass of photic vesicles around the nucleus may serve as storage for retinal in retinochrome and for newly synthesized aporetinochrome. The total amount of retinochrome in the retina was several times higher than that of rhodopsin, distinguishing the gastropod eye from the cephalopod eye.

  10. Rhodopsin and retinochrome in the retina of a marine gastropod, Conomulex luhuanus.

    PubMed

    Ozaki, K; Terakita, A; Hara, R; Hara, T

    1986-01-01

    Photopigments in the conch retina were examined with special attention given to the photic vesicles characteristic of gastropod photoreceptors. Three different fractions of visual cell fragments were prepared from the retina: the MV-fraction containing the rhabdomal microvilli, and the PVH- and PVL-fractions containing the photic vesicles located in the visual cell body. Rhodopsin was found in the MV-fraction (lambda max = 474 nm), and yielded a photoequilibrium mixture with metarhodopsin (lambda max = 512 nm) on irradiation with blue light. Retinochrome was found in both of the PVH- and PVL-fractions (lambda max = approximately 510 nm), and was bleached into metaretinochrome by exposure to orange light, showing no marked shift of the absorption peak. Unlike the PVH-fraction, the PVL-fraction contains much aporetinochrome in addition to retinochrome, suggesting that the large mass of photic vesicles around the nucleus may serve as storage for retinal in retinochrome and for newly synthesized aporetinochrome. The total amount of retinochrome in the retina was several times higher than that of rhodopsin, distinguishing the gastropod eye from the cephalopod eye. PMID:3750849

  11. Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

    PubMed

    Smith, Adam W

    2015-01-01

    Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These

  12. Structured Sensory Trauma Interventions

    ERIC Educational Resources Information Center

    Steele, William; Kuban, Caelan

    2010-01-01

    This article features the National Institute of Trauma and Loss in Children (TLC), a program that has demonstrated via field testing, exploratory research, time series studies, and evidence-based research studies that its Structured Sensory Intervention for Traumatized Children, Adolescents, and Parents (SITCAP[R]) produces statistically…

  13. Recording Sensory Words

    ERIC Educational Resources Information Center

    Ashbrook, Peggy

    2007-01-01

    From children's viewpoints, what they experience in the world is what the world is like--for everyone. "What do others experience with their senses when they are in the same situation?" is a question that young children can explore by collecting data as they use a "feely box," or take a "sensory walk." There are many ways to focus the children's…

  14. Studying Sensory Perception.

    ERIC Educational Resources Information Center

    Ackerly, Spafford C.

    2001-01-01

    Explains the vestibular organ's role in balancing the body and stabilizing the visual world using the example of a hunter. Describes the relationship between sensory perception and learning. Recommends using optical illusions to illustrate the distinctions between external realities and internal perceptions. (Contains 13 references.) (YDS)

  15. Environmental Awareness (Sensory Awareness).

    ERIC Educational Resources Information Center

    Carpenter, Marian

    Capitalizing on the resources available within a city block, this resource guide for the emotionally handicapped (K-6) describes methods and procedures for developing sensory awareness in the urban out-of-doors. Conceptual focus is on interdependency ("living things are interdependent"). Involvement in the environment (observing, thinking, doing)…

  16. Bovine rhodopsin: amino acid substitutions Asp-83----Asn and Glu-134----Gln prevent activation of cyclic GMP phosphodiesterase.

    PubMed

    Gurevich, V V; Zozulya, S A; Zerf, E P; Pokrovskaya, I D; Obukhova, T A; Garnovskaya, M N; Dumler, I L; Rychkova, M P

    1990-01-01

    Two bovine rhodopsin mutants with substitutions of negatively charged residues within transmembrane domains II and III by uncharged ones (Asp-83----Asn and Glu-134----Gln) were constructed. Both mutants stimulated transducin GTPase with slightly lowered efficiency, but were completely unable to activate cyclic GMP phosphodiesterase. PMID:1966786

  17. Sensory analysis of lipstick.

    PubMed

    Yap, K C S; Aminah, A

    2011-06-01

    Sensory analysis of lipstick product by trained panellists started with recruiting female panels who are lipstick users, in good health condition and willing to be a part of sensory members. This group of people was further scrutinized with duo-trio method using commercial lipstick samples that are commonly used among them. About 40% of the 15 panels recruited were unable to differentiate the lipstick samples they usually use better than chance. The balance of nine panels that were corrected at least with 65% across all trials in panels screening process was formed a working group to develop sensory languages as a means of describing product similarities and differences and a scoring system. Five sessions with each session took about 90 min were carried out using 10 types of lipsticks with different waxes mixture ratio in the formulation together with six commercial lipsticks that are the most common to the panels. First session was focus on listing out the panels' perception towards the characteristic of the lipstick samples after normal application on their lips. Second session was focus on the refining and categorizing the responses gathered from the first session and translated into sensory attributes with its definition. Third session was focus on the scoring system. Fourth and fifth sessions were repetition of the third session to ensure consistency. In a collective effort of the panels, sensory attributes developed for lipstick were Spreadability, Off flavour, Hardness, Smoothness, Moist, Not messy, Glossy and Greasy. Analysis of variance was able to provide ample evidence on gauging the panel performance. A proper panels selecting and training was able to produce a reliable and sensitive trained panel for evaluating the product based on the procedures being trained.

  18. Sensory analysis of lipstick.

    PubMed

    Yap, K C S; Aminah, A

    2011-06-01

    Sensory analysis of lipstick product by trained panellists started with recruiting female panels who are lipstick users, in good health condition and willing to be a part of sensory members. This group of people was further scrutinized with duo-trio method using commercial lipstick samples that are commonly used among them. About 40% of the 15 panels recruited were unable to differentiate the lipstick samples they usually use better than chance. The balance of nine panels that were corrected at least with 65% across all trials in panels screening process was formed a working group to develop sensory languages as a means of describing product similarities and differences and a scoring system. Five sessions with each session took about 90 min were carried out using 10 types of lipsticks with different waxes mixture ratio in the formulation together with six commercial lipsticks that are the most common to the panels. First session was focus on listing out the panels' perception towards the characteristic of the lipstick samples after normal application on their lips. Second session was focus on the refining and categorizing the responses gathered from the first session and translated into sensory attributes with its definition. Third session was focus on the scoring system. Fourth and fifth sessions were repetition of the third session to ensure consistency. In a collective effort of the panels, sensory attributes developed for lipstick were Spreadability, Off flavour, Hardness, Smoothness, Moist, Not messy, Glossy and Greasy. Analysis of variance was able to provide ample evidence on gauging the panel performance. A proper panels selecting and training was able to produce a reliable and sensitive trained panel for evaluating the product based on the procedures being trained. PMID:21272038

  19. Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Napolitano, Mauro; Rubio, Miguel Ángel; Santamaría-Gómez, Javier; Olmedo-Verd, Elvira; Robinson, Nigel J; Luque, Ignacio

    2012-05-01

    Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium. PMID:22389488

  20. Acyl-acyl carrier protein: Lysomonogalactosyldiacylglycerol acyl transferase in Anabaena variabilis

    SciTech Connect

    Chen, H.H.

    1989-01-01

    Monogalactosyldiacylglycerol was produced when membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were incubated with ({sup 14}C)acyl-acyl carrier protein. This enzymatic synthesis of monogalactosyldiacylglycerol localized in the membranes was not dependent on any added cofactors, such as ATP, coenzyme A, and dithiothreitol. Palmitoyl-, stearoyl-, and oleoyl-acyl carrier proteins were approximately equally active as substrates with Km of 0.37, 0.36, and 0.23 {mu}M, respectively. The ({sup 14}C)acyl group was exclusively transferred to the sn-1 hydroxyl of the glycerol backbone of monogalactosyldiacylglycerol as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. Using a double labelled ({sup 14}C)acyl-({sup 14}C)acyl carrier protein, this enzyme catalyzed the direct transfer of the acyl group from acyl-acyl carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by the increased activity with the addition of the lysomonogalactosyldiacylglycerol suspension. A specific galactolipid acyl hydrolase activity was released into the soluble protein fraction when the membranes of Anabaena variabilis were treated with 2% Triton X-100. The positional specificity of this acyl hydrolase was demonstrated to be similar to that of Rhizopus lipase, i.e. only the acyl group at the sn-1 position was hydrolyzed. The acyl hydrolase which was also localized in the membrane fraction of Anabaena variabilis was presumably responsible for producing endogenous lysomonogalactosyldiacylglycerol used by the acyltransferase.

  1. H2, N2, and O2 metabolism by isolated heterocysts from Anabaena sp. strain CA.

    PubMed Central

    Smith, R L; Kumar, D; Zhang, X K; Tabita, F R; Van Baalen, C

    1985-01-01

    Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution. These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp. strain CA-V possessing fragile vegetative cell walls. Hydrogen production was observed with isolated heterocysts. The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production. This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients. The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA. H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished. A phenotypic Hup- mutant (N9AR) of Anabaena sp. strain CA was isolated which did not respond to nickel. In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+. Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts. PMID:3921524

  2. Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

    PubMed Central

    Khudyakov, Ivan Y.; Golden, James W.

    2001-01-01

    Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria. PMID:11673438

  3. Inhibitory effect of petroleum oil on photosynthetic electron transport system in the cyanobacterium Anabaena doliolum

    SciTech Connect

    Singh, A.K.; Kumar, H.D. )

    1991-12-01

    Virtually nothing is known about the site of action of oil and the mechanism of inhibition of photosynthetic electron transport, a process responsible for the generation of ATP and NADPH, which are essential for carbon fixation. The present study was an attempt to learn something about these aspects. The influence of diesel on photosynthetic O{sub 2}-evolution, {sup 14}CO{sub 2} fixation, and electron transport system has been examined in Anabaena doliolum, a heterocystous cyanobacterium. A. doliolum and other heterocystous cyanobacteria are widely distributed in soil and aquatic ecosystems, and represent an important group of free-living nitrogen fixing microorganisms.

  4. Reactive oxygen species and antioxidant enzymes activity of Anabaena sp. PCC 7120 (Cyanobacterium) under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Li, Gen-bao; Liu, Yong-ding; Wang, Gao-hong; Song, Li-rong

    2004-12-01

    It was found that reactive oxygen species in Anabaena cells increased under simulated microgravity provided by clinostat. Activities of intracellular antioxidant enzymes, such as superoxide dismutase, catalase were higher than those in the controlled samples during the 7 days' experiment. However, the contents of gluathione, an intracellular antioxidant, decreased in comparison with the controlled samples. The results suggested that microgravity provided by clinostat might break the oxidative/antioxidative balance. It indicated a protective mechanism in algal cells, that the total antioxidant system activity increased, which might play an important role for algal cells to adapt the environmental stress of microgravity.

  5. Growth, photosynthesis and nitrogen fixation of Anabaena doliolum exposed to Assam crude extract

    SciTech Connect

    Gaur, J.P.; Singh, A.K. )

    1990-03-01

    Petroleum contaminants pose serious threat to the structure and functioning of aquatic ecosystems. Although their effects on various algal species have been studied before, heterocystous blue-green algae (cyanobacteria) have been little explored in this regard. Inadequate information concerning the effects of petroleum oils on them, especially on their nitrogen fixing system, prompted the authors to undertake this study. In this communication the authors report the influence of Assam crude on growth, photosynthesis ({sup 14}C incorporation), nitrogen fixation (nitrogenase activity) and heterocyst differentiation in Anabaena doliolum. This species and other heterocystous cyanobacteria occur widely in soil and aquatic ecosystems.

  6. Aerobic hydrogen accumulation by a nitrogen-fixing Cyanobacterium, Anabaena sp

    SciTech Connect

    Asada, Y.; Kawamura, S.

    1986-05-01

    Hydrogen evolution by a nitrogen-fixing cyanobacterium, Anabaena sp. strain N-7363, was tested in order to develop a water biophotolysis system under aerobic conditions. A culture of the strain supplemented with carbon dioxide under an air atmosphere evolved hydrogen and oxygen gas, which reached final concentrations of 9.7 and 69.8%, respectively, after 12 days of incubation. Hydrogen uptake activity was not observed during incubation, and nitrogenase was thought to be the sole enzyme responsible for the hydrogen evolution.

  7. Cloning and sequencing of the ferredoxin gene of blue-green alga Anabaena siamensis

    NASA Astrophysics Data System (ADS)

    Li, Shou-Dong; Song, Li-Rong; Liu, Yong-Ding; Zhao, Jin-Dong

    1998-03-01

    The structure gene for ferredoxin, petFI, from Anabaena siamensis has been amplified by polymerase chain reaction(PCR) and cloned into cloning vector pGEM-3zf(+). The nucleotide sequence of petFI has been determined with silver staining sequencing method. There is 96.8% homology between coding region of petFI from A. siamensis and that of petFI from A. sp. 7120. Amino acid sequences of seven strains of blue-green algae are compared.

  8. Effects of Aminooxyacetate and Aminoacetonitrile on Glycolate and Ammonia Release by the Cyanobacterium Anabaena cylindrica1

    PubMed Central

    Bergman, Birgitta; Renström, Eva; Hällbom, Lars; Codd, Geoffrey A.

    1985-01-01

    Aminooxyacetate and aminoacetonitrile cause increased excretion of glycolate by the cyanobacterium Anabaena cylindrica. Both compounds also reduce NH4-N release induced by methionine sulfoximine in non-nitrogen-fixing cultures. Changes in amino acid pool sizes together with changes in activities of some enzymes related to glycolate metabolism show that glyoxylate to glycine conversion and glycine to serine conversion are inhibited by aminooxyacetate and aminoacetonitrile, respectively. The results also verify that photorespiratory glycolate metabolism via amination of glyoxylate is operative in A. cylindrica. PMID:16664093

  9. CRYPTOGENIC SENSORY POLYNEUROPATHY

    PubMed Central

    Pasnoor, Mamatha; Dimachkie, Mazen M.; Barohn, Richard J.

    2014-01-01

    Chronic sensory or sensorimotor polyneuropathy is a common cause for referral to neurologists. Despite extensive diagnostic testing, up to one-third of these patients remain without a known cause. They are referred to as having cryptogenic sensory peripheral neuropathy (CSPN). The age of onset is variable but usually in the sixth to seventh decade of life, affecting men and women equally. CSPN symptoms progress slowly, most patients present with distal leg paresthesias or pain that progressed over years to involve the hands. On examination, there may be additional mild toe flexion and extension weakness. Electrophysiologic testing and histology reveals axonal neuropathy. Prognosis is usually favorable as most patients maintain independent ambulation. Besides patient education and reassurance, management is focused on pharmacotherapy of neuropathic pain (see Treatment of Painful Peripheral Neuropathy chapter) and physical therapy for balance training and occasionally assistive devices. PMID:23642719

  10. Instabilities in sensory processes

    NASA Astrophysics Data System (ADS)

    Balakrishnan, J.

    2014-07-01

    In any organism there are different kinds of sensory receptors for detecting the various, distinct stimuli through which its external environment may impinge upon it. These receptors convey these stimuli in different ways to an organism's information processing region enabling it to distinctly perceive the varied sensations and to respond to them. The behavior of cells and their response to stimuli may be captured through simple mathematical models employing regulatory feedback mechanisms. We argue that the sensory processes such as olfaction function optimally by operating in the close proximity of dynamical instabilities. In the case of coupled neurons, we point out that random disturbances and fluctuations can move their operating point close to certain dynamical instabilities triggering synchronous activity.

  11. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-04-23

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.

  12. Mn-catalase (Alr0998) protects the photosynthetic, nitrogen-fixing cyanobacterium Anabaena PCC7120 from oxidative stress.

    PubMed

    Banerjee, Manisha; Ballal, Anand; Apte, Shree Kumar

    2012-11-01

    Role of the non-haem, manganese catalase (Mn-catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn-catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat(+). The Alr0998 protein could be immunodetected in AnKat(+) cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat(+) cells but not in the wild-type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn-catalase. In response to oxidative stress, the AnKat(+) showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress-inducible 2-Cys-Prx protein. Treatment of wild-type Anabaena PCC7120 with H(2)O(2) caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO(2) fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat(+) strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn-catalase.

  13. BIOTRANSFORMATION OF 2,4,6-TRINITROTOLUENE IN A CONTINUOUS-FLOW ANABAENA SP. SYSTEM. (R825513C013)

    EPA Science Inventory

    Reductive transformation of 2,4,6-trinitrotoluene (TNT) was observed in a continuous-flow system of Anabaena sp. operated for 33 d with a 5.7 d hydraulic retention time and a range of influent TNT concentrations of 1–58 mg/l. The TNT removal effici...

  14. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen

    SciTech Connect

    Wolk, C.P.; Cai, Y.; Cardemil, L.; Flores, E.; Hohn, B.; Murry, M.; Schmetterer, G.; Schrautemeier, B.; Wilson, R.

    1988-03-01

    Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

  15. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

    PubMed Central

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-01-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. PMID:25915115

  16. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-01-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion. PMID:25915115

  17. Sensory cilia in arthropods.

    PubMed

    Keil, Thomas A

    2012-11-01

    In arthropods, the modified primary cilium is a structure common to all peripheral sensory neurons other than photoreceptors. Since its first description in 1958, it has been investigated in great detail in numerous sense organs (sensilla) of many insect species by means of electron microscopy and electrophysiology. The perfection of molecular biological methods has led to an enormous advance in our knowledge about development and function of sensory cilia in the fruitfly since the end of the last century. The cilia show a wealth of adaptations according to their different physiological roles: chemoreception, mechanoreception, hygroreception, and thermoreception. Divergent types of receptors and channels have evolved fulfilling these tasks. The number of olfactory receptor genes can be close to 300 in ants, whereas in crickets slightest mechanical stimuli are detected by the interaction of extremely sophisticated biomechanical devices with mechanosensory cilia. Despite their enormous morphological and physiological divergence, sensilla and sensory cilia develop according to a stereotyped pattern. Intraflagellar transport genes have been found to be decisive for proper development and function.

  18. Sensory Perception: Lessons from Synesthesia

    PubMed Central

    Harvey, Joshua Paul

    2013-01-01

    Synesthesia, the conscious, idiosyncratic, repeatable, and involuntary sensation of one sensory modality in response to another, is a condition that has puzzled both researchers and philosophers for centuries. Much time has been spent proving the condition’s existence as well as investigating its etiology, but what can be learned from synesthesia remains a poorly discussed topic. Here, synaesthesia is presented as a possible answer rather than a question to the current gaps in our understanding of sensory perception. By first appreciating the similarities between normal sensory perception and synesthesia, one can use what is known about synaesthesia, from behavioral and imaging studies, to inform our understanding of “normal” sensory perception. In particular, in considering synesthesia, one can better understand how and where the different sensory modalities interact in the brain, how different sensory modalities can interact without confusion ― the binding problem ― as well as how sensory perception develops. PMID:23766741

  19. The Anabaena sp. PCC 7120 Exoproteome: Taking a Peek outside the Box

    PubMed Central

    Oliveira, Paulo; Martins, Nuno M.; Santos, Marina; Couto, Narciso A. S.; Wright, Phillip C.; Tamagnini, Paula

    2015-01-01

    The interest in examining the subset of proteins present in the extracellular milieu, the exoproteome, has been growing due to novel insights highlighting their role on extracellular matrix organization and biofilm formation, but also on homeostasis and development. The cyanobacterial exoproteome is poorly studied, and the role of cyanobacterial exoproteins on cell wall biogenesis, morphology and even physiology is largely unknown. Here, we present a comprehensive examination of the Anabaena sp. PCC 7120 exoproteome under various growth conditions. Altogether, 139 proteins belonging to 16 different functional categories have been identified. A large fraction (48%) of the identified proteins is classified as “hypothetical”, falls into the “other categories” set or presents no similarity to other proteins. The evidence presented here shows that Anabaena sp. PCC 7120 is capable of outer membrane vesicle formation and that these vesicles are likely to contribute to the exoproteome profile. Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space. Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins. PMID:25782455

  20. The transient catalytically competent coenzyme allocation into the active site of Anabaena ferredoxin NADP+ -reductase.

    PubMed

    Peregrina, José Ramón; Lans, Isaías; Medina, Milagros

    2012-01-01

    Ferredoxin-NADP(+) reductase (FNR) catalyses the electron transfer from ferredoxin to NADP(+) via its flavin FAD cofactor. A molecular dynamics theoretical approach is applied here to visualise the transient catalytically competent interaction of Anabaena FNR with its coenzyme, NADP(+). The particular role of some of the residues identified as key in binding and accommodating the 2'P-AMP moiety of the coenzyme is confirmed in molecular terms. Simulations also indicate that the architecture of the active site precisely contributes to the orientation of the N5 of the FAD isoalloxazine ring and the C4 of the coenzyme nicotinamide ring in the conformation of the catalytically competent hydride transfer complex and, therefore, contributes to the efficiency of the process. In particular, the side chain of the C-terminal Y303 in Anabaena FNR appears key to providing the optimum geometry by reducing the stacking probability between the isoalloxazine and nicotinamide rings, thus providing the required co-linearity and distance among the N5 of the flavin cofactor, the C4 of the coenzyme nicotinamide and the hydride that has to be transferred between them. All these factors are highly related to the reaction efficiency, mechanism and reversibility of the process.

  1. Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120

    SciTech Connect

    Wolk, C. Peter Wolk; Fan, Qing; Zhou, Ruanbao; Huang, Guocun; Lechno-Yossef, Sigal; Kuritz, Tanya; Wojciuch, Elizabeth

    2007-01-01

    The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF{sub A}) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.

  2. Effects of lead accumulation on the Azolla caroliniana-Anabaena association.

    PubMed

    Roberts, Anne E; Boylen, Charles W; Nierzwicki-Bauer, Sandra A

    2014-04-01

    The effect of lead accumulation on photopigment production, mineral nutrition, and Anabaena vegetative cell size and heterocyst formation in Azolla caroliniana was investigated. Plants were exposed to 0, 1, 5, 10, and 20 mg L(-1) lead acetate for ten days. Lead accumulation increased when plants were treated with higher lead concentrations. Results revealed a statistically significant decline in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids in 5, 10, and 20 mg Pb L(-1) treatment groups as compared to plants with 0 or 1 mg Pb L(-1) treatments. No statistically significant change in anthocyanin production was observed. Calcium, magnesium, and zinc concentrations in plants decreased in increasing treatment groups, whereas sodium and potassium concentrations increased. Nitrogen and carbon were also found to decrease in plant tissue. Anabaena vegetative cells decreased in size and heterocyst frequency declined rapidly in a Pb dose-dependent manner. These results indicate that, while A. caroliniana removes lead from aqueous solution, the heavy metal causes physiological and biochemical changes by impairing photosynthesis, changing mineral nutrition, and impeding the growth and formation of heterocysts of the symbiotic cyanobacteria that live within leaf cavities of the fronds.

  3. Glycolipid biosynthesis in cyanobacteria. [Anabaena variabilis; Chlorogloeopsis sp. ; Schizothrix calcicola; Anacystis nidulans; Anacystis marina

    SciTech Connect

    Van Dusen, W.J.; Jaworski, J.G.

    1987-05-01

    The biosynthesis of monogalactosyldiacyl-glycerol (MGDG) was studied in five different cyanobacteria. Previous work has shown Anabaena variabilis to synthesize both MGDG and monoglucosyl-diacylglycerol (MG1cDG) with MG1cDG being the precursor of MGDG. They have examined four other cyanobacteria to determine if a similar relationship exists. The cyanobacteria studied were Anabaena variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis nidulans, and Anacystis marina. Each were grown in liquid culture and lipids were labeled with /sup 14/C)CO/sub 2/ for 20 min., 1.0 hr, 1.0 hr + 10 hr chase. Glycolipids were analyzed by initial separation of MGDG and MG1cDG by TLC followed by further analysis by HPLC. Complete separation of molecular species was obtained isocratically on an ODS column. All of the cyanobacteria labeled 16-C and 18-C fatty acids except for A. marina which labeled only 14-C and 16-C fatty acids. Desaturation of the fatty acids could be observed in the 1.0 hr and chase experiments. All were capable of labeling both MG1cDG and MGDG with the precursor-product relationship being observed. There does not appear to be a direct relationship between the epimerization of the sugar moiety and fatty acid desaturation.

  4. Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Happe, Thomas; Schütz, Kathrin; Böhme, Herbert

    2000-01-01

    A 10-kb DNA region of the cyanobacterium Anabaena variabilis ATCC 29413 containing the structural genes of the uptake hydrogenase (hupSL) was cloned and sequenced. In contrast to the hupL gene of Anabaena sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the hupL gene during the heterocyst differentiation of A. variabilis. The hupSL genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The hupSL operon in A. variabilis was interrupted by an interposon deletion (mutant strain AVM13). Under N2-fixing conditions, the mutant strain exhibited significantly increased rates in H2 accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H2 developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type. PMID:10692368

  5. Effects of lead accumulation on the Azolla caroliniana-Anabaena association.

    PubMed

    Roberts, Anne E; Boylen, Charles W; Nierzwicki-Bauer, Sandra A

    2014-04-01

    The effect of lead accumulation on photopigment production, mineral nutrition, and Anabaena vegetative cell size and heterocyst formation in Azolla caroliniana was investigated. Plants were exposed to 0, 1, 5, 10, and 20 mg L(-1) lead acetate for ten days. Lead accumulation increased when plants were treated with higher lead concentrations. Results revealed a statistically significant decline in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids in 5, 10, and 20 mg Pb L(-1) treatment groups as compared to plants with 0 or 1 mg Pb L(-1) treatments. No statistically significant change in anthocyanin production was observed. Calcium, magnesium, and zinc concentrations in plants decreased in increasing treatment groups, whereas sodium and potassium concentrations increased. Nitrogen and carbon were also found to decrease in plant tissue. Anabaena vegetative cells decreased in size and heterocyst frequency declined rapidly in a Pb dose-dependent manner. These results indicate that, while A. caroliniana removes lead from aqueous solution, the heavy metal causes physiological and biochemical changes by impairing photosynthesis, changing mineral nutrition, and impeding the growth and formation of heterocysts of the symbiotic cyanobacteria that live within leaf cavities of the fronds. PMID:24509077

  6. Energy transfer in Anabaena variabilis filaments under nitrogen depletion, studied by time-resolved fluorescence.

    PubMed

    Onishi, Aya; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

    2015-08-01

    Some filamentous cyanobacteria (including Anabaena) differentiate into heterocysts under nitrogen-depleted conditions. During differentiation, the phycobiliproteins and photosystem II in the heterocysts are gradually degraded. Nitrogen depletion induces changes in the pigment composition of both vegetative cells and heterocysts, which affect the excitation energy transfer processes. To investigate the changes in excitation energy transfer processes of Anabaena variabilis filaments grown in standard medium (BG11) and a nitrogen-free medium (BG110), we measured their steady-state absorption spectra, steady-state fluorescence spectra, and time-resolved fluorescence spectra (TRFS) at 77 K. TRFS were measured with a picosecond time-correlated single photon counting system. The pigment compositions of the filaments grown in BG110 changed throughout the growth period; the relative phycocyanin levels monotonically decreased, whereas the relative carotenoid (Car) levels decreased and then recovered to their initial value (at day 0), with formation of lower-energy Cars. Nitrogen starvation also altered the fluorescence kinetics of PSI; the fluorescence maximum of TRFS immediately after excitation occurred at 735, 740, and 730 nm after 4, 8, and 15 days growth in BG110, respectively. Based on these results, we discuss the excitation energy transfer dynamics of A. variabilis filaments under the nitrogen-depleted condition throughout the growth period. PMID:25596847

  7. The Anabaena sp. PCC 7120 Exoproteome: Taking a Peek outside the Box.

    PubMed

    Oliveira, Paulo; Martins, Nuno M; Santos, Marina; Couto, Narciso A S; Wright, Phillip C; Tamagnini, Paula

    2015-01-01

    The interest in examining the subset of proteins present in the extracellular milieu, the exoproteome, has been growing due to novel insights highlighting their role on extracellular matrix organization and biofilm formation, but also on homeostasis and development. The cyanobacterial exoproteome is poorly studied, and the role of cyanobacterial exoproteins on cell wall biogenesis, morphology and even physiology is largely unknown. Here, we present a comprehensive examination of the Anabaena sp. PCC 7120 exoproteome under various growth conditions. Altogether, 139 proteins belonging to 16 different functional categories have been identified. A large fraction (48%) of the identified proteins is classified as "hypothetical", falls into the "other categories" set or presents no similarity to other proteins. The evidence presented here shows that Anabaena sp. PCC 7120 is capable of outer membrane vesicle formation and that these vesicles are likely to contribute to the exoproteome profile. Furthermore, the activity of selected exoproteins associated with oxidative stress has been assessed, suggesting their involvement in redox homeostasis mechanisms in the extracellular space. Finally, we discuss our results in light of other cyanobacterial exoproteome studies and focus on the potential of exploring cyanobacteria as cell factories to produce and secrete selected proteins. PMID:25782455

  8. Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes.

    PubMed

    González López, C V; Acién Fernández, F G; Fernández Sevilla, J M; Sánchez Fernández, J F; Cerón García, M C; Molina Grima, E

    2009-12-01

    In this paper the utilization of the cyanobacteria Anabaena sp. in carbon dioxide removal processes is evaluated. For this, continuous cultures of this strain were performed at different dilution rates; alternatives for the recovery of the organic matter produced being also studied. A maximum CO(2) fixation rate of 1.45 g CO(2) L(-1) day(-1) was measured experimentally, but it can be increased up to 3.0 g CO(2) L(-1) day(-1) outdoors. The CO(2) is mainly transformed into exopolysaccharides, biomass representing one third of the total organic matter produced. Organic matter can be recovered by sedimentation with efficiencies higher than 90%, the velocity of sedimentation being 2.10(-4) s(-1). The major compounds were carbohydrates and proteins with productivities of 0.70 and 0.12 g L(-1) day(-1), respectively. The behaviour of the cultures of Anabaena sp. has been modelized, also the characteristics parameters requested to design separation units being reported. Finally, to valorizate the organic matter as biofertilizers and biofuels is proposed.

  9. Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

    PubMed Central

    Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

    2012-01-01

    Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

  10. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    PubMed Central

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C.H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  11. Wavelength Discrimination in Drosophila Suggests a Role of Rhodopsin 1 in Color Vision

    PubMed Central

    Garbers, Christian; Wachtler, Thomas

    2016-01-01

    Among the five photoreceptor opsins in the eye of Drosophila, Rhodopsin 1 (Rh1) is expressed in the six outer photoreceptors. In a previous study that combined behavioral genetics with computational modeling, we demonstrated that flies can use the signals from Rh1 for color vision. Here, we provide an in-depth computational analysis of wildtype Drosophila wavelength discrimination specifically considering the consequences of different choices of computations in the preprocessing of the behavioral data. The results support the conclusion that Drosophila wavelength discrimination behavior can best be explained by a contribution of Rh1. These findings are corroborated by results of an information-theoretical analysis that shows that Rh1 provides information for discrimination of natural reflectance spectra. PMID:27258000

  12. Wavelength Discrimination in Drosophila Suggests a Role of Rhodopsin 1 in Color Vision.

    PubMed

    Garbers, Christian; Wachtler, Thomas

    2016-01-01

    Among the five photoreceptor opsins in the eye of Drosophila, Rhodopsin 1 (Rh1) is expressed in the six outer photoreceptors. In a previous study that combined behavioral genetics with computational modeling, we demonstrated that flies can use the signals from Rh1 for color vision. Here, we provide an in-depth computational analysis of wildtype Drosophila wavelength discrimination specifically considering the consequences of different choices of computations in the preprocessing of the behavioral data. The results support the conclusion that Drosophila wavelength discrimination behavior can best be explained by a contribution of Rh1. These findings are corroborated by results of an information-theoretical analysis that shows that Rh1 provides information for discrimination of natural reflectance spectra. PMID:27258000

  13. Modulation of the Absorption Maximum of Rhodopsin by Amino Acids in the C-terminus†

    PubMed Central

    Yokoyama, Shozo; Tada, Takashi; Yamato, Takahisa

    2008-01-01

    Vision begins when light is absorbed by visual pigments. It is commonly believed that the absorption spectra of visual pigments are modulated by interactions between the retinal and amino acids within or near 4.5 Å of the retinal in the transmembrane (TM) segments. However, this dogma has not been rigorously tested. In this study, we show that the retinal-opsin interactions extend well beyond the retinal binding pocket. We found that, although it is positioned outside of TM segments, the C-terminus of the rhodopsin in the rockfish longspine thornyhead (Sebastolobus altivelis) modulates its λmax by interacting mainly with the last TM segment. Our results illustrate how amino acids in the C-terminus are likely to interact with the retinal. We anticipate our analyses to be a starting point for viewing the spectral tuning of visual pigments as interactions between the retinal and key amino acids that are distributed throughout the entire pigment. PMID:16922606

  14. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

    PubMed

    Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

  15. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

    PubMed

    Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  16. Retinal conformation governs pKa of protonated Schiff base in rhodopsin activation.

    PubMed

    Zhu, Shengshuang; Brown, Michael F; Feller, Scott E

    2013-06-26

    We have explored the relationship between conformational energetics and the protonation state of the Schiff base in retinal, the covalently bound ligand responsible for activating the G protein-coupled receptor rhodopsin, using quantum chemical calculations. Guided by experimental structural determinations and large-scale molecular simulations on this system, we examined rotation about each bond in the retinal polyene chain, for both the protonated and deprotonated states that represent the dark and photoactivated states, respectively. Particular attention was paid to the torsional degrees of freedom that determine the shape of the molecule, and hence its interactions with the protein binding pocket. While most torsional degrees of freedom in retinal are characterized by large energetic barriers that minimize structural fluctuations under physiological temperatures, the C6-C7 dihedral defining the relative orientation of the β-ionone ring to the polyene chain has both modest barrier heights and a torsional energy surface that changes dramatically with protonation of the Schiff base. This surprising coupling between conformational degrees of freedom and protonation state is further quantified by calculations of the pKa as a function of the C6-C7 dihedral angle. Notably, pKa shifts of greater than two units arise from torsional fluctuations observed in molecular dynamics simulations of the full ligand-protein-membrane system. It follows that fluctuations in the protonation state of the Schiff base occur prior to forming the activated MII state. These new results shed light on important mechanistic aspects of retinal conformational changes that are involved in the activation of rhodopsin in the visual process.

  17. Novel cyanobacterial bioreporters of phosphorus bioavailability based on alkaline phosphatase and phosphate transporter genes of Anabaena sp. PCC 7120.

    PubMed

    Muñoz-Martín, M Angeles; Mateo, Pilar; Leganés, Francisco; Fernández-Piñas, Francisca

    2011-07-01

    There is heterogeneity in the way cyanobacteria respond to P starvation and subsequently how they adapt to environments with low or fluctuating P concentrations. In this study, we have fused the promoterless lux operon luxCDABE to the promoter regions of Anabaena sp. PCC 7120 phoA genes putatively encoding alkaline phosphatases, phoA (all2843) and phoA-like (alr5291) and to the promoter region of one operon putatively encoding a high affinity phosphate transporter pst1 (all4575-4572). The self-bioluminescent strains constructed in this way, Anabaena AP (phoA promoter), Anabaena AP-L (phoA-like promoter), and Anabaena PST (pst1 promoter) have been used to study the expression of these genes in response to P starvation and P re-feeding with inorganic and organic phosphate sources. Our data showed that the pst1 promoter was activated at much higher level than the phoA-like promoter following P starvation; however, we did not observe activation of the phoA promoter. The P re-feeding experiments revealed that both strains, Anabaena (A.) PST and A. AP-L could be used as novel bioreporters of P availability in environmental samples. Both strains were used to estimate bioavailable P in environmental samples (fresh- and wastewaters) with a wide range of soluble P concentrations. The results indicated that most of the P in the water samples was in chemical forms available to the cyanobacterium; however there were some differences in the estimates given by both strains as A. PST appeared to be more adequate for the samples with the lowest P load while A. AP-L gave similar or even higher values of P concentrations than those chemically measured in samples with higher P load.

  18. Proteomics combines morphological, physiological and biochemical attributes to unravel the survival strategy of Anabaena sp. PCC7120 under arsenic stress.

    PubMed

    Pandey, Sarita; Rai, Rashmi; Rai, Lal Chand

    2012-01-01

    Proteomics in conjunction with morphological, physiological and biochemical variables has been employed for the first time to unravel survival strategies of the diazotrophic cyanobacterium Anabaena sp. PCC7120 under Arsenic (As) stress. Significant reduction in growth, carbon fixation, nitrogenase activity and chlorophyll content after 1 day (1d) and recovery after 15 days (15d) of As exposure indicates the acclimation of the test organism against As stress. The formation of akinete like structures is a novel observation never reported before in Anabaena sp. PCC7120. Proteomic characterization using 2-DE showed average 537, 422 and 439 spots in control, 1 and 15d treatment respectively. MALDI-TOF and LC-MS of As-treated Anabaena revealed a total of 45 differentially expressed proteins, of which 13 were novel (hypothetical) ones. Down-regulation of phosphoglycerate kinase (PGK), fructose bisphosphate aldolase II (FBA II), fructose 1,6 bisphosphatase (FBPase), transketolase (TK), and ATP synthase on day 1 and their significant recovery on the 15th day presumably maintained the glycolysis, pentose phosphate pathway (PPP) and turnover rate of Calvin cycle, hence survival of the test organism. Up-regulation of catalase (CAT), peroxiredoxin (Prx), thioredoxin (Trx) and oxidoreductase appears to protect the cells from oxidative stress. Appreciable induction in phytochelatin content (2.4 fold), GST activity (2.3 fold), and transcripts of phytochelatin synthase (5.0 fold), arsenate reductase (8.5 fold) and arsenite efflux genes - asr1102 (5.0 fold), alr1097 (4.7 fold) reiterates their role in As sequestration and shielding of the organism from As toxicity. While up-regulated metabolic and antioxidative defense proteins, phytochelatin and GST work synchronously, the ars genes play a central role in detoxification and survival of Anabaena under As stress. The proposed hypothetical model explains the interaction of metabolic proteins associated with the survival of Anabaena sp

  19. From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats.

    PubMed

    Jones, Gareth; Teeling, Emma C; Rossiter, Stephen J

    2013-01-01

    Great advances have been made recently in understanding the genetic basis of the sensory biology of bats. Research has focused on the molecular evolution of candidate sensory genes, genes with known functions [e.g., olfactory receptor (OR) genes] and genes identified from mutations associated with sensory deficits (e.g., blindness and deafness). For example, the FoxP2 gene, underpinning vocal behavior and sensorimotor coordination, has undergone diversification in bats, while several genes associated with audition show parallel amino acid substitutions in unrelated lineages of echolocating bats and, in some cases, in echolocating dolphins, representing a classic case of convergent molecular evolution. Vision genes encoding the photopigments rhodopsin and the long-wave sensitive opsin are functional in bats, while that encoding the short-wave sensitive opsin has lost functionality in rhinolophoid bats using high-duty cycle laryngeal echolocation, suggesting a sensory trade-off between investment in vision and echolocation. In terms of olfaction, bats appear to have a distinctive OR repertoire compared with other mammals, and a gene involved in signal transduction in the vomeronasal system has become non-functional in most bat species. Bitter taste receptors appear to have undergone a "birth-and death" evolution involving extensive gene duplication and loss, unlike genes coding for sweet and umami tastes that show conservation across most lineages but loss in vampire bats. Common vampire bats have also undergone adaptations for thermoperception, via alternative splicing resulting in the evolution of a novel heat-sensitive channel. The future for understanding the molecular basis of sensory biology is promising, with great potential for comparative genomic analyses, studies on gene regulation and expression, exploration of the role of alternative splicing in the generation of proteomic diversity, and linking genetic mechanisms to behavioral consequences.

  20. From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats.

    PubMed

    Jones, Gareth; Teeling, Emma C; Rossiter, Stephen J

    2013-01-01

    Great advances have been made recently in understanding the genetic basis of the sensory biology of bats. Research has focused on the molecular evolution of candidate sensory genes, genes with known functions [e.g., olfactory receptor (OR) genes] and genes identified from mutations associated with sensory deficits (e.g., blindness and deafness). For example, the FoxP2 gene, underpinning vocal behavior and sensorimotor coordination, has undergone diversification in bats, while several genes associated with audition show parallel amino acid substitutions in unrelated lineages of echolocating bats and, in some cases, in echolocating dolphins, representing a classic case of convergent molecular evolution. Vision genes encoding the photopigments rhodopsin and the long-wave sensitive opsin are functional in bats, while that encoding the short-wave sensitive opsin has lost functionality in rhinolophoid bats using high-duty cycle laryngeal echolocation, suggesting a sensory trade-off between investment in vision and echolocation. In terms of olfaction, bats appear to have a distinctive OR repertoire compared with other mammals, and a gene involved in signal transduction in the vomeronasal system has become non-functional in most bat species. Bitter taste receptors appear to have undergone a "birth-and death" evolution involving extensive gene duplication and loss, unlike genes coding for sweet and umami tastes that show conservation across most lineages but loss in vampire bats. Common vampire bats have also undergone adaptations for thermoperception, via alternative splicing resulting in the evolution of a novel heat-sensitive channel. The future for understanding the molecular basis of sensory biology is promising, with great potential for comparative genomic analyses, studies on gene regulation and expression, exploration of the role of alternative splicing in the generation of proteomic diversity, and linking genetic mechanisms to behavioral consequences. PMID:23755015

  1. Sensory syndromes in parietal stroke.

    PubMed

    Bassetti, C; Bogousslavsky, J; Regli, F

    1993-10-01

    We studied 20 patients with an acute parietal stroke with hemisensory disturbances but no visual field deficit and no or only slight motor weakness, without thalamic involvement on CT or MRI and found three main sensory syndromes. (1) The pseudothalamic sensory syndrome consists of a faciobrachiocrural impairment of elementary sensation (touch, pain, temperature, vibration). All patients have an inferior-anterior parietal stroke involving the parietal operculum, posterior insula, and, in all but one patient, underlying white matter. (2) The cortical sensory syndrome consists of an isolated loss of discriminative sensation (stereognosis, graphesthesia, position sense) involving one or two parts of the body. These patients show a superior-posterior parietal stroke. (3) The atypical sensory syndrome consists of a sensory loss involving all modalities of sensation in a partial distribution. Parietal lesions of different topography are responsible for this clinical picture, which probably represents a minor variant of the two previous sensory syndromes. Neuropsychological dysfunction was present in 17 patients. The only constant association was between conduction aphasia and right-sided pseudothalamic sensory deficit. We conclude that parietal stroke can cause different sensory syndromes depending on the topography of the underlying lesion. Sensory deficits can be monosymptomatic but never present as a "pure sensory stroke" involving face, arm, leg, and trunk together.

  2. In silico and wet-lab study revealed cadmium is the potent inhibitor of HupL in Anabaena sp. PC C 7120.

    PubMed

    Singh, Shilpi; Shrivastava, Alok Kumar

    2016-01-01

    The hupL of Anabaena sp. PCC 7120 encodes the large subunit of uptake hydrogenase found in all diazotrophic cyanobacteria and boosts up the nitrogen-fixing potential by catalyzing the removal of the molecular hydrogen produced as a by-product of dinitrogen fixation. Bioinformatics analysis revealed that HupL from Anabaena sp. PCC7120 is a 60.2 kDa, thermostable, glycine-rich protein having highest structural similarity with NiFeSe hydrogenase of Desulfomicrobium baculatumis. Toxicity of selected abiotic stresses like arsenic, cadmium, copper, and salt with HupL was further reconciled by wet-lab approaches like qRT-PCR, hydrogenase and nitrogenase activity assay as hydrogenases unintendedly affect the nitrogenase activity in Anabaena. Down-regulated transcript along with highly inhibited hydrogenase and nitrogenase activities under cadmium stress revealed that cadmium is a potent inhibitor of hydrogenases in Anabaena which indirectly affects its nitrogen-fixing capabilities

  3. Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Ning, Degang; Qian, Yaru; Miao, Xiaogang; Wen, Chongwei

    2011-06-01

    The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.

  4. Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120.

    PubMed

    Raghavan, Prashanth S; Rajaram, Hema; Apte, Shree K

    2011-11-01

    The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.

  5. Rhodopsin mutations in a Scottish retinitis pigmentosa population, including a novel splice site mutation in intron four.

    PubMed Central

    Bell, C; Converse, C A; Hammer, H M; Osborne, A; Haites, N E

    1994-01-01

    Retinitis pigmentosa (RP) is the name given to a group of disorders, both clinically and genetically heterogeneous, that primarily affect the photoreceptor function of the eye. Mutations in the genes encoding for rhodopsin, RDS-peripherin, or the beta subunit of the cGMP phosphodiesterase enzyme can be responsible for the phenotype. In this study the rhodopsin gene has been screened for mutations in a panel of RP individuals and five different sequence changes have been detected to date in three dominantly inherited and two unclassified families. One of these, a base substitution in the 3'UTR, has not yet been confirmed as disease specific, while three missense substitutions have previously been reported and are likely to be responsible for the phenotype. The fifth change, a base substitution at the intron 4 acceptor splice site, represents a novel mutation and is assumed to be the causative mutation. Images PMID:7819178

  6. Differentiation of free-living Anabaena and Nostoc cyanobacteria on the basis of fatty acid composition.

    PubMed

    Caudales, R; Wells, J M

    1992-04-01

    The cellular fatty acids of free-living, nitrogen-fixing cyanobacteria belonging to the genera Anabaena and Nostoc were analyzed to differentiate the genera. The fatty acid compositions of 10 Anabaena strains and 10 Nostoc strains that were grown for 12 days on BG-11o medium were determined by gas-liquid chromatography-mass spectroscopy. Of the 53 fatty acids detected, 17 were major components; the average level for each of these 17 fatty acids was at least 0.9% of the total fatty acids (in at least one of the genera). These fatty acids included (with mean percentages in the Anabaena and Nostoc strains, respectively) the saturated fatty acids 16:0 (30.55 and 23.23%) and 18:0 (0.77 and 1.27%); several unsaturated fatty acids, including 14:1 cis-7 (2.50 and 0.11%), 14:1 cis-9 (3.10 and 3.41%), a polyunsaturated 16-carbon (sites undetermined) fatty acid with an equivalent chain length of 15.30 (1.20 and 1.03%), 16:4 cis-4 (0.95 and 0.87%), 16:3 cis-6 (2.16 and 1.51%), 16:1 cis-7 (1.44 and 0.36%), 16:1 cis-9 (6.53 and 18.76%), 16:1 trans-9 (4.02 and 1.35%), 16:1 cis-11 (1.62 and 0.42%), 18:2 cis-9 (10.16 and 12.44%), 18:3 cis-9 (18.19 and 17.25%), 18:1 cis-9 (4.01 and 5.10%), and 18:1 trans-9 (0.92 and 1.94%); and the branched-chain fatty acids iso-16:0 (2.50 and 1.14%) and iso-15:1 (0.34 and 2.05%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1581185

  7. Small GTPases Rab8a and Rab11a Are Dispensable for Rhodopsin Transport in Mouse Photoreceptors

    PubMed Central

    Ying, Guoxin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Boye, Sanford L.; Hauswirth, William W.; Baehr, Wolfgang

    2016-01-01

    Rab11a and Rab8a are ubiquitous small GTPases shown as required for rhodopsin transport in Xenopus laevis and zebrafish photoreceptors by dominant negative (dn) disruption of function. Here, we generated retina-specific Rab11a (retRab11a) and Rab8a (retRab8a) single and double knockout mice to explore the consequences in mouse photoreceptors. Rhodopsin and other outer segment (OS) membrane proteins targeted correctly to OS and electroretinogram (ERG) responses in all three mutant mouse lines were indistinguishable from wild-type (WT). Further, AAV (adeno-associated virus)-mediated expression of dnRab11b in retRab11a-/- retina, or expression of dnRab8b in retRab8a-/- retina did not cause OS protein mislocalization. Finally, a retRab8a-/- retina injected at one month of age with AAVs expressing dnRab11a, dnRab11b, dnRab8b, and dnRab10 (four dn viruses on Rab8a-/- background) and harvested three months later exhibited normal OS protein localization. In contrast to results obtained with dnRab GTPases in Xenopus and zebrafish, mouse Rab11a and Rab8a are dispensable for proper rhodopsin and outer segment membrane protein targeting. Absence of phenotype after expression of four dn Rab GTPases in a Rab8a-/- retina suggests that Rab8b and Rab11b paralogs maybe dispensable as well. Our data thus demonstrate significant interspecies variation in photoreceptor membrane protein and rhodopsin trafficking. PMID:27529348

  8. Spectral methods for study of the G-protein-coupled receptor rhodopsin: I. Vibrational and electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2015-05-01

    Here we review the application of modern spectral methods for the study of G-protein-coupled receptors (GPCRs) using rhodopsin as a prototype. Because X-ray analysis gives us immobile snapshots of protein conformations, it is imperative to apply spectroscopic methods for elucidating their function: vibrational (Raman, FTIR), electronic (UV-visible absorption, fluorescence) spectroscopies, and magnetic resonance (electron paramagnetic resonance, EPR), and nuclear magnetic resonance (NMR). In the first of the two companion articles, we discuss the application of optical spectroscopy for studying rhodopsin in a membrane environment. Information is obtained regarding the time-ordered sequence of events in rhodopsin activation. Isomerization of the chromophore and deprotonation of the retinal Schiff base leads to a structural change of the protein involving the motion of helices H5 and H6 in a pH-dependent process. Information is obtained that is unavailable from X-ray crystallography, which can be combined with spectroscopic studies to achieve a more complete understanding of GPCR function.

  9. Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning*

    PubMed Central

    Zheng, Lijun; Farrell, David M.; Fulton, Ruth M.; Bagg, Eve E.; Salcedo, Ernesto; Manino, Meridee; Britt, Steven G.

    2015-01-01

    The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residueat this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant. PMID:26195627

  10. Analysis of Conserved Glutamate and Aspartate Residues in Drosophila Rhodopsin 1 and Their Influence on Spectral Tuning.

    PubMed

    Zheng, Lijun; Farrell, David M; Fulton, Ruth M; Bagg, Eve E; Salcedo, Ernesto; Manino, Meridee; Britt, Steven G

    2015-09-01

    The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residue at this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant. PMID:26195627

  11. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

    PubMed

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2013-08-01

    Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy. PMID:23851421

  12. Rhodopsin and Melanopsin Contributions to the Early Redilation Phase of the Post-Illumination Pupil Response (PIPR)

    PubMed Central

    Adhikari, Prakash; Feigl, Beatrix; Zele, Andrew J.

    2016-01-01

    Melanopsin expressing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) entirely control the post-illumination pupil response (PIPR) from 6 s post-stimulus to the plateau during redilation after light offset. However, the photoreceptor contributions to the early redilation phase of the PIPR (< 6 s post-stimulus) have not been reported. Here, we evaluated the photoreceptor contributions to the early phase PIPR (0.6 s to 5.0 s) by measuring the spectral sensitivity of the criterion PIPR amplitude in response to 1 s light pulses at five narrowband stimulus wavelengths (409, 464, 508, 531 and 592 nm). The retinal irradiance producing a criterion PIPR was normalised to the peak and fitted by either a single photopigment nomogram or the combined melanopsin and rhodopsin spectral nomograms with the +L+M cone photopic luminous efficiency (Vλ) function. We show that the PIPR spectral sensitivity at times ≥ 1.7 s after light offset is best described by the melanopsin nomogram. At times < 1.7 s, the peak PIPR sensitivity shifts to longer wavelengths (range: 482 to 498 nm) and is best described by the combined photoreceptor nomogram, with major contributions from melanopsin and rhodopsin. This first report of melanopsin and rhodopsin contributions to the early phase PIPR is in line with the electrophysiological findings of ipRGC and rod signalling after the cessation of light stimuli and provides a cut-off time for isolating photoreceptor specific function in healthy and diseased eyes. PMID:27548480

  13. Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

    PubMed

    Furuse, Munenori; Tamogami, Jun; Hosaka, Toshiaki; Kikukawa, Takashi; Shinya, Naoko; Hato, Masakatsu; Ohsawa, Noboru; Kim, So Young; Jung, Kwang Hwan; Demura, Makoto; Miyauchi, Seiji; Kamo, Naoki; Shimono, Kazumi; Kimura-Someya, Tomomi; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2015-11-01

    Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

  14. Reductive transformation of methyl parathion by the cyanobacterium Anabaena sp. strain PCC7120.

    PubMed

    Barton, J W; Kuritz, T; O'Connor, L E; Ma, C Y; Maskarinec, M P; Davison, B H

    2004-08-01

    Organophosphorus compounds are toxic chemicals that are applied worldwide as household pesticides and for crop protection, and they are stockpiled for chemical warfare. As a result, they are routinely detected in air and water. Methods and routes of biodegradation of these compounds are being sought. We report that under aerobic, photosynthetic conditions, the cyanobacterium Anabaena sp. transformed methyl parathion first to o,o-dimethyl o-p-nitrosophenyl thiophosphate and then to o,o-dimethyl o-p-aminophenyl thiophosphate by reducing the nitro group. The process of methyl parathion transformation occurred in the light, but not in the dark. Methyl parathion was toxic to cyanobacteria in the dark but did not affect their viability in the light. Methyl parathion transformation was not affected by mutations in the genes involved in nitrate reduction in cyanobacteria. PMID:14758519

  15. The regulation of HanA during heterocyst development in cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Lu, Jing-Jing; Shi, Lei; Chen, Wen-Li; Wang, Li

    2014-10-01

    In response to deprivation of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops heterocyst, which is specifically involved in the nitrogen fixation. In this study, we focused on the regulation of HanA, a histone-like protein, in heterocyst development. Electrophoretic mobility shift assay results showed that NtcA, a global nitrogen regulator necessary for heterocyst differentiation, could bind to two NtcA-binding motifs in the hanA promoter region. qPCR results also showed that NtcA may regulate the expression of hanA. By using the hanA promoter-controlled gfp as a reporter gene and performing western blot we found that the amount of HanA in mature heterocysts was decreased gradually.

  16. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    PubMed

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively. PMID:25272755

  17. Stimulation of pigment accumulation in Anabaena azollae strains: effect of light intensity and sugars.

    PubMed

    Venugopal, V; Prasanna, R; Sood, A; Jaiswal, P; Kaushik, B D

    2006-01-01

    The influence of high light intensity on the growth and pigment accumulating ability of Anabaena azollae was investigated. A. azollae responded positively to high light intensity (6 klx) and was further evaluated at higher intensities (10 and 15 klx), in the presence of glucose, sucrose and jaggery +/- DCMU. Significant enhancement in phycobiliproteins and carotenoids was observed in the sugar supplemented cultures at high light intensities. SDS-PAGE profiles of whole cell proteins revealed the presence of unique bands in such treatments. Sucrose supplementation induced a 30-90 % increase in carotenoids, phycocyanin and phycoerythrin content at 10 klx. Molecular analysis of the stimulatory and interactive role of sugars on pigment enhancement at high light intensity may aid in better exploitation of cyanobacteria as a source of pigments.

  18. The electronic structure of the neutral isoalloxazine semiquinone within Anabaena flavodoxin: New insights from HYSCORE experiments

    NASA Astrophysics Data System (ADS)

    Martínez, Jesús I.; Alonso, Pablo J.; Medina, Milagros

    2012-05-01

    A complete study of Anabaena flavodoxin in the neutral semiquinone state by means of the EPR pulse technique HYSCORE is here presented. The results provide new information about the hyperfine interactions of the unpaired electronic spin and the nuclei in the isoalloxazine ring. This allows a better knowledge of the electronic structure of the neutral flavin radical within the protein. Combination of these results with other previously obtained by using other EPR related techniques allowed producing a very precise mapping of the flavin spin distribution in the neutral semiquinone state. This information can be very useful for determining the relationship between the electronic structure and mechanisms in flavoproteins. An experimental protocol for measuring the electronic structure details available to date is suggested.

  19. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

    SciTech Connect

    Owttrim, G.W.; Coleman, J.R.

    1987-05-01

    A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system.

  20. Purification and some properties of Fe protein of nitrogenase from. Anabaena cylindrica

    NASA Astrophysics Data System (ADS)

    Du, Daixian; Lin, Huimin; He, Zhenrong; Dai, Lingfen; Xin, Wusheng; Li, Shanghao

    1990-12-01

    The Fe protein of Anabaena cylindrica was first separated and purified by chromatography through DEAE-cellulose columns then by gel electrophoresis. The specific activity was up to 142.46 nmol C2H4/mg protein · min. It was homogeneous as shown by 1) a single band in the gel electrophorogram; 2) absence of Mo and tryptophan; 3) content of about 3.4 atoms of Fe per mole protein. The molecular weight of the Fe protein of A. cylindrica was about 61,000 daltons as estimated by SDS-gel electrophoresis and calculated from the amino acid composition. The residues of aspartate and glutamate were about 2.6 times that of arginine and lysine in the Fe protein. Crossing Fe protein of A. cylindrica with Mo-Fe protein of Azotobacter vinelandii gave positive result. The reciprocal crossing also showed activity.

  1. Effect of Assam crude on photosynthesis and associated electron transport system in Anabaena doliolum

    SciTech Connect

    Singh, A.K.; Gaur, J.P.

    1988-11-01

    It has been suggested that the toxic effects of oils possibly occur either on photosystem I (PS I) or II (PS II) of the Z scheme, or perhaps but less likely, on the CO/sub 2/ fixation side of photosynthesis. The primary effect of petroleum may be through direct action on the energy yielding electron transport system. Since no information is available establishing the action of oils on PS I or II, or on the redox coupling between the two photosystems, the present study was undertaken. The influence of Assam crude on photosynthetic O/sub 2/ evolution as well as upon its electron transport system in Anabaena doliolum, a heterocystous blue-green alga (cyanobacterium), has been examined. A. doliolum and other heterocystous cyanobacteria are widely distributed in soil and aquatic ecosystems, and represent an important group of free-living, nitrogen fixing microorganisms.

  2. Metabolism of sulfur compounds by whole filaments and heterocysts of Anabaena variabilis

    SciTech Connect

    Giddings, T.H. Jr.; Wolk, C.P.; Shomer-Ilan, A.

    1981-06-01

    Filaments of the heterocyst-forming cyanobacterium Anabaena variabilis reduced /sup 35/SO/sub 4//sup 2 -/, incorporating /sup 35/S into cysteine, methionine, glutathione, sulfolipid, and several unidentified metabolites. The majority of the incorporated label accumulated in reduced glutathione. Heterocysts isolated from labeled filaments contained the same major labeled products. Isolated, metabolically active heterocysts were unable to reduce /sup 35/SO/sub 4//sup 2 -/, but were able to incorporate /sup 35/S/sup 2 -/ into cysteine and glutathione. The results suggest that the initial activation of SO/sub 4//sup 2 -/ occurs in vegetative cells and that some reduced forms, possibly including S/sup 2 -/, are translocated into heterocysts.

  3. Purification and properties of glutathione reductase from the cyanobacterium Anabaena sp. strain 7119

    SciTech Connect

    Serrano, A.; Rivas, J.; Losada, M.

    1984-04-01

    An NADPH-glutathione reductase (EC 1.6.4.2) has been purified 6000-fold to electrophoretic homogeneity from the filamentous cyanobacterium Anabaena sp. strain 7119. The purified enzyme exhibits a specific activity of 249 U/mg and is characterized by being a dimeric flavin adenine dinucleotide-containing protein with a ratio of absorbance at 280 nm to absorbance at 462 nm of 5.8, a native molecular weight of 104,000, a Stokes radius of 4.13 nm, and a pI of 4.02. The enzyme activity is inhibited by sulfhydryl reagents and heavy-metal ions, especially in the presence of NADPH, with oxidized glutathione behaving as a protective agent. As is the case with the same enzyme from other sources, the kinetic data are consistent with a branched mechanism. Nevertheless, the cyanobacterial enzyme presents three distinctive

  4. Aerobic hydrogen production by the heterocystous cyanobacteria Anabaena spp. strains CA and 1F.

    PubMed Central

    Zhang, X K; Haskell, J B; Tabita, F R; Van Baalen, C

    1983-01-01

    Aerobic photoproduction of H2 was demonstrated in Anabaena spp. strains CA and 1F when cells were growing under nitrogen-fixing conditions. The rates of production, measured either by the hydrogen electrode or in a flow system by gas chromatography, were 10 to 15% of the rate of photosynthetic O2 evolution or 50 to 80% of the rates of acetylene reduction. Strains CA and 1F differed in several respects. In strain CA, H2 production was immediately partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, whereas strain 1F was not immediately affected. Strain CA also showed a consistently higher rate of H2 production than did strain 1F. H2 production in strain CA was also markedly influenced by the light intensity used for growth, although the growth rates indicated that the light intensities used were essentially saturating. PMID:6417109

  5. The regulation of HanA during heterocyst development in cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Lu, Jing-Jing; Shi, Lei; Chen, Wen-Li; Wang, Li

    2014-10-01

    In response to deprivation of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops heterocyst, which is specifically involved in the nitrogen fixation. In this study, we focused on the regulation of HanA, a histone-like protein, in heterocyst development. Electrophoretic mobility shift assay results showed that NtcA, a global nitrogen regulator necessary for heterocyst differentiation, could bind to two NtcA-binding motifs in the hanA promoter region. qPCR results also showed that NtcA may regulate the expression of hanA. By using the hanA promoter-controlled gfp as a reporter gene and performing western blot we found that the amount of HanA in mature heterocysts was decreased gradually. PMID:24980942

  6. FurA influences heterocyst differentiation in Anabaena sp. PCC 7120.

    PubMed

    González, Andrés; Valladares, Ana; Peleato, M Luisa; Fillat, María F

    2013-08-19

    In Anabaena sp. PCC 7120, FurA is a global transcriptional regulator whose expression is strongly induced by NtcA in proheterocysts and remains stably expressed in mature heterocysts. In the present study, overexpression of furA partially suppressed heterocyst differentiation by impairing morphogenesis at an early stage. Recombinant purified FurA specifically bound in vitro to the promoter regions of ntcA, while quantitative RT-PCR analyses indicated that furA overexpression strongly affected the transient increase of ntcA expression that occurs shortly after nitrogen step-down. Overall, the results suggest a connection between iron homeostasis and heterocyst differentiation via FurA, by modulating the expression of ntcA. PMID:23851073

  7. EXPRESSION OF THE GEOSMIN SYNTHASE GENE IN THE CYANOBACTERIUM ANABAENA CIRCINALIS AWQC318(1).

    PubMed

    Giglio, Steven; Saint, Christopher P; Monis, Paul T

    2011-12-01

    The occurrence of taste and odor episodes attributed to geosmin continues to trouble water utilities worldwide, and only recently have advances been made in our fundamental understanding of the biochemical and genetic mechanisms responsible for the production of geosmin in microorganisms. For the first time, we have examined the expression of the geosmin synthase gene and corresponding geosmin production by Anabaena circinalis Rabenh. ex Bornet et Flahault AWQC318 under conditions of continuous light illumination and the removal of light as a stimulus and demonstrate that the expression of geosmin synthase appears to be constitutive under these conditions. The decrease in geosmin synthase transcription post maximum cell numbers and stationary phase suggests that a decrease in isoprenoid synthesis may occur before a decrease in the transcription of ribosomal units as the process of cell death is initiated.

  8. Sensory receptors in monotremes.

    PubMed

    Proske, U; Gregory, J E; Iggo, A

    1998-07-29

    This is a summary of the current knowledge of sensory receptors in skin of the bill of the platypus, Ornithorhynchus anatinus, and the snout of the echidna, Tachyglossus aculeatus. Brief mention is also made of the third living member of the monotremes, the long-nosed echidna, Zaglossus bruijnii. The monotremes are the only group of mammals known to have evolved electroreception. The structures in the skin responsible for the electric sense have been identified as sensory mucous glands with an expanded epidermal portion that is innervated by large-diameter nerve fibres. Afferent recordings have shown that in both platypuses and echidnas the receptors excited by cathodal (negative) pulses and inhibited by anodal (positive) pulses. Estimates give a total of 40,000 mucous sensory glands in the upper and lower bill of the platypus, whereas there are only about 100 in the tip of the echidna snout. Recording of electroreceptor-evoked activity from the brain of the platypus have shown that the largest area dedicated to somatosensory input from the bill, S1, shows alternating rows of mechanosensory and bimodal neurons. The bimodal neurons respond to both electrosensory and mechanical inputs. In skin of the platypus bill and echidna snout, apart from the electroreceptors, there are structures called push rods, which consist of a column of compacted cells that is able to move relatively independently of adjacent regions of skin. At the base of the column are Merkel cell complexes, known to be type I slowly adapting mechanoreceptors, and lamellated corpuscles, probably vibration receptors. It has been speculated that the platypus uses its electric sense to detect the electromyographic activity from moving prey in the water and for obstacle avoidance. Mechanoreceptors signal contact with the prey. For the echidna, a role for the electrosensory system has not yet been established during normal foraging behaviour, although it has been shown that it is able to detect the presence

  9. Expanding the direct HetR regulon in Anabaena sp. strain PCC 7120.

    PubMed

    Videau, Patrick; Ni, Shuisong; Rivers, Orion S; Ushijima, Blake; Feldmann, Erik A; Cozy, Loralyn M; Kennedy, Michael A; Callahan, Sean M

    2014-03-01

    In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation.

  10. DL-7-azatryptophan and citrulline metabolism in the cyanobacterium Anabaena sp. strain 1F

    SciTech Connect

    Chen, C.H.; Van Baalen, C.; Tabita, F.R.

    1987-03-01

    An alternative route for the primary assimilation of ammonia proceeds via glutamine synthetase-carbamyl phosphate synthetase and its inherent glutaminase activity in Anabaena sp. strain 1F, a marine filamentous, heterocystous cyanobacterium. Evidence for the presence of this possible alternative route to glutamate was provided by the use of amino acid analogs as specific enzyme inhibitors, enzymological studies, and radioistopic labeling experiments. The amino acid pool patterns of continuous cultures of Anabaena sp. strain 1F were markedly influenced by the nitrogen source. A relatively high concentration of glutamate was maintained in the amino acid pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamate in nitrogen metabolism. The addition of 1.0 microM azaserine increased the intracellular pools of glutamate and glutamine. All attempts to detect any enzymatic activity for glutamate synthase by measuring the formation of L-(/sup 14/C)glutamate from 2-keto-(1-/sup 14/C)glutarate and glutamine failed. The addition of 10 microM DL-7-azatryptophan caused a transient accumulation of intracellular citrulline and alanine which was not affected by the presence of chloramphenicol. The in vitro activity of carbamyl phosphate synthetase and glutaminase increased severalfold in the presence of azatryptophan. Results from radioisotopic labeling experiments with (/sup 14/C)bicarbonate and L-(1-/sup 14/C)ornithine also indicated that citrulline was formed via carbamyl phosphate synthetase and ornithine transcarbamylase. In addition to its effects on nitrogen metabolism, azatryptophan also affected carbon metabolism by inhibiting photosynthetic carbon assimilation and photosynthetic oxygen evolution.

  11. Expanding the direct HetR regulon in Anabaena sp. strain PCC 7120.

    PubMed

    Videau, Patrick; Ni, Shuisong; Rivers, Orion S; Ushijima, Blake; Feldmann, Erik A; Cozy, Loralyn M; Kennedy, Michael A; Callahan, Sean M

    2014-03-01

    In response to a lack of environmental combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocyst cells in a periodic pattern. HetR is a transcription factor that coordinates the regulation of this developmental program. An inverted repeat-containing sequence in the hepA promoter required for proheterocyst-specific transcription was identified based on sequence similarity to a previously characterized binding site for HetR in the promoter of hetP. The binding affinity of HetR for the hepA site is roughly an order of magnitude lower than that for the hetP binding site. A BLAST search of the Anabaena genome identified 166 hepA-like sites that occur as single or tandem sites (two binding sites separated by 13 bp). The vast majority of these sites are present in predicted intergenic regions. HetR bound five representative single binding sites in vitro, and binding was abrogated by transversions in the binding sites that conserved the inverted repeat nature of the sites. Binding to four representative tandem sites was not observed. Transcriptional fusions of the green fluorescent protein gene gfp with putative promoter regions associated with the representative binding sites indicated that HetR could function as either an activator or repressor and that activation was cell-type specific. Taken together, we have expanded the direct HetR regulon and propose a model in which three categories of HetR binding sites, based on binding affinity and nucleotide sequence, contribute to three of the four phases of differentiation. PMID:24375104

  12. Multiplicity and specificity of siderophore uptake in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Rudolf, Mareike; Stevanovic, Mara; Kranzler, Chana; Pernil, Rafael; Keren, Nir; Schleiff, Enrico

    2016-09-01

    Many cyanobacteria secrete siderophores to sequester iron. Alternatively, mechanisms to utilize xenosiderophores have evolved. The overall uptake systems are comparable to that of other bacteria involving outer membrane transporters energized by TonB as well as plasma membrane-localized transporters. However, the function of the bioinformatically-inferred components is largely not established and recent studies showed a high diversity of the complexity of the uptake systems in different cyanobacteria. Thus, we approached the systems of the filamentous Anabaena sp. PCC 7120 as a model of a siderophore-secreting cyanobacterium. Anabaena sp. produces schizokinen and uptake of Fe-schizokinen involves the TonB-dependent transporter, schizokinen transporter (SchT), and the ABC-type transport system FhuBCD. We confirm that this system is also relevant for the uptake of structurally similar Fe-siderophore complexes like Fe-aerobactin. Moreover, we demonstrate a function of the TonB-dependent transporter IutA2 in Fe-schizokinen uptake in addition to SchT. The iutA2 mutant shows growth defects upon iron limitation, alterations in Fe-schizokinen uptake and in the transcription profile of the Fe-schizokinen uptake system. The physiological properties of the mutant confirm the importance of iron uptake for cellular function, e.g. for the Krebs cycle. Based on the relative relation of expression of schT and iutA2 as well as of the iron uptake rate to the degree of starvation, a model for the need of the co-existence of two different outer membrane transporters for the same substrate is discussed. PMID:27325117

  13. Sensory adaptation for timing perception.

    PubMed

    Roseboom, Warrick; Linares, Daniel; Nishida, Shin'ya

    2015-04-22

    Recent sensory experience modifies subjective timing perception. For example, when visual events repeatedly lead auditory events, such as when the sound and video tracks of a movie are out of sync, subsequent vision-leads-audio presentations are reported as more simultaneous. This phenomenon could provide insights into the fundamental problem of how timing is represented in the brain, but the underlying mechanisms are poorly understood. Here, we show that the effect of recent experience on timing perception is not just subjective; recent sensory experience also modifies relative timing discrimination. This result indicates that recent sensory history alters the encoding of relative timing in sensory areas, excluding explanations of the subjective phenomenon based only on decision-level changes. The pattern of changes in timing discrimination suggests the existence of two sensory components, similar to those previously reported for visual spatial attributes: a lateral shift in the nonlinear transducer that maps relative timing into perceptual relative timing and an increase in transducer slope around the exposed timing. The existence of these components would suggest that previous explanations of how recent experience may change the sensory encoding of timing, such as changes in sensory latencies or simple implementations of neural population codes, cannot account for the effect of sensory adaptation on timing perception. PMID:25788590

  14. Sensory aspects of movement disorders

    PubMed Central

    Patel, Neepa; Jankovic, Joseph; Hallett, Mark

    2016-01-01

    Movement disorders, which include disorders such as Parkinson’s disease, dystonia, Tourette’s syndrome, restless legs syndrome, and akathisia, have traditionally been considered to be disorders of impaired motor control resulting predominantly from dysfunction of the basal ganglia. This notion has been revised largely because of increasing recognition of associated behavioural, psychiatric, autonomic, and other non-motor symptoms. The sensory aspects of movement disorders include intrinsic sensory abnormalities and the effects of external sensory input on the underlying motor abnormality. The basal ganglia, cerebellum, thalamus, and their connections, coupled with altered sensory input, seem to play a key part in abnormal sensorimotor integration. However, more investigation into the phenomenology and physiological basis of sensory abnormalities, and about the role of the basal ganglia, cerebellum, and related structures in somatosensory processing, and its effect on motor control, is needed. PMID:24331796

  15. FurA from Anabaena PCC 7120: New insights on its regulation and the interaction with DNA

    NASA Astrophysics Data System (ADS)

    Hernández, J. A.; López-Gomollón, S.; Pellicer, S.; Martín, B.; Sevilla, E.; Bes, M. T.; Peleato, M. L.; Fillat, M. F.

    2006-08-01

    Fur (ferric uptake regulator) proteins are global regulatory proteins involved in the maintenance of iron homeostasis. They recognize specific DNA sequences denoted iron boxes. It is assumed that Fur proteins act as classical repressors. Under iron-rich conditions, Fur dimers complexed with ferrous ions bind to iron boxes, preventing transcription. In addition to iron homeostasis, Fur proteins control the concerted response to oxidative and acidic stresses in heterotrophic prokaryotes. Our group studies the interaction between Fur proteins and target DNA sequences. Moreover, the regulation of FurA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, whose genome codes for three fur homologues has been investigated. We present an overview about the different factors involved in the regulation of FurA and analyze the parameters that influence FurA-DNA interaction in the cyanobacterium Anabaena PCC 7120.

  16. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; Florencio, Francisco J

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

  17. Characterization of two naturally truncated, Ssb-like proteins from the nitrogen-fixing cyanobacterium, Anabaena sp. PCC7120.

    PubMed

    Kirti, Anurag; Rajaram, Hema; Apte, Shree Kumar

    2013-11-01

    Single-stranded (ss) DNA-binding (Ssb) proteins are vital for all DNA metabolic processes and are characterized by an N-terminal OB-fold followed by P/G-rich spacer region and a C-terminal tail. In the genome of the heterocystous, nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC 7120, two genes alr0088 and alr7579 are annotated as ssb, but the corresponding proteins have only the N-terminal OB-fold and no P/G-rich region or acidic tail, thereby rendering them unable to interact with genome maintenance proteins. Both the proteins were expressed under normal growth conditions in Anabaena PCC7120 and regulated differentially under abiotic stresses which induce DNA damage, indicating that these are functional genes. Constitutive overexpression of Alr0088 in Anabaena enhanced the tolerance to DNA-damaging stresses which caused formation of DNA adducts such as UV and MitomycinC, but significantly decreased the tolerance to γ-irradiation, which causes single- and double-stranded DNA breaks. On the other hand, overexpression of Alr7579 had no significant effect on normal growth or stress tolerance of Anabaena. Thus, of the two truncated Ssb-like proteins, Alr0088 may be involved in protection of ssDNA from damage, but due to the absence of acidic tail, it may not aid in repair of damaged DNA. These two proteins are present across cyanobacterial genera and unique to them. These initial studies pave the way to the understanding of DNA repair in cyanobacteria, which is not very well documented.

  18. The interaction of boron with glycolipids is required to increase tolerance to stresses in Anabaena PCC 7120.

    PubMed

    Abreu, Isidro; Orús, Isabel; Bolaños, Luis; Bonilla, Ildefonso

    2014-10-01

    Boron (B) is an essential nutrient for heterocystous cyanobacteria growing under diazotrophic conditions. Under B-deficient conditions, the heterocyst envelope is highly disorganized, and the glycolipid layer is predominantly lost. Therefore, we examined whether B is implicated in the regulation of synthesis or processing and/or stability of glycolipids in Anabaena PCC 7120. RT-PCR analysis indicated that the expression of hglE was not significantly changed under B deficiency, suggesting that the synthesis of glycolipids during heterocyst formation was not compromised. In contrast, the overexpression of devB and hepA, encoding a glycolipid and a carbohydrate transporter, respectively, results in the instability of the envelope under B-deficient conditions. The capacity of borate to bind and stabilize molecules is considered the basis of any B biological function. Using a borate-binding-specific resin and thin layer chromatography, we detected the glycolipids that interact with B. Several heterocyst-specific glycolipids were detected as putative B ligands, suggesting a role for B in stabilizing the heterocyst envelope. Moreover, the glycolipids of Anabaena growing in non-diazotrophic conditions were also detected as putative B ligands. Although B is not essential for Anabaena under non-N2-fixing conditions, the presence of this micronutrient increased the tolerance of Anabaena to detergent treatment, salinity and hyperosmotic conditions. Taken together, the results of the present experiment suggest a beneficial role for B in environmental adaptation. Furthermore, we discuss the nutrient requirement for living organisms growing in nature and not under laboratory conditions.

  19. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120

    PubMed Central

    Sánchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, Carla V.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments. PMID:27588019

  20. The interaction of boron with glycolipids is required to increase tolerance to stresses in Anabaena PCC 7120.

    PubMed

    Abreu, Isidro; Orús, Isabel; Bolaños, Luis; Bonilla, Ildefonso

    2014-10-01

    Boron (B) is an essential nutrient for heterocystous cyanobacteria growing under diazotrophic conditions. Under B-deficient conditions, the heterocyst envelope is highly disorganized, and the glycolipid layer is predominantly lost. Therefore, we examined whether B is implicated in the regulation of synthesis or processing and/or stability of glycolipids in Anabaena PCC 7120. RT-PCR analysis indicated that the expression of hglE was not significantly changed under B deficiency, suggesting that the synthesis of glycolipids during heterocyst formation was not compromised. In contrast, the overexpression of devB and hepA, encoding a glycolipid and a carbohydrate transporter, respectively, results in the instability of the envelope under B-deficient conditions. The capacity of borate to bind and stabilize molecules is considered the basis of any B biological function. Using a borate-binding-specific resin and thin layer chromatography, we detected the glycolipids that interact with B. Several heterocyst-specific glycolipids were detected as putative B ligands, suggesting a role for B in stabilizing the heterocyst envelope. Moreover, the glycolipids of Anabaena growing in non-diazotrophic conditions were also detected as putative B ligands. Although B is not essential for Anabaena under non-N2-fixing conditions, the presence of this micronutrient increased the tolerance of Anabaena to detergent treatment, salinity and hyperosmotic conditions. Taken together, the results of the present experiment suggest a beneficial role for B in environmental adaptation. Furthermore, we discuss the nutrient requirement for living organisms growing in nature and not under laboratory conditions. PMID:25092228

  1. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Sánchez-Riego, Ana M; Mata-Cabana, Alejandro; Galmozzi, Carla V; Florencio, Francisco J

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx) as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however, nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (ΔntrC), apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species) in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments. PMID:27588019

  2. Protection from UV-B damage of mosquito larvicidal toxins from Bacillus thuringiensis subsp. israelensis expressed in Anabaena PCC 7120.

    PubMed

    Manasherob, Robert; Ben-Dov, Eitan; Xiaoqiang, Wu; Boussiba, Sammy; Zaritsky, Arieh

    2002-09-01

    A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed delta-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent.

  3. Identification of a rhodopsin gene mutation in a large family with autosomal dominant retinitis pigmentosa.

    PubMed

    Yu, Xinping; Shi, Wei; Cheng, Lulu; Wang, Yanfang; Chen, Ding; Hu, Xuting; Xu, Jinling; Xu, Limin; Wu, Yaming; Qu, Jia; Gu, Feng

    2016-01-01

    Retinitis pigmentosa (RP) is a genetically highly heterogeneous retinal disease and one of the leading causes of blindness in the world. Next-generation sequencing technology has enormous potential for determining the genetic etiology of RP. We sought to identify the underlying genetic defect in a 35-year-old male from an autosomal-dominant RP family with 14 affected individuals. By capturing next-generation sequencing (CNGS) of 144 genes associated with retinal diseases, we identified eight novel DNA variants; however, none of them cosegregated for all the members of the family. Further analysis of the CNGS data led to identification of a recurrent missense mutation (c.403C > T, p.R135W) in the rhodopsin (RHO) gene, which cosegregated with all affected individuals in the family and was not observed in any of the unaffected family members. The p.R135W mutation has a reference single nucleotide polymorphism (SNP) ID (rs104893775), and it appears to be responsible for the disease in this large family. This study highlights the importance of examining NGS data with reference SNP IDs. Thus, our study is important for data analysis of NGS-based clinical genetic diagnoses. PMID:26794436

  4. Molecular dynamics investigation of primary photoinduced events in the activation of rhodopsin.

    PubMed Central

    Saam, Jan; Tajkhorshid, Emad; Hayashi, Shigehiko; Schulten, Klaus

    2002-01-01

    Retinal cis-trans isomerization and early relaxation steps have been studied in a 10-ns molecular dynamics simulation of a fully hydrated model of membrane-embedded rhodopsin. The isomerization, induced by transiently switching the potential energy function governing the C(11)==C(12) dihedral angle of retinal, completes within 150 fs and yields a strongly distorted retinal. The most significant conformational changes in the binding pocket are straightening of retinal's polyene chain and separation of its beta-ionone ring from Trp-265. In the following 500 ps, transition of 6s-cis to 6s-trans retinal and dramatic changes in the hydrogen bonding network of the binding pocket involving the counterion for the protonated Schiff base, Glu-113, occur. Furthermore, the energy initially stored internally in the distorted retinal is transformed into nonbonding interactions of retinal with its environment. During the following 10 ns, increased mobilities of some parts of the protein, such as the kinked regions of the helices, mainly helix VI, and the intracellular loop I2, were observed, as well as transient structural changes involving the conserved salt bridge between Glu-134 and Arg-135. These features prepare the protein for major structural transformations achieved later in the photocycle. Retinal's motion, in particular, can be compared to an opening turnstile freeing the way for the proposed rotation of helix VI. This was demonstrated by a steered molecular dynamics simulation in which an applied torque enforced the rotation of helix VI. PMID:12496081

  5. The roles of Syx5 in Golgi morphology and Rhodopsin transport in Drosophila photoreceptors

    PubMed Central

    Satoh, Takunori; Nakamura, Yuri

    2016-01-01

    ABSTRACT SNAREs (SNAP receptors) are the key components of protein complexes that drive membrane fusion. Here, we report the function of a SNARE, Syntaxin 5 (Syx5), in the development of photoreceptors in Drosophila. In wild-type photoreceptors, Syx5 localizes to cis-Golgi, along with cis-Golgi markers: Rab1 and GM130. We observed that Syx5-deficient photoreceptors show notable accumulation of these cis-Golgi markers accompanying drastic accumulation of vesicles between endoplasmic reticulum (ER) and Golgi cisternae. Extensive analysis of Rh1 (rhodopsin 1) trafficking revealed that in Syx5-deficient photoreceptors, Rh1 is exported from the ER with normal kinetics, retained in the cis-Golgi region along with GM130 for a prolonged period, and then subsequently degraded presumably by endoplasmic reticulum-associated protein degradation (ERAD) after retrieval to the ER. Unlike our previous report of Rab6-deficient photoreceptors – where two apical transport pathways are specifically inhibited – vesicle transport pathways to all plasma membrane domains are inhibited in Syx5-deficient photoreceptors, implying that Rab6 and Syx5 are acting in different steps of intra-Golgi transport. These results indicate that Syx5 is crucial for membrane protein transport, presumably during ER-derived vesicle fusion to form cis-Golgi cisternae. PMID:27591190

  6. Gene Therapy to Rescue Retinal Degeneration Caused by Mutations in Rhodopsin

    PubMed Central

    Rossmiller, Brian P.; Ryals, Renee C.; Lewin, Alfred S.

    2015-01-01

    Retinal gene therapy has proven safe and at least partially successful in clinical trials and in numerous animal models. Gene therapy requires characterization of the progression of the disease and understanding of its genetic cause. Testing gene therapies usually requires an animal model that recapitulates the key features of the human disease, though photoreceptors and cells of the retinal pigment epithelium produced from patient-derived stem cells may provide an alternative test system for retinal gene therapy. Gene therapy also requires a delivery system that introduces the therapeutic gene to the correct cell type and does not cause unintended damage to the tissue. Current systems being tested in the eye are nanoparticles, pseudotyped lentiviruses, and adeno-associated virus (AAV) of various serotypes. Here, we describe the techniques of AAV vector design as well as the in vivo and ex vivo tests necessary for assessing the efficacy of retinal gene therapy to treat retinal degeneration caused by mutations in the rhodopsin gene. PMID:25697537

  7. The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.

    PubMed

    Hontani, Yusaku; Inoue, Keiichi; Kloz, Miroslav; Kato, Yoshitaka; Kandori, Hideki; Kennis, John T M

    2016-09-21

    Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 μs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2. PMID:27550793

  8. Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization.

    PubMed

    Mittelmeier, Telsa M; Boyd, Joseph S; Lamb, Mary Rose; Dieckmann, Carol L

    2011-05-16

    The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells. PMID:21555459

  9. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    DOE PAGESBeta

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2016-04-12

    Here, serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solvedmore » with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.« less

  10. The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.

    PubMed

    Hontani, Yusaku; Inoue, Keiichi; Kloz, Miroslav; Kato, Yoshitaka; Kandori, Hideki; Kennis, John T M

    2016-09-21

    Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 μs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.

  11. Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools.

    PubMed

    AzimiHashemi, N; Erbguth, K; Vogt, A; Riemensperger, T; Rauch, E; Woodmansee, D; Nagpal, J; Brauner, M; Sheves, M; Fiala, A; Kattner, L; Trauner, D; Hegemann, P; Gottschalk, A; Liewald, J F

    2014-12-15

    Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.

  12. Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin.

    PubMed

    Yan, Elsa C Y; Kazmi, Manija A; Ganim, Ziad; Hou, Jian-Min; Pan, Douhai; Chang, Belinda S W; Sakmar, Thomas P; Mathies, Richard A

    2003-08-01

    The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110-Cys187); this process may be a general element of G protein-coupled receptor activation.

  13. Analysis of proteins involved in the production of MAA׳s in two Cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica

    PubMed Central

    Rahman, Md Akhlaqur; Sinha, Sukrat; Sachan, Shephali; Kumar, Gaurav; Singh, Shailendra Kumar; Sundaram, Shanthy

    2014-01-01

    Mycosporine- like amino acids (MAAs) are small (<400Da), colourless, water soluble compounds composed of cyclohexenone or cyclohexinimine chromophere conjugated with the nitrogen substituent of amino acid or its amino alcohol. These compounds are known for their UV- absorbing role in various organisms and seem to have evolutionary significance. The biosynthesis of MAAs is presumed to occur via the first part of shikimate pathway. In the present work two cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica were tested for their ability to synthesize MAAs and protein involved in the production of MAAs. It was found that protein sequence 3-phosphoshikimate 1-carboxyvinyltransferase is involved in producing mycosporine glycine in Synechocystis PCC 6803 and 3-dehydroquinate synthase is involved for producing shinorine in Anabaena cylindrica. Phylogenetic and bioinformatic analysis of Mycosporine like amino acid producing protein sequence of both cyanobacterial species Synechocystis PCC 6803 and Anabaena cylindrica provide a useful framework to understand the relationship of the different forms and how they have evolved from a common ancestor. These products seem to be conserved but the residues are prone to variation which might be due the fact that different cyanobacteria show different physiological process in response of Ultraviolet stress. PMID:25187686

  14. Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena.

    PubMed

    Mariscal, Vicente; Nürnberg, Dennis J; Herrero, Antonia; Mullineaux, Conrad W; Flores, Enrique

    2016-09-01

    Filamentous, N2 -fixing, heterocyst-forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ-GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ-related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation.

  15. The influence of humic acid on the toxicity of nano-ZnO and Zn2+ to the Anabaena sp.

    PubMed

    Tang, Yulin; Li, Shuyan; Lu, Yao; Li, Qian; Yu, Shuili

    2015-07-01

    This study explored the effects of humic acid (HA) on the toxicity of ZnO nanoparticles (nano-ZnO) and Zn(2+) to Anabaena sp. Typical chlorophyll fluorescence parameters, including effective quantum yield, photosynthetic efficiency and maximal electron transport rate, were measured by a pulse-amplitude modulated fluorometer. Results showed that nano-ZnO and Zn(2+) could inhibit Anabaena sp. growth with the EC50 (concentration for 50% of maximal effect) of 0.74 ± 0.01 and 0.3 ± 0.01 mg/L, respectively. In the presence of 3.0 mg/L of HA, EC50 of nano-ZnO increased to 1.15 ± 0.04 mg/L and EC50 of Zn(2+) was still 0.3 ± 0.01 mg/L. Scanning electron microscopy observation revealed that HA prevented the adhesion of nano-ZnO on the algae cells due to the increased electrostatic repulsion. The generation of intracellular reactive oxygen species and cellular lipid peroxidation were significantly limited by HA. Nano-ZnO had more damage to the cell membrane than Zn(2+) did, which could be proven by the malondialdehyde content in Anabaena sp. cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 895-903, 2015.

  16. Assessment of the CO2 fixation capacity of Anabaena sp. ATCC 33047 outdoor cultures in vertical flat-panel reactors.

    PubMed

    Clares, Marta E; Moreno, José; Guerrero, Miguel G; García-González, Mercedes

    2014-10-10

    The extent of biological CO2 fixation was evaluated for outdoor cultures of the cyanobacterium Anabaena sp. ATCC 33047. Culture conditions were optimized indoors in bubble-column photochemostats operating in continuous mode, subjected to irradiance cycles mimicking the light regime outdoors. Highest values achieved for CO2 fixation rate and biomass productivity were 1 and 0.6 g L(-1) day(-1), respectively. The comparison among different reactors operating simultaneously - open pond, horizontal tubular reactor and vertical flat-panel - allowed to assess their relative efficiency for the outdoor development of Anabaena cultures. Despite the higher volumetric CO2 fixation capacity (and biomass productivity) exhibited by the tubular photobioreactor, yield of the flat-panel reactor was 50% higher than that of the tubular option on a per area basis, reaching values over 35 g CO2 fixed m(-2) d(-1). The flat-panel reactor actually represents a most suitable system for CO2 capture coupled to the generation of valuable biomass by Anabaena cultures.

  17. A novel alkyl hydroperoxidase (AhpD) of Anabaena PCC7120 confers abiotic stress tolerance in Escherichia coli.

    PubMed

    Shrivastava, Alok Kumar; Singh, Shilpi; Singh, Prashant Kumar; Pandey, Sarita; Rai, L C

    2015-01-01

    In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

  18. Overexpression of SepJ alters septal morphology and heterocyst pattern regulated by diffusible signals in Anabaena.

    PubMed

    Mariscal, Vicente; Nürnberg, Dennis J; Herrero, Antonia; Mullineaux, Conrad W; Flores, Enrique

    2016-09-01

    Filamentous, N2 -fixing, heterocyst-forming cyanobacteria grow as chains of cells that are connected by septal junctions. In the model organism Anabaena sp. strain PCC 7120, the septal protein SepJ is required for filament integrity, normal intercellular molecular exchange, heterocyst differentiation, and diazotrophic growth. An Anabaena strain overexpressing SepJ made wider septa between vegetative cells than the wild type, which correlated with a more spread location of SepJ in the septa as observed with a SepJ-GFP fusion, and contained an increased number of nanopores, the septal peptidoglycan perforations that likely accommodate septal junctions. The septa between heterocysts and vegetative cells, which are narrow in wild-type Anabaena, were notably enlarged in the SepJ-overexpressing mutant. Intercellular molecular exchange tested with fluorescent tracers was increased for the SepJ-overexpressing strain specifically in the case of calcein transfer between vegetative cells and heterocysts. These results support an association between calcein transfer, SepJ-related septal junctions, and septal peptidoglycan nanopores. Under nitrogen deprivation, the SepJ-overexpressing strain produced an increased number of contiguous heterocysts but a decreased percentage of total heterocysts. These effects were lost or altered in patS and hetN mutant backgrounds, supporting a role of SepJ in the intercellular transfer of regulatory signals for heterocyst differentiation. PMID:27273832

  19. Assessment of the CO2 fixation capacity of Anabaena sp. ATCC 33047 outdoor cultures in vertical flat-panel reactors.

    PubMed

    Clares, Marta E; Moreno, José; Guerrero, Miguel G; García-González, Mercedes

    2014-10-10

    The extent of biological CO2 fixation was evaluated for outdoor cultures of the cyanobacterium Anabaena sp. ATCC 33047. Culture conditions were optimized indoors in bubble-column photochemostats operating in continuous mode, subjected to irradiance cycles mimicking the light regime outdoors. Highest values achieved for CO2 fixation rate and biomass productivity were 1 and 0.6 g L(-1) day(-1), respectively. The comparison among different reactors operating simultaneously - open pond, horizontal tubular reactor and vertical flat-panel - allowed to assess their relative efficiency for the outdoor development of Anabaena cultures. Despite the higher volumetric CO2 fixation capacity (and biomass productivity) exhibited by the tubular photobioreactor, yield of the flat-panel reactor was 50% higher than that of the tubular option on a per area basis, reaching values over 35 g CO2 fixed m(-2) d(-1). The flat-panel reactor actually represents a most suitable system for CO2 capture coupled to the generation of valuable biomass by Anabaena cultures. PMID:25068618

  20. The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination.

    PubMed

    García-Martínez, Jorge; Brunk, Michael; Avalos, Javier; Terpitz, Ulrich

    2015-01-01

    Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO(-) mutant and carO(+) control strains showed a faster development of light-exposed carO(-) germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.

  1. The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination

    PubMed Central

    García-Martínez, Jorge; Brunk, Michael; Avalos, Javier; Terpitz, Ulrich

    2015-01-01

    Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin. PMID:25589426

  2. Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter

    PubMed Central

    Escudero, Leticia; Mariscal, Vicente

    2015-01-01

    ABSTRACT In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. IMPORTANCE Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular

  3. LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity.

    PubMed

    Kumar, Arvind; Kirti, Anurag; Rajaram, Hema

    2015-02-01

    The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca(2+) and in the temperature range of 30-57°C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, (86)GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.

  4. Assessment of the effect of azo dye RP2B on the growth of a nitrogen fixing cyanobacterium--Anabaena sp.

    PubMed

    Hu, T L; Wu, S C

    2001-03-01

    Certain nitrogen fixing cyanobacteria are diazotrophic, which profoundly impacts the aquatic ecosystem chemically and biologically. Although certain types are banned due to their carcinogenicity, azo dyes are commonly used in the dyeing or textile industry. This work investigates the effect of azo dye on the growth of cyanobacteria. Anabaena sp. isolated from the Da Jia Brook is an odor producing, nitrogen fixing cyanobacterium. The growth rates of Anabaena sp. in the media with or without nitrogen source were 3.56 x 10(-2) mg/ml day and 2.44 x 10(-2) mg/ml day, respectively. Anabaena sp. could not use azo dye RP2B as the nitrogen source. Experimental results indicated that the growth of Anabaena sp. was inhibited in the medium containing RP2B. The degree of inhibition increased from 50% to 81% with an increasing concentration of RP2B (0-50 mg/l). The IC-50 (inhibitory concentration) of RP2B on the growth of Anabaena sp. was 5 mg/l (as based on dry weight) or 7 mg/l (as measured by chlorophyll a).

  5. Histochemical demonstration of a rhodopsin-like substance in the eye of the arrow-worm, Spadella schizoptera (Chaetognatha).

    PubMed

    Goto, T; Yoshida, M

    1988-01-01

    The presumed photoreceptive region of the arrow-worms of the species Sagitta crassa and Spadella schizoptera consists of perforated lamellae which are unique as the photoreceptive structure. The existence of a visual pigment in this region was demonstrated by a histofluorescent technique using Spadella schizoptera, whose presumed photoreceptive region was much larger than in Sagitta crassa. A specific fluorescence, indicative of the presence of retinal-based proteins, appeared only in the perforated lamellar region. The result suggests that the perforated lamellae contain a rhodopsin-like substance and could be the primary photoreceptive site. PMID:3268423

  6. Histochemical demonstration of a rhodopsin-like substance in the eye of the arrow-worm, Spadella schizoptera (Chaetognatha).

    PubMed

    Goto, T; Yoshida, M

    1988-01-01

    The presumed photoreceptive region of the arrow-worms of the species Sagitta crassa and Spadella schizoptera consists of perforated lamellae which are unique as the photoreceptive structure. The existence of a visual pigment in this region was demonstrated by a histofluorescent technique using Spadella schizoptera, whose presumed photoreceptive region was much larger than in Sagitta crassa. A specific fluorescence, indicative of the presence of retinal-based proteins, appeared only in the perforated lamellar region. The result suggests that the perforated lamellae contain a rhodopsin-like substance and could be the primary photoreceptive site.

  7. Opsin Effect on the Electronic Structure of the Retinylidene Chromophore in Rhodopsin.

    PubMed

    Sproviero, Eduardo M

    2015-03-10

    Direct examination of experimental NMR parameters combined with electronic structure analysis was used to provide a first-principle interpretation of NMR experiments and give a precise evaluation of how the electronic perturbation of the protein environment affects the electronic properties of the retinylidene chromophere in rhodopsin. To this end, we pursued a theoretical analysis using a combination of tools including quantum mechanics/molecular mechanics (QM/MM) at the Density Functional Theory (DFT) level, in conjunction with gauge independent atomic orbital (GIAO) calculations of (13)C NMR chemical shieldings and (1)J(CC) spin-spin coupling constants obtained with the Coupled Perturbed DFT (CPDFT) method. The opsin effect on the retinylidene chromophere is interpreted as an inductive effect of Glu-113 which readjusts the weighting factors of resonance substructures of the conjugated chain of the chromophere. These changes give a rationalization to the alternating effect of the (13)C chemical shifts magnitudes when comparing the retinylidene chromophere in the presence and absence of the protein environment. Conversely, perturbation of π orbitals has little to no effect over (1)J (13)C-(13)C spin-spin coupling constants, as they are mainly dominated by the Fermi contact term, and hence the counteraion effect is restricted to the vicinity of the perturbation. Thus, the apparent contradiction between experimental findings based on chemical shifts (deep penetration) and one-bond J-couplings (localized effects of the protonated Schiff base at the chain terminus) is in fact a consequence of different properties responding differently to the same external perturbation.

  8. Agonists and partial agonists of rhodopsin: retinal polyene methylation affects receptor activation.

    PubMed

    Vogel, Reiner; Lüdeke, Steffen; Siebert, Friedrich; Sakmar, Thomas P; Hirshfeld, Amiram; Sheves, Mordechai

    2006-02-14

    Using Fourier transform infrared (FTIR) difference spectroscopy, we have studied the impact of sites and extent of methylation of the retinal polyene with respect to position and thermodynamic parameters of the conformational equilibrium between the Meta I and Meta II photoproducts of rhodopsin. Deletion of methyl groups to form 9-demethyl and 13-demethyl analogues, as well as addition of a methyl group at C10 or C12, shifted the Meta I/Meta II equilibrium toward Meta I, such that the retinal analogues behaved like partial agonists. This equilibrium shift resulted from an apparent reduction of the entropy gain of the transition of up to 65%, which was only partially offset by a concomitant reduction of the enthalpy increase. The analogues produced Meta II photoproducts with relatively small alterations, while their Meta I states were significantly altered, which accounted for the aberrant transitions to Meta II. Addition of a methyl group at C14 influenced the thermodynamic parameters but had little impact on the position of the Meta I/Meta II equilibrium. Neutralization of the residue 134 in the E134Q opsin mutant increased the Meta II content of the 13-demethyl analogue, but not of the 9-demethyl analogue, indicating a severe impairment of the allosteric coupling between the conserved cytoplasmic ERY motif involved in proton uptake and the Schiff base/Glu 113 microdomain in the 9-demethyl analogue. The 9-methyl group appears therefore essential for the correct positioning of retinal to link protonation of the cytoplasmic motif with protonation of Glu 113 during receptor activation.

  9. Photocycle and vectorial proton transfer in a rhodopsin from the eukaryote Oxyrrhis marina.

    PubMed

    Janke, Christian; Scholz, Frank; Becker-Baldus, Johanna; Glaubitz, Clemens; Wood, Phillip G; Bamberg, Ernst; Wachtveitl, Josef; Bamann, Christian

    2013-04-23

    Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist Oxyrrhis marina (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1's features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the pK(a) value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low pK(a) of the proton acceptor is not dominated by this interaction, but by other electrostatic factors.

  10. Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors.

    PubMed

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2014-05-01

    Current methods of G protein coupled receptors (GPCRs) phylogenetic classification are sequence based and therefore inappropriate for highly divergent sequences, sharing low sequence identity. In this study, sequence structure profile based alignment generated by PROMALS3D was used to understand the GPCR Class A Rhodopsin superfamily evolution using the MEGA 5 software. Phylogenetic analysis included a combination of Neighbor-Joining method and Maximum Likelihood method, with 1000 bootstrap replicates. Our study was able to identify potential ligand association for Class A Orphans and putative/unclassified Class A receptors with no cognate ligand information: GPR21 and GPR52 with fatty acids; GPR75 with Neuropeptide Y; GPR82, GPR18, GPR141 with N-arachidonylglycine; GPR176 with Free fatty acids, GPR10 with Tachykinin & Neuropeptide Y; GPR85 with ATP, ADP & UDP glucose; GPR151 with Galanin; GPR153 and GPR162 with Adrenalin, Noradrenalin; GPR146, GPR139, GPR142 with Neuromedin, Ghrelin, Neuromedin U-25 & Thyrotropin-releasing hormone; GPR171 with ATP, ADP & UDP Glucose; GPR88, GPR135, GPR161, GPR101with 11-cis-retinal; GPR83 with Tackykinin; GPR148 with Prostanoids, GPR109b, GPR81, GPR31with ATP & UTP and GPR150 with GnRH I & GnRHII. Furthermore, we suggest that this study would prove useful in re-classification of receptors, selecting templates for homology modeling and identifying ligands which may show cross reactivity with other GPCRs as signaling via multiple ligands play a significant role in disease modulation.

  11. An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

    PubMed

    Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

    2005-04-29

    We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

  12. Dynamics of cellular and extracellular cAmp in Anabaena flos-aquae (cyanophyta): intrinsic culture variability and correlation with metabolic variables

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1981-01-01

    The production and extracellular release of cyclic adenosine 3':5'-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anabaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intracellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to (/sup 14/C)-bicarbonate uptake by an exponential relationship.

  13. Acyl-acyl-carrier protein: lysomonogalactosyldiacylglycerol acyltransferase from the cyanobacterium Anabaena variabilis.

    PubMed

    Chen, H H; Wickrema, A; Jaworski, J G

    1988-12-16

    Membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were capable of catalyzing the direct transfer of the acyl group from acyl-acyl-carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. Other glycolipids including monoglucosyldiacylglycerol and digalactosyldiacylglycerol were not products of this reaction. The transfer was not dependent on any added cofactors. Palmitoyl-, stearoyl- and oleoyl-acyl-carrier protein were approximately equally active as substrates. Transfer was exclusively to the C-1 of the glycerol, as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. In addition to the single galactolipid, a second minor reaction product was free fatty acid, presumably due to hydrolysis of the acyl-acyl-carrier protein. Using a double-labelled [14C]acyl-[14C]acyl-carrier protein, the reaction was demonstrated to be a transfer reaction, rather than a simple exchange of acyl groups with endogenous monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by increasing activity with the addition of liposomes of lysomonogalactosyldiacylglycerol.

  14. Differential sensitivity of Anabaena doliolum to Cu and Zn in batch and semicontinuous cultures.

    PubMed

    Tripathi, B N; Mehta, S K; Gaur, J P

    2003-10-01

    Elevated concentrations of Cu and Zn inhibited Anabaena doliolum more severely in semicontinuous culture than in batch culture with growth and protein, chlorophyll a, and carotenoid contents generally more than two-fold more sensitive in the former culture system. The greater sensitivity of A. doliolum to test metals in semicontinuous culture was associated with their greater accumulation. The level of inhibition of various parameters of the test organism remained almost constant in semicontinuous culture, but considerable alleviation of the inhibitory effect occurred in batch culture with time concomitant with a regular decline in metal content of cells. However, metal content of cells in semicontinuous culture remained more or less constant with time, thereby causing no change in the level of inhibition. Unlike semicontinuous culture, batch culture showed considerable depletion of phosphate from the medium and a rise in pH (from 7 to 7.8). In conclusion, batch culture is not appropriate for long-term assessment of metal toxicity as it might substantially underestimate toxic effects of heavy metals. PMID:12927563

  15. HesF, an exoprotein required for filament adhesion and aggregation in Anabaena sp. PCC 7120.

    PubMed

    Oliveira, Paulo; Pinto, Filipe; Pacheco, Catarina C; Mota, Rita; Tamagnini, Paula

    2015-05-01

    Here, we report on the identification and characterization of a protein (Alr0267) named HesF, found in the extracellular milieu of Anabaena sp. PCC 7120 grown diazotrophically. hesF was found to be highly upregulated upon transition from non-nitrogen-fixing to nitrogen-fixing conditions, and the highest transcript levels were detected towards the end of the heterocyst differentiation process. The hesF promoter drives transcription of the gene in heterocysts only, and both NtcA and HetR are essential for the gene's in vivo activation. An examination of HesF's translocation showed that the secretion system is neither heterocyst-specific nor dependent on nitrogen-fixing conditions. Furthermore, HesF was found to be a type I secretion system substrate, since an HgdD mutant failed to secrete HesF. Several analyses revealed that a HesF minus mutant strain lacks the heterocyst-specific polysaccharide fibrous layer, accumulates high amounts of polysaccharides in the medium and that HesF is essential for the typical aggregation phenotype in diazotrophic conditions. Thus, we propose that HesF is a carbohydrate-binding exoprotein that plays a role in maintaining the heterocyst cell wall structure. A combination of and possibly interaction between HesF and heterocyst-specific polysaccharides seems to be responsible for filament adhesion and culture aggregation in heterocyst-forming cyanobacteria.

  16. Effects of recombinated Anabaena sp. lipoxygenase on the protein component and dough property of wheat flour.

    PubMed

    Wang, Xiaoming; Lu, Fengxia; Zhang, Chong; Lu, Yingjian; Bie, Xiaomei; Xie, Yajuan; Lu, Zhaoxin

    2014-10-01

    The improvement effect of recombinated Anabaena sp. lipoxygenase (ana-rLOX) on the rheological property of dough was investigated with a farinograph and an extensograph. When 30 U/g ana-rLOX was added to wheat flour, the dough stability time extended from 7 to 9.5 min, the degree of softening increased about 31.1%, and the farinograph index also ascended. The dough with added ana-rLOX showed stronger resistance to extension throughout 135 min of resting time as compared to the dough without ana-rLOX. In addition, the protein component in the dough was varied with ana-rLOX. The glutenin in the dough was increased, whereas the gliadin, albumin, and globulin were decreased after the additino of ana-rLOX to the flours. Ana-rLOX could make globulin-3A, globulin 1a, and S48186 grain softness protein cross-link with gliadin and low-molecular-weight (LMW) glutenin, leading to the formation of the protein polymer. These results based on proteomic analysis might provide evidence that ana-rLOX could affect the gluten protein component and explain why it improved the farinograph and extensograph parameters of wheat flour.

  17. Calcium impacts carbon and nitrogen balance in the filamentous cyanobacterium Anabaena sp. PCC 7120

    PubMed Central

    Walter, Julia; Lynch, Fiona; Battchikova, Natalia; Aro, Eva-Mari

    2016-01-01

    Calcium is integral to the perception, communication and adjustment of cellular responses to environmental changes. However, the role of Ca2+ in fine-tuning cellular responses of wild-type cyanobacteria under favourable growth conditions has not been examined. In this study, extracellular Ca2+ has been altered, and changes in the whole transcriptome of Anabaena sp. PCC 7120 have been evaluated under conditions replete of carbon and combined nitrogen. Ca2+ induced differential expression of many genes driving primary cellular metabolism, with transcriptional regulation of carbon- and nitrogen-related processes responding with opposing trends. However, physiological effects of these transcriptional responses on biomass accumulation, biomass composition, and photosynthetic activity over the 24h period following Ca2+ adjustment were found to be minor. It is well known that intracellular carbon:nitrogen balance is integral to optimal cell growth and that Ca2+ plays an important role in the response of heterocystous cyanobacteria to combined-nitrogen deprivation. This work adds to the current knowledge by demonstrating a signalling role of Ca2+ for making sensitive transcriptional adjustments required for optimal growth under non-limiting conditions. PMID:27012282

  18. Prediction of nitrogen metabolism-related genes in Anabaena by kernel-based network analysis.

    PubMed

    Okamoto, Shinobu; Yamanishi, Yoshihiro; Ehira, Shigeki; Kawashima, Shuichi; Tonomura, Koichiro; Kanehisa, Minoru

    2007-03-01

    Prediction of molecular interaction networks from large-scale datasets in genomics and other omics experiments is an important task in terms of both developing bioinformatics methods and solving biological problems. We have applied a kernel-based network inference method for extracting functionally related genes to the response of nitrogen deprivation in cyanobacteria Anabaena sp. PCC 7120 integrating three heterogeneous datasets: microarray data, phylogenetic profiles, and gene orders on the chromosome. We obtained 1348 predicted genes that are somehow related to known genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. While this dataset contained previously known genes related to the nitrogen deprivation condition, it also contained additional genes. Thus, we attempted to select any relevant genes using the constraints of Pfam domains and NtcA-binding sites. We found candidates of nitrogen metabolism-related genes, which are depicted as extensions of existing KEGG pathways. The prediction of functional relationships between proteins rather than functions of individual proteins will thus assist the discovery from the large-scale datasets.

  19. Salt and UV-B induced changes in Anabaena PCC 7120: physiological, proteomic and bioinformatic perspectives.

    PubMed

    Rai, Snigdha; Singh, Shilpi; Shrivastava, Alok Kumar; Rai, L C

    2013-11-01

    This study examines response of Anabaena sp. PCC 7120 to salt and UV-B stress by combining physiological, biochemical, proteomics and bioinformatics approaches. Sixty five significantly altered protein spots corresponding to 51 protein genes identified using MALDI-TOF MS/MS were divided into nine functional categories. Based on relative abundance, these proteins were grouped into four major sets. Of these, 27 and 5 proteins were up- and downregulated, respectively, both under salt and UV-B while 8 and 11 proteins showed accumulation in salt and UV-B applied singly. Some responses common to salt and UV-B included (i) enhanced expression of FeSOD, alr3090 and accumulation of MDA indicating oxidative stress, (ii) accumulation of PDH, G6P isomerase, FBPaldolase, TK, GAPDH and PGK suggesting enhanced glycolysis, (iii) upregulation of 6-PGD, 6PGL and NADPH levels signifying operation of pentose phosphate pathway, (iv) upregulation of Dps, NDK and alr3199 indicating DNA damage, and (v) accumulation of proteins of ribosome assembly, transcriptional and translational processing. In contrast, enhanced expression of RUBISCO, increased glycolate oxidase activity and ammonium content under salt signify the difference. Salt was found to be more damaging than UV-B probably due to a cumulative effect of ionic, osmotic and oxidative damage. A group of proteins having common expression represent decreased toxicity of salt and UV-B when applied in combination.

  20. Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7210 expressed in Escherichia coli

    SciTech Connect

    Gurevitz, M.; Somerville, C.R.; McIntosh, L.

    1985-10-01

    The authors have placed the genes encoding ribulosebisphosphate carboxylase/oxygenase from the Anabaena 7120 operon under transcriptional control of the lac promoter carried on the Escherichia coli plasmid pUC19. The genes encoding both the large and small subunit polypeptides (rbcL and rbcS) are transcribed and translated so that approx. = 0.6% of the soluble protein in E. coli extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the rbcL and rbcS genes. As a result, excess large subunit is synthesized and accumulates in E. coli as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, the authors propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.

  1. Oxygen-dependent proton efflux in cyanobacteria (blue-green algae). [Anabaena variabilis

    SciTech Connect

    Scherer, S.; Stuerzl, E.; Boeger, P.

    1984-05-01

    The oxygen-dependent proton efflux (in the dark) of intact cells of Anabaena variabilis and four other cyanobacteria (blue-green algae) was investigated. In contrast to bacteria and isolated mitochondria, an H/sup +//e ratio (= protons translocated per electron transported) of only 0.23 to 0.35 and a P/e ratio of 0.8 to 1.5 were observed, indicative of respiratory electron transport being localized essentially on the thylakoids, not on the cytoplasmic membrane. Oxygen-induced acidification of the medium was sensitive to cyanide and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Inhibitors such as 2,6-dinitrophenol and vanadate exhibited a significant decrease in the H/sup +//e ratio. After the oxygen pulse, electron transport started immediately, but proton efflux lagged 40 to 60 s behind, a period also needed before maximum ATP pool levels were attained. The authors suggest that proton efflux in A. variabilis is due to a proton-translocating ATP hydrolase (ATP-consuming ATPase) rather than to respiratory electron transport located on the cytoplasmic membrane.

  2. Functional expression and purification of Anabaena PCC 7120 XisA protein.

    PubMed

    Trivedi, Ujwal; Kaushik, Shubham; Kunjadia, Prashant; Saravanan, Matheshwaran; Nagaraja, Valakunja; Archana, Gattupalli; Nareshkumar, Gattupalli

    2016-02-01

    Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 (°)C on LB under static condition.

  3. Effects of supplements on the bioaccumulation of lead in Anabaena spp

    SciTech Connect

    Bender, J.; Ibeanusi, V.

    1987-08-01

    Heavy metals are known to be insidious toxic pollutants in the natural environment and their presence in aquatic systems is a current major concern. Since the primary producers, green algae and cyanobacteria, are able to accumulate large quantities of metals, these contaminants can be efficiently passed along in the food chain. However, the ability to accumulate metals from solution also indicates their potential use as sequestering agents in metal recovery systems. Previous studies on the mechanisms of metals uptake have demonstrated that the process generally takes place in two distinct stages. The first stage has been described as a passive adsorption of ions and it is likely that a number of different functional groups are involved in this process. The second stage of uptake is slower and involves active transport mechanisms requiring cellular energy. A descriptive model of long-term metal uptake, including experiments in media enrichments, has been lacking in previous research. Understanding the process of metal uptake and its relation to cell growth over longer periods of time is important to both the development of recovery systems and the production of metal transport through the ecosystem. The objectives of this research are to investigate (1) the effects of carbon dioxide and waste water enrichments on the simultaneous culture and lead uptake in Anabaena during a 30-day culture period and (2) the effects of energy supply on the lead uptake.

  4. Anabaena sp. DyP-type peroxidase is a tetramer consisting of two asymmetric dimers.

    PubMed

    Yoshida, Toru; Ogola, Henry Joseph Oduor; Amano, Yoshimi; Hisabori, Toru; Ashida, Hiroyuki; Sawa, Yoshihiro; Tsuge, Hideaki; Sugano, Yasushi

    2016-01-01

    DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.

  5. Aluminum effects on uptake and metabolism of phosphorus by the Cyanobacterium Anabaena cylindrica

    SciTech Connect

    Pettersson, A.; Haellbom, L. Bergman, B.

    1988-01-01

    Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Pre- or post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AlPO/sub 4/ never exceeded 10% of the total phosphate concentration. The uptake of /sup 32/P-phosphorus is not disturbed by aluminium either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica.

  6. Cryo-imaging of photosystems and phycobilisomes in Anabaena sp. PCC 7120 cells.

    PubMed

    Steinbach, Gábor; Schubert, Félix; Kaňa, Radek

    2015-11-01

    Primary photosynthetic reactions take place inside thylakoid membrane where light-to-chemical energy conversion is catalyzed by two pigment-protein complexes, photosystem I (PSI) and photosystem II (PSII). Light absorption in cyanobacteria is increased by pigment-protein supercomplexes--phycobilisomes (PBSs) situated on thylakoid membrane surfaces that transfer excitation energy into both photosystems. We have explored the localization of PSI, PSII and PBSs in thylakoid membrane of native cyanobacteria cell Anabaena sp. 7120 by means of cryogenic confocal microscopy. We have adapted a conventional temperature controlling stage to an Olympus FV1000 confocal microscope. The presence of red shifted emission of chlorophylls from PSI has been confirmed by spectral measurements. Confocal fluorescence images of PSI (in a spectral range 710-750 nm), PSII (in a spectral range 690-705 nm) and PBSs (in a spectral range 650-680 nm) were recorded at low temperature. Co-localization of images showed spatial heterogeneity of PSI, PSII and PBSs over the thylakoid membrane, and three dominant areas were identified: PSI-PSII-PBS supercomplex area, PSII-PBS supercomplex area and PSI area. The observed results were discussed with regard to light-harvesting regulation in cyanobacteria.

  7. Flavodoxin from Anabaena 7120: uniform N-15 enrichment and one- and two-dimensional NMR investigations

    SciTech Connect

    Stockman, B.J.; Westler, W.M.; Mooberry, E.S.; Markley, J.L.

    1987-05-01

    The flavodoxin isolated from Anabaena 7120 grown under iron-limiting conditions has been studied in its oxidized form. Flavodoxin 95%+ enriched in VN was obtained by growing the cyanobacterium with 98% VN nitrate as the sole nitrogen source. A one-dimensional H NMR (500 MHz) spectrum has been collected, as well as a double-quantum-filtered COSY spectrum in D2O. One-dimensional VN NMR (50.68 MHz) spectra have been obtained by observing nitrogen directly and by using the INEPT pulse sequence. Results of a two-dimensional H-detected VN experiment allowed the correlation of VN and H resonances of VN-/sup I/H groups. The four nitrogen resonance of the FMN cofactor have been assigned: N(1), 188.0 ppm; N(3), 162.5 ppm; N(5) 335.0 ppm; and N(10), 163.5 ppm. The resonance assigned to N(3) is coupled to a proton at 10.9 ppm. Shifts in the positions of these VN resonances, compared to corresponding ones in free FMN, suggest that these positions are strongly hydrogen-bonded in the oxidized state as has been observed with lower molecular weight flavodoxins.

  8. Aluminum Effects on Uptake and Metabolism of Phosphorus by the Cyanobacterium Anabaena cylindrica

    PubMed Central

    Pettersson, Annette; Hällbom, Lars; Bergman, Birgitta

    1988-01-01

    Aluminum severely affects the growth of the cyanobacterium Anabaena cylindrica and induces symptoms indicating phosphorus starvation. Preor post-treating the cells with high (90 micromolar) phosphorus reduces the toxicity of aluminum compared to cells receiving a lower orthophosphate concentration. In this study aluminum (ranging from 9 to 36 micromolar) and phosphorus concentrations were chosen so that the precipitation of insoluble AIPO4 never exceeded 10% of the total phosphate concentration. The uptake of 32P-phosphorus is not disturbed by aluminum either at high (100 micromolar) or low (10 micromolar) concentrations of phosphate. Also, the rapid accumulation of polyphosphate granules in cells exposed to aluminum indicates that the incorporation of phosphate is not disturbed. However, a significant decrease in the mobilization of the polyphosphates is observed, as is a lowered activity of the enzyme acid phosphatase, in aluminum treated cells. We conclude that aluminum acts on the intracellular metabolism of phosphate, which eventually leads to phosphorus starvation rather than on its uptake in the cyanobacterium A. cylindrica. PMID:16665849

  9. HesF, an exoprotein required for filament adhesion and aggregation in Anabaena sp. PCC 7120.

    PubMed

    Oliveira, Paulo; Pinto, Filipe; Pacheco, Catarina C; Mota, Rita; Tamagnini, Paula

    2015-05-01

    Here, we report on the identification and characterization of a protein (Alr0267) named HesF, found in the extracellular milieu of Anabaena sp. PCC 7120 grown diazotrophically. hesF was found to be highly upregulated upon transition from non-nitrogen-fixing to nitrogen-fixing conditions, and the highest transcript levels were detected towards the end of the heterocyst differentiation process. The hesF promoter drives transcription of the gene in heterocysts only, and both NtcA and HetR are essential for the gene's in vivo activation. An examination of HesF's translocation showed that the secretion system is neither heterocyst-specific nor dependent on nitrogen-fixing conditions. Furthermore, HesF was found to be a type I secretion system substrate, since an HgdD mutant failed to secrete HesF. Several analyses revealed that a HesF minus mutant strain lacks the heterocyst-specific polysaccharide fibrous layer, accumulates high amounts of polysaccharides in the medium and that HesF is essential for the typical aggregation phenotype in diazotrophic conditions. Thus, we propose that HesF is a carbohydrate-binding exoprotein that plays a role in maintaining the heterocyst cell wall structure. A combination of and possibly interaction between HesF and heterocyst-specific polysaccharides seems to be responsible for filament adhesion and culture aggregation in heterocyst-forming cyanobacteria. PMID:25142951

  10. Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets.

    PubMed

    Kim, Youngchang; Ye, Zi; Joachimiak, Grazyna; Videau, Patrick; Young, Jasmine; Hurd, Kathryn; Callahan, Sean M; Gornicki, Piotr; Zhao, Jindong; Haselkorn, Robert; Joachimiak, Andrzej

    2013-05-01

    HetR is an essential regulator of heterocyst development in cyanobacteria. Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein binds to a DNA palindrome upstream of hetP and other genes. We have determined the crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at 2.50-, 3.00-, and 3.25-Å resolution, respectively. The highest-resolution structure shows fine details of specific protein-DNA interactions. The lower-resolution structures with longer DNA duplexes have similar interaction patterns and show how the flap domains interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein-DNA contacts predicted on the basis of the structure were confirmed by single amino acid mutations that abolished binding in vitro and complementation in vivo. A striking feature of the structure is the association of glutamate 71 from each subunit of the HetR dimer with three successive cytosines in each arm of the palindromic target, a feature that is conserved among all known heterocyst-forming cyanobacteria sequenced to date. PMID:23610410

  11. Lipopolysaccharide containing L-acofriose in the filamentous blue-green alga Anabaena variabilis.

    PubMed

    Weckesser, J; Katz, A; Drews, G; Mayer, H; Fromme, I

    1974-11-01

    For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga (Anabaena variabilis). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l-rhamnose, its 3-O-methyl ether l-acofriose, d-mannose, d-glucose, and d-galactose. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d-glucosamine. Three hydroxy fatty acids were identified, namely, beta-hydroxymyristic, beta-hydroxypalmitic and beta-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.

  12. Dynamic transcriptional changes in response to rehydration in Anabaena sp. PCC 7120.

    PubMed

    Higo, Akiyoshi; Suzuki, Takayuki; Ikeuchi, Masahiko; Ohmori, Masayuki

    2007-11-01

    Global transcriptional responses to dehydration and rehydration were determined in Anabaena sp. PCC 7120. Nearly 300 genes were up- or downregulated during both dehydration and rehydration. While as many as 133 genes showed dehydration-specific downregulation, only 29 genes showed dehydration-specific upregulation. In contrast, while only 13 genes showed rehydration-specific downregulation, as many as 259 genes showed rehydration-specific upregulation. The genes upregulated during rehydration responded rapidly and transiently, whereas those upregulated during dehydration did so gradually and persistently. The expression of various genes involved in DNA repair, protein folding and NAD synthesis, as well as genes responding to nitrogen depletion and CO2 limitation, was upregulated during rehydration. Although no genes for transcriptional regulators showed dehydration-specific upregulation, eight showed rehydration-specific upregulation. Among them, two genes, ancrpB and alr0618, encode putative transcriptional activators of the cAMP receptor protein (CRP) family. DNA microarray analysis using gene disruptants revealed that AnCrpB and Alr0618 regulate the genes induced by nitrogen depletion and by CO2 limitation, respectively. We conclude that rehydration is a complex process in which the expression of certain genes, particularly those for metabolism, is dramatically induced. PMID:17975076

  13. Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

    PubMed

    Singh, Prashant Kumar; Shrivastava, Alok Kumar; Chatterjee, Antra; Pandey, Sarita; Rai, Snigdha; Singh, Shilpi; Rai, L C

    2015-09-01

    Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

  14. Sensory Transduction in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Brown, Austin L.; Ramot, Daniel; Goodman, Miriam B.

    The roundworm Caenorhabditis elegans has a well-defined and comparatively simple repertoire of sensory-guided behaviors, all of which rely on its ability to detect chemical, mechanical or thermal stimuli. In this chapter, we review what is known about the ion channels that mediate sensation in this remarkable model organism. Genetic screens for mutants defective in sensory-guided behaviors have identified genes encoding channel proteins, which are likely transducers of chemical, thermal, and mechanical stimuli. Such classical genetic approaches are now being coupled with molecular genetics and in vivo cellular physiology to elucidate how these channels are activated in specific sensory neurons. The ion channel superfamilies implicated in sensory transduction in C. elegans - CNG, TRP, and DEG/ENaC - are conserved across phyla and also appear to contribute to sensory transduction in other organisms, including vertebrates. What we learn about the role of these ion channels in C. elegans sensation is likely to illuminate analogous processes in other animals, including humans.

  15. Crossmodal plasticity in sensory loss.

    PubMed

    Frasnelli, Johannes; Collignon, Olivier; Voss, Patrice; Lepore, Franco

    2011-01-01

    In this review, we describe crossmodal plasticity following sensory loss in three parts, with each section focusing on one sensory system. We summarize a wide range of studies showing that sensory loss may lead, depending of the affected sensory system, to functional changes in other, primarily not affected senses, which range from heightened to lowered abilities. In the first part, the effects of blindness on mainly audition and touch are described. The latest findings on brain reorganization in blindness are reported, with a particular emphasis on imaging studies illustrating how nonvisual inputs recruit the visually deafferented occipital cortex. The second part covers crossmodal processing in deafness, with a special focus on the effects of deafness on visual processing. In the last portion of this review, we present the effects that the loss of a chemical sense have on the sensitivity of the other chemical senses, that is, smell, taste, and trigeminal chemosensation. We outline how the convergence of the chemical senses to the same central processing areas may lead to the observed reduction in sensitivity of the primarily not affected senses. Altogether, the studies reviewed herein illustrate the fascinating plasticity of the brain when coping with sensory deprivation. PMID:21741555

  16. dPob/EMC is essential for biosynthesis of rhodopsin and other multi-pass membrane proteins in Drosophila photoreceptors

    PubMed Central

    Satoh, Takunori; Ohba, Aya; Liu, Ziguang; Inagaki, Tsuyoshi; Satoh, Akiko K

    2015-01-01

    In eukaryotes, most integral membrane proteins are synthesized, integrated into the membrane, and folded properly in the endoplasmic reticulum (ER). We screened the mutants affecting rhabdomeric expression of rhodopsin 1 (Rh1) in the Drosophila photoreceptors and found that dPob/EMC3, EMC1, and EMC8/9, Drosophila homologs of subunits of ER membrane protein complex (EMC), are essential for stabilization of immature Rh1 in an earlier step than that at which another Rh1-specific chaperone (NinaA) acts. dPob/EMC3 localizes to the ER and associates with EMC1 and calnexin. Moreover, EMC is required for the stable expression of other multi-pass transmembrane proteins such as minor rhodopsins Rh3 and Rh4, transient receptor potential, and Na+K+-ATPase, but not for a secreted protein or type I single-pass transmembrane proteins. Furthermore, we found that dPob/EMC3 deficiency induces rhabdomere degeneration in a light-independent manner. These results collectively indicate that EMC is a key factor in the biogenesis of multi-pass transmembrane proteins, including Rh1, and its loss causes retinal degeneration. DOI: http://dx.doi.org/10.7554/eLife.06306.001 PMID:25715730

  17. Overexpression of AhpC enhances stress tolerance and N2-fixation in Anabaena by upregulating stress responsive genes.

    PubMed

    Shrivastava, Alok Kumar; Pandey, Sarita; Dietz, Karl Josef; Singh, Prashant Kumar; Singh, Shilpi; Rai, Ruchi; Rai, Lal Chand

    2016-11-01

    The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40-60% compared to a 33-40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena. PMID:27487031

  18. Overexpression of AhpC enhances stress tolerance and N2-fixation in Anabaena by upregulating stress responsive genes.

    PubMed

    Shrivastava, Alok Kumar; Pandey, Sarita; Dietz, Karl Josef; Singh, Prashant Kumar; Singh, Shilpi; Rai, Ruchi; Rai, Lal Chand

    2016-11-01

    The study explores the significance of peroxides in regulating the CO2- and N2-fixation capacities in Anabaena sp. PCC7120. To this end Anabaena strains were generated carrying an extra copy of ahpC (An+ahpC) or by deleting from their endogenous functional ahpC (AnΔahpC). AhpC levels were 2.2- to 6.0-fold higher in An+ahpC than in wild type. An+ahpC revealed 1.4- to 2-fold upregulation of photosystems I and II, nitrogenase, superoxide dismutase and catalase activities while same activities were 1.3- to 2.5-fold downregulated in the insertional mutant (AnΔahpC) compared to the wild type. Peroxide, superoxide and malondialdehyde contents were low in An+ahpC and high in AnΔahpC. Growth was inhibited in AnΔahpC by approximately 40-60% compared to a 33-40% enhanced growth in An+ahpC under selected stresses. Most interestingly, heterocyst frequency was increased in An+ahpC. In order to address transcriptional and posttranscriptional effects, transcripts of genes including groEL, fld, kat, gor, gst, dps, bfr, tf, sodA, dnaK, prx, uspA, pcs and apx were quantified and found to be increased 1.33- to 7.70-fold in unstressed and 1.76- to 13.80-fold in stressed An+ahpC. In a converse manner, they were downregulated by 1.20- to 7.50-fold in unstressed and 1.23 to 10.20-fold in stressed AnΔahpC. It is concluded that the level of AhpC controls a major set of metabolic and developmental genes in normal and stress conditions and thus likely is in the core of the redox regulatory system of Anabaena.

  19. Utilization of Anabaena sp. in CO₂ removal processes: modelling of biomass, exopolysaccharides productivities and CO₂ fixation rate.

    PubMed

    Sánchez Fernández, J F; González-López, C V; Acién Fernández, F G; Fernández Sevilla, J M; Molina Grima, E

    2012-05-01

    This paper focuses on modelling the growth rate and exopolysaccharides production of Anabaena sp. ATCC 33047, to be used in carbon dioxide removal and biofuels production. For this, the influence of dilution rate, irradiance and aeration rate on the biomass and exopolysaccharides productivity, as well as on the CO(2) fixation rate, have been studied. The productivity of the cultures was maximum at the highest irradiance and dilution rate assayed, resulting to 0.5 g(bio) l(-1) day(-1) and 0.2 g(eps) l(-1) day(-1), and the CO(2) fixation rate measured was 1.0 gCO(2) l(-1) day(-1). The results showed that although Anabaena sp. was partially photo-inhibited at irradiances higher than 1,300 μE m(-2) s(-1), its growth rate increases hyperbolically with the average irradiance inside the culture, and so does the specific exopolysaccharides production rate. The latter, on the other hand, decreases under high external irradiances, indicating that the exopolysaccharides metabolism hindered by photo-damage. Mathematical models that consider these phenomena have been proposed. Regarding aeration, the yield of the cultures decreased at rates over 0.5 v/v/min or when shear rates were higher than 60 s(-1), demonstrating the existence of thus existence of stress damage by aeration. The behaviour of the cultures has been verified outdoors in a pilot-scale airlift tubular photobioreactor. From this study it is concluded that Anabaena sp. is highly recommended to transform CO(2) into valuable products as has been proved capable of metabolizing carbon dioxide at rates of 1.2 gCO(2) l(-1) day(-1) outdoors. The adequacy of the proposed equations is demonstrated, resulting to a useful tool in the design and operation of photobioreactors using this strain.

  20. Establishment of quantitative PCR methods for the quantification of geosmin-producing potential and Anabaena sp. in freshwater systems.

    PubMed

    Su, Ming; Gaget, Virginie; Giglio, Steven; Burch, Michael; An, Wei; Yang, Min

    2013-06-15

    Geosmin has often been associated with off-flavor problems in drinking water with Anabaena sp. as the major producer. Rapid on-site detection of geosmin-producers as well as geosmin is important for a timely management response to potential off-flavor events. In this study, quantitative polymerase chain reaction (qPCR) methods were developed to detect the levels of Anabaena sp. and geosmin, respectively, by designing two PCR primer sets to quantify the rpoC1 gene (ARG) and geosmin synthase one (GSG) in Anabaena sp. in freshwater systems. The ARG density determined by qPCR assay is highly related to microscopic cell count (r(2) = 0.726, p < 0.001), and the limit of detection (LOD) and limit of quantification (LOQ) of the qPCR method were 0.02 pg and 0.2 pg of DNA, respectively. At the same time, the relationship between geosmin concentrations measured by gas chromatography-mass spectrometry (GC-MS) and GSG copies was also established (r(2) = 0.742, p < 0.001) with similar LOD and LOQ values. Using the two qPCR protocols, we succeeded in measuring different levels of ARG and GSG copies in different freshwater systems with high incidence environmental substrata and diverse ecological conditions, showing that the methods developed could be applied for environmental monitoring. Moreover, comparing to the microscopic count and GC-MS analytical methods, the qPCR methods can reduce the time-to-results from several days to a few hours and require considerably less traditional algal identification and taxonomic expertise.

  1. ppGpp metabolism is involved in heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Zhang, Shao-Ran; Lin, Gui-Ming; Chen, Wen-Li; Wang, Li; Zhang, Cheng-Cai

    2013-10-01

    When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173-176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117-122, 1987). Here we show that all1549 (here designated relana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of relana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of relana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation.

  2. ppGpp metabolism is involved in heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Zhang, Shao-Ran; Lin, Gui-Ming; Chen, Wen-Li; Wang, Li; Zhang, Cheng-Cai

    2013-10-01

    When deprived of a combined-nitrogen source in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) can form heterocysts capable of nitrogen fixation. The process of heterocyst differentiation takes about 20 to 24 h, during which extensive metabolic and morphological changes take place. Guanosine tetraphosphate (ppGpp) is the signal of the stringent response that ensures cell survival by adjusting major cellular activities in response to nutrient starvation in bacteria, and ppGpp accumulates at the early stage of heterocyst differentiation (J. Akinyanju, R. J. Smith, FEBS Lett. 107:173-176, 1979; J Akinyanju, R. J. Smith, New Phytol. 105:117-122, 1987). Here we show that all1549 (here designated relana) in Anabaena, homologous to relA/spoT, is upregulated in response to nitrogen deprivation and predominantly localized in vegetative cells. The disruption of relana strongly affects the synthesis of ppGpp, and the resulting mutant, all1549Ωsp/sm, fails to form heterocysts and to grow in the absence of a combined-nitrogen source. This phenotype can be complemented by a wild-type copy of relana. Although the upregulation of hetR is affected in the mutant, ectopic overexpression of hetR cannot rescue the phenotype. However, we found that the mutant rapidly loses its viability, within a time window of 3 to 6 h, following the deprivation of combined nitrogen. We propose that ppGpp plays a major role in rebalancing the metabolic activities of the cells in the absence of the nitrogen source supply and that this regulation is necessary for filament survival and consequently for the success of heterocyst differentiation. PMID:23935047

  3. Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway

    SciTech Connect

    Ganter, U.M.; Longstaff, C.; Pajares, M.A.; Rando, R.R.; Siebert, F. )

    1991-03-01

    Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.

  4. Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.

    PubMed

    Berger, Adeline; Lorain, Stéphanie; Joséphine, Charlène; Desrosiers, Melissa; Peccate, Cécile; Voit, Thomas; Garcia, Luis; Sahel, José-Alain; Bemelmans, Alexis-Pierre

    2015-05-01

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

  5. Sequence, Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

    PubMed Central

    Wolf, Steffen; Grünewald, Stefan

    2015-01-01

    G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs. PMID:25881057

  6. Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

    PubMed

    Ganter, U M; Longstaff, C; Pajares, M A; Rando, R R; Siebert, F

    1991-03-01

    Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species. PMID:2049524

  7. Mechanism of Rhodopsin Activation as Examined with Ring-constrained Retinal Analogs and the Crystal Structure of the Ground State Protein*

    PubMed Central

    Jang, Geeng-Fu; Kuksa, Vladimir; Filipek, Stawomir; Bartl||, Franz; Ritter, Eglof; Gelb, Michael H.; Hofmann, Klaus Peter; Palczewski, Krzysztof

    2006-01-01

    The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the β-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the β-ionone ring which ultimately leads to helix movements and determines receptor activation. PMID:11316815

  8. Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

    PubMed

    Ganter, U M; Longstaff, C; Pajares, M A; Rando, R R; Siebert, F

    1991-03-01

    Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.

  9. Characterization of three putative xylulose 5-phosphate/fructose 6-phosphate phosphoketolases in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Moriyama, Takashi; Tajima, Naoyuki; Sekine, Kohsuke; Sato, Naoki

    2015-01-01

    Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) is a key enzyme in the central carbohydrate metabolism in heterofermentative bacteria, in which enzymatic property of Xfps is well characterized. This is not the case in other microbes. The cyanobacterium Anabaena sp. PCC 7120 possesses three putative genes encoding Xfp, all1483, all2567, and alr1850. We purified three putative Xfps as recombinant proteins. The results of gel filtration indicated that these proteins form homomultimer complex. All1483 and All2567 showed phosphoketolase activity, whereas Alr1850 did not show the activity. Kinetic analyses demonstrated that substrates, fructose 6-phosphate and inorganic phosphate, are cooperatively bound to enzymes positively and negatively, respectively.

  10. Ultrastructure of the fresh water cyanobacterium Anabaena variabilis SPU 003 and its application for oxygen-free hydrogen production.

    PubMed

    Shah, V; Garg, N; Madamwar, D

    2001-01-01

    Photoproduction of hydrogen has been studied as one of the ways to produce a clean, renewable energy source. Ultrastructure of the selected strain Anabaena variabilis SPU 003, a heterocystous cyanobacterium, has been done to understand the cell structure. The organism was found to be essentially a dark hydrogen producer. While pH had no significant effect on hydrogen production, optimum temperature was found to be 30 degrees C. Various sugars increased the production of hydrogen while the presence of various nitrogen sources inhibits the production. The production of hydrogen is highly sensitive to salinity and micronutrients.

  11. Combined effect of oil, oil products and dispersants on the blue-green algae Synechocystis aquatilis and Anabaena variabilis

    SciTech Connect

    Gapochka, L.D.; Brodskii, L.I.; Kravchenko, M.E.; Fedorov, V.D.

    1980-01-01

    The study of the combined effect of oil, oil products and dispersants on the growth of the blue-green algae Synechocystis aquatilis and Anabaena variabilis has shown that out of 12 studied oil-dispersant pairs 6 revealed a positive relationship, which provides evidence for a decrease in oil and oil products toxic effect in the presence of a dispersant. The positive interaction between oil and oil products was found. The negative oil and oil products effect on all studied indices of A. variabilis culture increases with time.

  12. Effect of retorted-oil shale leachate on a blue-green alga (Anabaena flos-aquae)

    SciTech Connect

    McKnight, D.M.; Pereira, W.E.; Rostad, C.E.; Stiles, E.A.

    1983-01-01

    Oil shale reserves in Piceance Creek and the White River, Colorado, contain retorted-shale piles which may pollute th eground water through leaching. Bioassay experiments were carried out to determine the effects of leachates on algae, particularly ANABAENA FLOSAQUAE and SCENEDESMUS. Tests were done to establish inhibitionary concentrations of the leachates from various sources of spent shale. Retorted-shale leachates had major effects on the algae papulations at a 40% concentration, minor effects at 8%, and no effects at 0.4%. Discrepancies between experimental results and actual retorted-shale piles are dixcussed. (JMT)

  13. Molecular modeling of A1 and A2A adenosine receptors: comparison of rhodopsin- and beta2-adrenergic-based homology models through the docking studies.

    PubMed

    Yuzlenko, Olga; Kieć-Kononowicz, Katarzyna

    2009-01-15

    Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. The homology models of adenosine A1 and A2A receptors were constructed. The high-resolution X-ray structure of bovine rhodopsin and crystal structure of beta2-adrenergic receptor were used as templates. The binding sites of the A1 and A2A ARs were constructed by using data obtained from mutagenesis experiments as well as docking simulations of the respective AR antagonsists DPCPX and XAC. To compare rhodopsin- and beta2-adrenergic-based models, the binding mode of A1 (KW-3902, LUF-5437) and A2A (KW-6002, ZM-241385) ARs antagonists were also examined. The differences in the binding ability of both models were noted during the study. The beta2-adrenergic-based A2A AR model was much more capable to stabilize the ligand in the binding site cavity than the corresponding rhodopsin-based A2A AR model, however, such differences were not so clear in case of A1 AR models. It was suggested that for the A1 AR it is possible to use the crystal structure of rhodopsin as a template as well as beta2-adrenergic receptor, but for A2A AR, with the now available beta2-adrenergic receptor X-ray structure, docking studies should be avoided on the rhodopsin-based model. However, taking into account that the beta2AR shares about 31% of the residues with the AR in comparison to 21% in case of bRho, we suggest using beta2-adrenergic-based models for the A1 and A2A ARs for further in silico ligand screening also because of their generally better ability to stabilize ligands inside the binding pocket.

  14. Anaerobic and Aerobic Hydrogen Gas Formation by the Blue-Green Alga Anabaena cylindrica

    PubMed Central

    Daday, Arlene; Platz, Rosalea A.; Smith, Geoffrey D.

    1977-01-01

    An investigation was made of certain factors involved in the formation of hydrogen gas, both in an anaerobic environment (argon) and in air, by the blue-green alga Anabaena cylindrica. The alga had not been previously adapted under hydrogen gas and hence the hydrogen evolution occurred entirely within the nitrogen-fixing heterocyst cells; organisms grown in a fixed nitrogen source, and which were therefore devoid of heterocysts, did not produce hydrogen under these conditions. Use of the inhibitor dichlorophenyl-dimethyl urea showed that hydrogen formation was directly dependent on photosystem I and only indirectly dependent on photosystem II, consistent with heterocysts being the site of hydrogen formation. The uncouplers carbonyl cyanide chlorophenyl hydrazone and dinitrophenol almost completely inhibited hydrogen formation, indicating that the process occurs almost entirely via the adenosine 5′-triphosphate-dependent nitrogenase. Salicylaldoxime also inhibited hydrogen formation, again illustrating the necessity of photophosphorylation. Whereas hydrogen formation could usually only be observed in anaerobic, dinitrogen-free environments, incubation in the presence of the dinitrogen-fixing inhibitor carbon monoxide plus the hydrogenase inhibitor acetylene resulted in significant formation of hydrogen even in air. Hydrogen formation was studied in batch cultures as a function of age of the cultures and also as a function of culture concentration, in both cases the cultures being harvested in logarithmic growth. Hydrogen evolution (and acetylene-reducing activity) exhibited a distinct maximum with respect to the age of the cultures. Finally, the levels of the protective enzyme, superoxide dismutase, were measured in heterocyst and vegetative cell fractions of the organism; the level was twice as high in heterocyst cells (2.3 units/mg of protein) as in vegetative cells (1.1 units/mg of protein). A simple procedure for isolating heterocyst cells is described. PMID

  15. Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

    PubMed

    Teikari, Jonna; Österholm, Julia; Kopf, Matthias; Battchikova, Natalia; Wahlsten, Matti; Aro, Eva-Mari; Hess, Wolfgang R; Sivonen, Kaarina

    2015-08-01

    Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

  16. Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120.

    PubMed

    Kawano, Yusuke; Sekine, Midori; Ihara, Masaki

    2014-05-01

    Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.

  17. Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

    PubMed Central

    Spiller, H; Latorre, C; Hassan, M E; Shanmugam, K T

    1986-01-01

    A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment. PMID:2867990

  18. Regulation of Three Nitrogenase Gene Clusters in the Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Thiel, Teresa; Pratte, Brenda S.

    2014-01-01

    The filamentous cyanobacterium Anabaena variabilis ATCC 29413 fixes nitrogen under aerobic conditions in specialized cells called heterocysts that form in response to an environmental deficiency in combined nitrogen. Nitrogen fixation is mediated by the enzyme nitrogenase, which is very sensitive to oxygen. Heterocysts are microxic cells that allow nitrogenase to function in a filament comprised primarily of vegetative cells that produce oxygen by photosynthesis. A. variabilis is unique among well-characterized cyanobacteria in that it has three nitrogenase gene clusters that encode different nitrogenases, which function under different environmental conditions. The nif1 genes encode a Mo-nitrogenase that functions only in heterocysts, even in filaments grown anaerobically. The nif2 genes encode a different Mo-nitrogenase that functions in vegetative cells, but only in filaments grown under anoxic conditions. An alternative V-nitrogenase is encoded by vnf genes that are expressed only in heterocysts in an environment that is deficient in Mo. Thus, these three nitrogenases are expressed differentially in response to environmental conditions. The entire nif1 gene cluster, comprising at least 15 genes, is primarily under the control of the promoter for the first gene, nifB1. Transcriptional control of many of the downstream nif1 genes occurs by a combination of weak promoters within the coding regions of some downstream genes and by RNA processing, which is associated with increased transcript stability. The vnf genes show a similar pattern of transcriptional and post-transcriptional control of expression suggesting that the complex pattern of regulation of the nif1 cluster is conserved in other cyanobacterial nitrogenase gene clusters. PMID:25513762

  19. Effects of nutrient loading on Anabaena flos-aquae biofilm: biofilm growth and nutrient removals.

    PubMed

    Li, Xiaowei; Wei, Qun; Tu, Xiaojie; Zhu, Yuxuan; Chen, Yanfei; Guo, Lina; Zhou, Jun; Sun, Hongyun

    2016-01-01

    Effects of three different nutrient loadings (low nutrient loading, medium nutrient loading and high nutrient loading, denoted as LNS, MNS and HNS, respectively) on the structure and functions of algal biofilm using Anabaena flos-aquae were investigated using synthetic wastewater. Nutrients removal efficiencies, biofilm thickness, microalgae dehydrogenase activity (DHA) and exopolysaccharide (EPS) productions were examined. Results showed that the changes of nutrient concentration were insignificant after 4 days of experiment for the case of HNS condition; 9 days for the case of MNS condition, and 6 days for the case of LNS condition, respectively. The biofilm thickness, nutrient removal efficiencies, algae DHA and EPS productions increased with the increase of nutrient loadings in synthetic wastewater. For the case of HNS condition, the microalgal biofilm exhibited the best performance in terms of C, N and P removal efficiencies, reaching the removal rates of 68.45, 3.56 and 1.61 mg·L(-1)·d(-1) for C, N, P, respectively. This was likely because, fact with the high nutrient loading, the high biological activity could be achieved, thus resulting in high nutrient removals. The thickness of the biofilm in HNS condition was 75 μm, which was closely related to EPS production. DHA and EPS concentrations were 7.24 and 1.8 × 10(-2) mg·mm(-2), respectively. It was also shown that apart from the nutrient loading, the structure and functions of microalgal biofilm were also influenced by other factors, such as illumination and temperature. PMID:27438243

  20. Bioremoval of heavy metals and nutrients from sewage plant by Anabaena oryzae and Cyanosarcina fontana.

    PubMed

    Fawzy, Mustafa A; Issa, Ahmed A

    2016-01-01

    The present study demonstrated the growth of two species of cyanobacteria on wastewater isolated from sewage plant in Aswan, Egypt. We evaluated their efficiency for eliminating nitrogen, phosphorus, chemical oxygen demand (COD) and heavy metals (Fe(2+), Pb(2+), Cu(2+), and Mn(2+)). The growth of Cyanosarcina fontana has supported wastewater as a growth medium than Anabaena oryzae compared to standard medium. The nutrients concentration such as COD, NO3-N and PO4-P were decreased by the growth of A. oryzae and C. fontana in the wastewater after primary settling and centrate. However, the reduction of COD was less efficient than the other nutrients. The reduction percentage of COD, NO3-N and PO4-P reached 39.3, 84.1 and 90.7% as well as 54.6, 83.1, and 89.8%, in cultures of A. oryzae and C. fontana grown in the wastewater after primary settling, respectively. The reduction amounted to 10.1, 76.8, and 63.0% by A. oryzae and 43.2, 62.1, and 74.8% by C. fontana, grown in the centrate, respectively. Cyanobacteria species have the ability to accumulate the heavy metals from the wastewater to level far than the exceeding metal level in the water. Whereas, the heavy metals biosorption performance of C. fontana was higher in accumulating Fe(2+) (93.95%), Pb(2+) (81.21%), Cu(2+) (63.9%), and Mn(2+) (48.49%) compared to A. oryzae. The biosorption ability is dependent on the nature of the adsorbent studied and the type of wastewater treated. Therefore, removal of heavy metals and nutrients by the tested algae is strongly recommended as a powerful technique for the removal of pollutants from wastewater.

  1. Regulation of nitrogenase gene expression by transcript stability in the cyanobacterium Anabaena variabilis.

    PubMed

    Pratte, Brenda S; Thiel, Teresa

    2014-10-01

    The nitrogenase gene cluster in cyanobacteria has been thought to comprise multiple operons; however, in Anabaena variabilis, the promoter for the first gene in the cluster, nifB1, appeared to be the primary promoter for the entire nif cluster. The structural genes nifHDK1 were the most abundant transcripts; however, their abundance was not controlled by an independent nifH1 promoter, but rather, by RNA processing, which produced a very stable nifH1 transcript and a moderately stable nifD1 transcript. There was also no separate promoter for nifEN1. In addition to the nifB1 promoter, there were weak promoters inside the nifU1 gene and inside the nifE1 gene, and both promoters were heterocyst specific. In an xisA mutant, which effectively separated promoters upstream of an 11-kb excision element in nifD1 from the downstream genes, the internal nifE1 promoter was functional. Transcription of the nif1 genes downstream of the 11-kb element, including the most distant genes, hesAB1 and fdxH1, was reduced in the xisA mutant, indicating that the nifB1 promoter contributed to their expression. However, with the exception of nifK1 and nifE1, which had no expression, the downstream genes showed low to moderate levels of transcription in the xisA mutant. The hesA1 gene also had a promoter, but the fdxH gene had a processing site just upstream of the gene. The processing of transcripts at sites upstream of nifH1 and fdxH1 correlated with increased stability of these transcripts, resulting in greater amounts than transcripts that were not close to processing sites. PMID:25092030

  2. Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress

    PubMed Central

    Teikari, Jonna; Österholm, Julia; Kopf, Matthias; Battchikova, Natalia; Wahlsten, Matti; Aro, Eva-Mari; Hess, Wolfgang R.

    2015-01-01

    Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone. PMID:26025890

  3. [Sensory Awareness through Outdoor Education].

    ERIC Educational Resources Information Center

    Farquhar, Carin; And Others

    Designed for instruction of emotionally handicapped children and youth, these seven articles present concepts and activities relative to sensory awareness and outdoor education. The first article presents definitions, concepts, detailed methodology, and over 50 activities designed to create awareness of man's five senses. Utilizing the art of…

  4. Sensory Aids for the Blind.

    ERIC Educational Resources Information Center

    National Academy of Sciences - National Research Council, Washington, DC. Committee on Prosthetics Research and Development.

    The problems of providing sensory aids for the blind are presented and a report on the present status of aids discusses direct translation and recognition reading machines as well as mobility aids. Aspects of required research considered are the following: assessment of needs; vision, audition, taction, and multimodal communication; reading aids,…

  5. Making Sense of Sensory Systems

    ERIC Educational Resources Information Center

    Hendrix, Marie

    2010-01-01

    The role of caregivers requires that they continuously assess the needs and performance of children and provide the support necessary for them to achieve their potential. A thorough understanding of child development, including the role and impact of sensory development, is critical for caregivers to properly evaluate and assist these children.…

  6. GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

    PubMed

    Potnis, Akhilesh A; Rajaram, Hema; Apte, Shree K

    2016-03-01

    The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP. This correlated with in vivo data. GroEL exhibited ATPase activity which was enhanced in the presence of GroES and absence of a denatured protein, contrary to that observed for bacterial GroEL. However, a significant role for ATP could not be ascertained during in vitro folding assays. The monomeric Cpn60 exhibited much lower refolding activity than GroEL, unaffected by GroES and ATP. In vitro studies revealed inhibition of the refolding activity of Anabaena GroEL by Cpn60, which could be due to their different oligomeric status. The role of GroES and ATP may have been added during the course of evolution from the ancient cyanobacteria to modern day bacteria enhancing the refolding ability and ensuring wider scope of substrates for GroEL.

  7. Identification of facultatively heterotrophic, N/sub 2/-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation. [Anabaena spp; Nostoc

    SciTech Connect

    Flores, E.; Wolk, C.P.

    1985-06-01

    Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

  8. Identification of the oriC region and its influence on heterocyst development in the filamentous cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Zhou, Yin; Chen, Wen-Li; Wang, Li; Zhang, Cheng-Cai

    2011-07-01

    Anabaena sp. strain PCC 7120 (Anabaena PCC 7120) is a filamentous, nitrogen-fixing cyanobacterium. Upon deprivation of combined nitrogen, about 5-10 % of the cells become heterocysts, i.e. cells devoted to N(2) fixation. Heterocysts are intercalated among vegetative cells and distributed in a semi-regular pattern, and adjacent heterocysts are rarely observed. Previously, we showed that the cell cycle could play a regulatory function during heterocyst development, although the mechanism involved remains unknown. As a further step to understand this phenomenon, we identified the oriC region for chromosomal DNA replication, located between dnaA and dnaN. The oriC region of Anabaena PCC 7120 was able to support the self-replication of a plasmid in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Surprisingly, integration of the oriC region into the chromosome of Anabaena PCC 7120 through homologous recombination led to much slower cell growth in the absence of a combined-nitrogen source and to multiple contiguous proheterocysts after prolonged incubation. Real-time RT-PCR showed that expression of two heterocyst-related genes, hetR and hetN, was altered in these strains: hetR expression remained high 48 h after induction, and hetN increased to high levels after induction for 12 h. These results suggest that the balance between oriC and DnaA could be important for heterocyst development.

  9. Characterization of a chromosomal type II toxin-antitoxin system mazEaFa in the Cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Ning, Degang; Jiang, Yan; Liu, Zhaoying; Xu, Qinggang

    2013-01-01

    Cyanobacteria have evolved to survive stressful environmental changes by regulating growth, however, the underlying mechanism for this is obscure. The ability of chromosomal type II toxin-antitoxin (TA) systems to modulate growth or cell death has been documented in a variety of prokaryotes. A chromosomal mazEaFa locus of Anabaena sp. PCC 7120 has been predicted as a putative mazEF TA system. Here we demonstrate that mazEaFa form a bicistronic operon that is co-transcribed under normal growth conditions. Overproduction of MazFa induced Anabaena growth arrest which could be neutralized by co-expression of MazEa. MazFa also inhibited the growth of Escherichia coli cells, and this effect could be overcome by simultaneous or subsequent expression of MazEa via formation of the MazEa-MazFa complex in vivo, further confirming the nature of the mazEaFa locus as a type II TA system. Interestingly, like most TA systems, deletion of mazEaFa had no effect on the growth of Anabaena during the tested stresses. Our data suggest that mazEaFa, or together with other chromosomal type II TA systems, may promote cells to cope with particular stresses by inducing reversible growth arrest of Anabaena.

  10. Alr5068, a Low-Molecular-Weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Tan, Hui; Wan, Shuang; Liu, Pi-Qiong; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2013-10-01

    The filamentous cyanobacterium Anabaena sp. PCC 7120 forms nitrogen-fixing heterocysts after deprivation of combined nitrogen. Under such conditions, vegetative cells provide heterocysts with photosynthate and receive fixed nitrogen from the latter. Heterocyst envelope contains a glycolipid layer and a polysaccharide layer to restrict the diffusion of oxygen into heterocysts. Low-Molecular-Weight protein tyrosine phosphatases (LMW-PTPs) are involved in the biosynthesis of exopolysaccharides in bacteria. Alr5068, a protein from Anabaena sp. PCC 7120, shows significant sequence similarity with LMW-PTPs. In this study we characterized the enzymatic properties of Alr5068 and showed that it can dephosphorylate several autophosphorylated tyrosine kinases (Alr2856, Alr3059 and All4432) of Anabaena sp. PCC 7120 in vitro. Several conserved residues among LMW-PTPs are shown to be essential for the phosphatase activity of Alr5068. Overexpression of alr5068 results in a strain unable to survive under diazotrophic conditions, with the formation of morphologically mature heterocysts detached from the filaments. Overexpression of an alr5068 allele that lost phosphatase activity led to the formation of heterocyst with an impaired polysaccharide layer. The alr5068 gene was upregulated after nitrogen step-down and its mutation affected the expression of hepA and hepC, two genes necessary for the formation of the heterocyst envelope polysaccharide (HEP) layer. Our results suggest that Alr5068 is associated with the production of HEP in Anabaena sp. PCC 7120.

  11. Characterization and Optimization of Bioflocculant Exopolysaccharide Production by Cyanobacteria Nostoc sp. BTA97 and Anabaena sp. BTA990 in Culture Conditions.

    PubMed

    Tiwari, Onkar Nath; Khangembam, Romi; Shamjetshabam, Minerva; Sharma, Aribam Subhalaxmi; Oinam, Gunapati; Brand, Jerry J

    2015-08-01

    Bioflocculant exopolysaccharide (EPS) production by 40 cyanobacterial strains during their photoautotrophic growth was investigated. Highest levels of EPS were produced by Nostoc sp. BTA97 and Anabaena sp. BTA990. EPS production was maximum during stationary growth phase, when nitrogenase activity was very low. Maximum EPS production occurred at pH 8.0 in the absence of any combined nitrogen source. The cyanobacterial EPS consisted of soluble protein and polysaccharide that included substantial amounts of neutral sugars and uronic acid. The EPS isolated from Anabaena sp. BTA990 and Nostoc sp. BTA97 demonstrated high flocculation capacity. There was a positive correlation between uronic acid content and flocculation activity. The flocculant bound a cationic dye, Alcian Blue, indicating it to be polyanionic. The 16S rRNA gene sequences for Nostoc sp. BTA97 and Anabaena sp. BTA990 were deposited at NCBI GenBank, and accession numbers were obtained as KJ830951 and KJ830948, respectively. The results of these experiments indicate that strains Anabaena sp. BTA990 and Nostoc sp. BTA97 are good candidates for the commercial production of EPS and might be utilized in industrial applications as an alternative to synthetic and abiotic flocculants.

  12. Alr5068, a Low-Molecular-Weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120.

    PubMed

    Tan, Hui; Wan, Shuang; Liu, Pi-Qiong; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2013-10-01

    The filamentous cyanobacterium Anabaena sp. PCC 7120 forms nitrogen-fixing heterocysts after deprivation of combined nitrogen. Under such conditions, vegetative cells provide heterocysts with photosynthate and receive fixed nitrogen from the latter. Heterocyst envelope contains a glycolipid layer and a polysaccharide layer to restrict the diffusion of oxygen into heterocysts. Low-Molecular-Weight protein tyrosine phosphatases (LMW-PTPs) are involved in the biosynthesis of exopolysaccharides in bacteria. Alr5068, a protein from Anabaena sp. PCC 7120, shows significant sequence similarity with LMW-PTPs. In this study we characterized the enzymatic properties of Alr5068 and showed that it can dephosphorylate several autophosphorylated tyrosine kinases (Alr2856, Alr3059 and All4432) of Anabaena sp. PCC 7120 in vitro. Several conserved residues among LMW-PTPs are shown to be essential for the phosphatase activity of Alr5068. Overexpression of alr5068 results in a strain unable to survive under diazotrophic conditions, with the formation of morphologically mature heterocysts detached from the filaments. Overexpression of an alr5068 allele that lost phosphatase activity led to the formation of heterocyst with an impaired polysaccharide layer. The alr5068 gene was upregulated after nitrogen step-down and its mutation affected the expression of hepA and hepC, two genes necessary for the formation of the heterocyst envelope polysaccharide (HEP) layer. Our results suggest that Alr5068 is associated with the production of HEP in Anabaena sp. PCC 7120. PMID:23827083

  13. GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

    PubMed

    Potnis, Akhilesh A; Rajaram, Hema; Apte, Shree K

    2016-03-01

    The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP. This correlated with in vivo data. GroEL exhibited ATPase activity which was enhanced in the presence of GroES and absence of a denatured protein, contrary to that observed for bacterial GroEL. However, a significant role for ATP could not be ascertained during in vitro folding assays. The monomeric Cpn60 exhibited much lower refolding activity than GroEL, unaffected by GroES and ATP. In vitro studies revealed inhibition of the refolding activity of Anabaena GroEL by Cpn60, which could be due to their different oligomeric status. The role of GroES and ATP may have been added during the course of evolution from the ancient cyanobacteria to modern day bacteria enhancing the refolding ability and ensuring wider scope of substrates for GroEL. PMID:26449235

  14. A review on intelligent sensory modelling

    NASA Astrophysics Data System (ADS)

    Tham, H. J.; Tang, S. Y.; Teo, K. T. K.; Loh, S. P.

    2016-06-01

    Sensory evaluation plays an important role in the quality control of food productions. Sensory data obtained through sensory evaluation are generally subjective, vague and uncertain. Classically, factorial multivariate methods such as Principle Component Analysis (PCA), Partial Least Square (PLS) method, Multiple Regression (MLR) method and Response Surface Method (RSM) are the common tools used to analyse sensory data. These methods can model some of the sensory data but may not be robust enough to analyse nonlinear data. In these situations, intelligent modelling techniques such as Fuzzy Logic and Artificial neural network (ANNs) emerged to solve the vagueness and uncertainty of sensory data. This paper outlines literature of intelligent sensory modelling on sensory data analysis.

  15. The Sakaguchi reaction product quenches phycobilisome fluorescence, allowing determination of the arginine concentration in cells of Anabaena strain PCC 7120.

    PubMed

    Ke, Shan; Haselkorn, Robert

    2013-01-01

    The filamentous cyanobacterium Anabaena fixes nitrogen in specialized cells called heterocysts. The immediate product of fixation, ammonia, is known to be assimilated by addition to glutamate to make glutamine. How fixed nitrogen is transported along the filament to the 10 to 20 vegetative cells that separate heterocysts is unknown. N-fixing heterocysts accumulate an insoluble polymer containing aspartate and arginine at the cell poles. Lockau's group has proposed that the polymer is degraded at the poles to provide a mobile carrier, arginine, to the vegetative cells (R. Richter, M. Hejazi, R. Kraft, K. Ziegler, and W. Lockau, Eur. J. Biochem. 263:163-169, 1999). We wished to use the Sakaguchi reaction for arginine to determine the relative cellular concentration of arginine along the filament. At present, the methods for measuring absorption of the Sakaguchi reaction product at 520 nm are insufficiently sensitive for that purpose. However, that product quenches the fluorescence of phycobiliproteins, which we have adapted to a determination of arginine. Our results are consistent with the proposal that arginine is a principal nitrogen carrier from heterocysts to vegetative cells in Anabaena.

  16. Peroxidation radical formation and regiospecificity of recombinated Anabaena sp. lipoxygenase and its effect on modifying wheat proteins.

    PubMed

    Wang, Xiaoming; Lu, Fengxia; Zhang, Chong; Lu, Yingjian; Bie, Xiaomei; Ren, Di; Lu, Zhaoxin

    2014-02-19

    Peroxidation radical formation and the regiospecificity of recombinated lipoxygenase from Anabaena sp. PCC7120 (ana-rLOX) were characterized by using ESR and HPLC-MS. It was found that ana-rLOX oxygenated at the C-13 position of the substrate linoleic acid (LA); at C-13 and C-16 of α-linolenic acid (ALA); at C-9, C-12, and C-15 of arachidonic acid (AA); at C-12, C-15, and C-18 of eicosapentaenoic acid (EPA); and at C-14 and C-16 of docosahexaenoic acid (DHA), respectively. A total of 7, 14, 30, 28, and 18 radical adducts for LA, ALA, AA, EPA, and DHA were respectively identified by HPLC-MS. The functional characteristics of wheat protein, such as foaming capacity (FC), foam stability (FS), emulsifying activity index (EAI), emulsifying stability index (ESI), increased with enzymatic reactions. However, the average particle size of wheat proteins decreased with addition of ana-rLOX/LA. The ana-rLOX was also positivele effective in improving dough properties. These results provided clear evidence that ana-rLOX from Anabaena sp. could effectively improve the quality of wheat flour, which suggested that the enzyme could be applied as flour improver.

  17. On-water remote monitoring robotic system for estimating the patch coverage of Anabaena sp. filaments in shallow water.

    PubMed

    Romero-Vivas, E; Von Borstel, F D; Pérez-Estrada, C J; Torres-Ariño, D; Villa-Medina, J F; Gutiérrez, J

    2015-06-01

    An on-water remote monitoring robotic system was developed for indirectly estimating the relative density of marine cyanobacteria blooms at the subtidal sandy-rocky beach in Balandra Cove, Baja California Sur, Mexico. The system is based on an unmanned surface vehicle to gather underwater videos of the seafloor for avoiding physical damage on Anabaena sp. cyanobacteria colonies, which grow in tufts of filaments weakly attached to rocks, seagrass, and macroalgae. An on-axis image stabilization mechanism was developed to support a camcorder and minimize wave perturbation while recording underwater digital images of the seafloor. Color image processing algorithms were applied to estimate the patch coverage area and density, since Anabaena sp. filaments exhibit a characteristic green tone. Results of field tests showed the feasibility of the robotic system to estimate the relative density, distribution, and coverage area of cyanobacteria blooms, preventing the possible impact of direct observation. The robotic system could also be used in surveys of other benthos in the sublittoral zone.

  18. Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family.

    PubMed

    Langdon, G M; Jiménez, M A; Genzor, C G; Maldonado, S; Sancho, J; Rico, M

    2001-06-01

    An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.

  19. Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

    PubMed

    Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

    2015-05-01

    Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.

  20. Time-dependent growth of crystalline Au0-nanoparticles in cyanobacteria as self-reproducing bioreactors: 2. Anabaena cylindrica

    PubMed Central

    Rösken, Liz M; Cappel, Felix; Körsten, Susanne; Fischer, Christian B; Schönleber, Andreas; van Smaalen, Sander; Geimer, Stefan; Beresko, Christian; Ankerhold, Georg

    2016-01-01

    Summary Microbial biosynthesis of metal nanoparticles as needed in catalysis has shown its theoretical ability as an extremely environmentally friendly production method in the last few years, even though the separation of the nanoparticles is challenging. Biosynthesis, summing up biosorption and bioreduction of diluted metal ions to zero valent metals, is especially ecofriendly, when the bioreactor itself is harmless and needs no further harmful reagents. The cyanobacterium Anabaena cylindrica (SAG 1403.2) is able to form crystalline Au0-nanoparticles from Au3+ ions and does not release toxic anatoxin-a. X-ray powder diffraction (XRD), transmission electron microscopy (TEM) and laser-induced breakdown spectroscopy (LIBS) are applied to monitor the time-dependent development of gold nanoparticles for up to 40 hours. Some vegetative cells (VC) are filled with nanoparticles within minutes, while the extracellular polymeric substances (EPS) of vegetative cells and the heterocyst polysaccharide layer (HEP) are the regions, where the first nanoparticles are detected on most other cells. The uptake of gold starts immediately after incubation and within four hours the average size remains constant around 10 nm. Analyzing the TEM images with an image processing program reveals a wide distribution for the diameter of the nanoparticles at all times and in all regions of the cyanobacteria. Finally, the nanoparticle concentration in vegetative cells of Anabaena cylindrica is about 50% higher than in heterocysts (HC). These nanoparticles are found to be located along the thylakoid membranes. PMID:27335727

  1. Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120

    SciTech Connect

    Yuping Cai )

    1991-09-01

    IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120, is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generated an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain. A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS 892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.

  2. Feasibility of /sup 55/Fe autoradiography as performed on N/sub 2/-fixing Anabaena spp. populations and associated bacteria

    SciTech Connect

    Paerl, H.W.

    1982-01-01

    /sup 55/Fe emits low-energy X rays and Auger electrons by electron capture decay. Auger electrons are useful for autoradiographic examination of /sup 55/Fe incorporation among microbial communities. Attainable resolution, in terms of silver grain deposition, is excellent and comparable to /sup 3/H. Two known Fe-demanding processes, photosynthetic CO/sub 2/ fixation and N/sub 2/ fixation, were examined by autoradiography of Anabaena populations. During photosynthetically active (illuminated) N/sub 2/-fixing periods, biological incorporation of /sup 55/FeCl/sub 3/ by vegetative cells and heterocysts was evident. When N/sub 2/ fixation was suppressed by NH/sub 4//sup +/ additions, heterocysts revaled no incorporation of /sup 55/Fe. Conversely, when N/sub 2/-fixing Anabaena filaments were placed in darkness, /sup 55/Fe incorporation decreased in vegetative cells, whereas heterocysts showed sustained rates of /sup 55/Fe incorporation. Bacteria actively incorporated /sup 55/Fe under both light and dark conditions. The chelated (by Na/sub 2/-ethylenediaminetetraacetate) form of /sup 55/FeCl/sub 3/ was more readily incorporated than the nonchelated form. Furthermore, abiotic adsorption of /sup 55/Fe to filters and nonliving particles proved lower when chelated /sup 55/Fe was used in experiments. /sup 55/Fe autoradiography is useful for observing the fate and cellular distribution of various forms of Fe among aquatic microbial communities.

  3. Purification, crystallization and preliminary crystallographic analysis of KatB, a manganese catalase from Anabaena PCC 7120.

    PubMed

    Bihani, Subhash Chandra; Chakravarty, Dhiman; Ballal, Anand

    2013-11-01

    Catalases are enzymes that play an important role in the detoxification of hydrogen peroxide (H2O2) in aerobic organisms. Among catalases, haem-containing catalases are ubiquitously distributed and their enzymatic mechanism is very well understood. On the other hand, manganese catalases that contain a bimanganese core in the active site have been less well characterized and their mode of action is not fully understood. The genome of Anabaena PCC 7120 does not show the presence of a haem catalase-like gene; instead, two ORFs encoding manganese catalases (Mn-catalases) are present. Here, the crystallization and preliminary X-ray crystallographic analysis of KatB, one of the two Mn-catalases from Anabaena, are reported. KatB was crystallized using the hanging-drop vapour-diffusion method with PEG 400 as a precipitant and calcium acetate as an additive. Diffraction data were collected in-house on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 2.2 Å resolution at 100 K. The tetragonal crystal belonged to space group P4(1)2(1)2 (or enantiomer), with unit-cell parameters a = b = 101.87, c = 138.86 Å. Preliminary X-ray diffraction analysis using the Matthews coefficient and self-rotation function suggests the presence of a trimer in the asymmetric unit.

  4. Polysaccharides from the envelopes of heterocysts and spores of the blue-green algae Anabaena variabilis and Cylindrospermum licheniforme

    SciTech Connect

    Cardemil, L.; Wolk, C.P.

    1981-09-01

    The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kutz., and of heterocysts and spores of Anabaena variabilis Kutz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.

  5. The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

    PubMed

    Picossi, Silvia; Flores, Enrique; Herrero, Antonia

    2015-09-01

    Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph.

  6. Time-dependent growth of crystalline Au(0)-nanoparticles in cyanobacteria as self-reproducing bioreactors: 2. Anabaena cylindrica.

    PubMed

    Rösken, Liz M; Cappel, Felix; Körsten, Susanne; Fischer, Christian B; Schönleber, Andreas; van Smaalen, Sander; Geimer, Stefan; Beresko, Christian; Ankerhold, Georg; Wehner, Stefan

    2016-01-01

    Microbial biosynthesis of metal nanoparticles as needed in catalysis has shown its theoretical ability as an extremely environmentally friendly production method in the last few years, even though the separation of the nanoparticles is challenging. Biosynthesis, summing up biosorption and bioreduction of diluted metal ions to zero valent metals, is especially ecofriendly, when the bioreactor itself is harmless and needs no further harmful reagents. The cyanobacterium Anabaena cylindrica (SAG 1403.2) is able to form crystalline Au(0)-nanoparticles from Au(3+) ions and does not release toxic anatoxin-a. X-ray powder diffraction (XRD), transmission electron microscopy (TEM) and laser-induced breakdown spectroscopy (LIBS) are applied to monitor the time-dependent development of gold nanoparticles for up to 40 hours. Some vegetative cells (VC) are filled with nanoparticles within minutes, while the extracellular polymeric substances (EPS) of vegetative cells and the heterocyst polysaccharide layer (HEP) are the regions, where the first nanoparticles are detected on most other cells. The uptake of gold starts immediately after incubation and within four hours the average size remains constant around 10 nm. Analyzing the TEM images with an image processing program reveals a wide distribution for the diameter of the nanoparticles at all times and in all regions of the cyanobacteria. Finally, the nanoparticle concentration in vegetative cells of Anabaena cylindrica is about 50% higher than in heterocysts (HC). These nanoparticles are found to be located along the thylakoid membranes. PMID:27335727

  7. Inactivation of a heterocyst-specific invertase indicates a principal role of sucrose catabolism in heterocysts of Anabaena sp.

    PubMed

    López-Igual, Rocío; Flores, Enrique; Herrero, Antonia

    2010-10-01

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that carries out N(2) fixation in specialized cells called heterocysts, which exchange nutrients and regulators with the filament's vegetative cells that perform the photosynthetic fixation of CO(2). The Anabaena genome carries two genes coding for alkaline/neutral invertases, invA and invB. As shown by Northern analysis, both genes were expressed monocistronically and induced under nitrogen deprivation, although induction was stronger for invB than for invA. Whereas expression of an InvA-N-GFP fusion (green fluorescent protein [GFP] fused to the N terminus of the InvA protein [InvA-N]) was homogeneous along the cyanobacterial filament, consistent with the lack of dependence on HetR, expression of an InvB-N-GFP fusion upon combined nitrogen deprivation took place mainly in differentiating and mature heterocysts. In an hetR genetic background, the InvB-N-GFP fusion was strongly expressed all along the filament. An insertional mutant of invA could grow diazotrophically but was impaired in nifHDK induction and exhibited an increased frequency of heterocysts, suggesting a regulatory role of the invertase-mediated carbon flux in vegetative cells. In contrast, an invB mutant was strongly impaired in diazotrophic growth, showing a crucial role of sucrose catabolism mediated by the InvB invertase in the heterocysts.

  8. Profenofos induced modulation in physiological indices, genomic template stability and protein banding patterns of Anabaena sp. PCC 7120.

    PubMed

    Tiwari, Balkrishna; Chakraborty, Sindhunath; Singh, Savita; Mishra, Arun K

    2016-11-01

    To understand the mechanism underlying organophosphate pesticide toxicity, cyanobacterium Anabaena PCC 7120 was subjected to varied concentrations (0, 5, 10, 20 and 30 mg L(-1)) of profenofos and the effects were investigated in terms of changes in cellular physiology, genomic template stability and protein expression pattern. The supplementation of profenofos reduced the growth, total pigment content and photosynthetic efficiency of the test organism in a dose dependent manner with maximum toxic effect at 30 mg L(-1). The high fluorescence intensity of 2', 7' -dichlorofluorescin diacetate and increased production of malondialdehyde confirmed the prevalence of acute oxidative stress condition inside the cells of the cyanobacterium. Rapid amplified polymorphic DNA (RAPD) fingerprinting and SDS-PAGE analyses showed a significant alteration in the banding patterns of DNA and proteins respectively. A marked increase in superoxide dismutase, catalase, peroxidase activity and a concomitant reduction in glutathione content indicated their possible role in supporting the growth of Anabaena 7120 up to 20 mg L(-1). These findings suggest that the uncontrolled use of profenofos in the agricultural fields may not only lead to the destruction of the cyanobacterial population, but it would also disturb the nutrient dynamics and energy flow. PMID:27428931

  9. The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

    PubMed

    Picossi, Silvia; Flores, Enrique; Herrero, Antonia

    2015-09-01

    Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph. PMID:25684321

  10. Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

    PubMed

    Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

    2015-05-01

    Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity. PMID:25644579

  11. Response to Vestibular Sensory Events in Autism

    ERIC Educational Resources Information Center

    Kern, Janet K.; Garver, Carolyn R.; Grannemann, Bruce D.; Trivedi, Madhukar H.; Carmody, Thomas; Andrews, Alonzo A.; Mehta, Jyutika A.

    2007-01-01

    The purpose of this study was to examine the response to vestibular sensory events in persons with autism. The data for this study was collected as part of a cross-sectional study that examined sensory processing (using the Sensory Profile) in 103 persons with autism, 3-43 years of age, compared to age- and gender-matched community controls. The…

  12. Multi-Sensory Intervention Observational Research

    ERIC Educational Resources Information Center

    Thompson, Carla J.

    2011-01-01

    An observational research study based on sensory integration theory was conducted to examine the observed impact of student selected multi-sensory experiences within a multi-sensory intervention center relative to the sustained focus levels of students with special needs. A stratified random sample of 50 students with severe developmental…

  13. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

    SciTech Connect

    Cai, Yuping; Wolk, C.P. )

    1990-06-01

    Use of the sacB gene provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, they mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

  14. Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

    PubMed

    Burnat, Mireia; Flores, Enrique

    2014-10-01

    Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

  15. Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena

    PubMed Central

    Burnat, Mireia; Flores, Enrique

    2014-01-01

    Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [14C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The Δalr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [14C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the Δalr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium. PMID:25209059

  16. Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

    PubMed

    Burnat, Mireia; Flores, Enrique

    2014-10-01

    Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium. PMID:25209059

  17. Quantitative modeling of the molecular steps underlying shut-off of rhodopsin activity in rod phototransduction

    PubMed Central

    Kraft, Timothy W.

    2016-01-01

    Purpose To examine the predictions of alternative models for the stochastic shut-off of activated rhodopsin (R*) and their implications for the interpretation of experimentally recorded single-photon responses (SPRs) in mammalian rods. Theory We analyze the transitions that an activated R* molecule undergoes as a result of successive phosphorylation steps and arrestin binding. We consider certain simplifying cases for the relative magnitudes of the reaction rate constants and derive the probability distributions for the time to arrestin binding. In addition to the conventional model in which R* catalytic activity declines in a graded manner with successive phosphorylations, we analyze two cases in which the activity is assumed to occur not via multiple small steps upon each phosphorylation but via a single large step. We refer to these latter two cases as the binary R* shut-off and three-state R* shut-off models. Methods We simulate R*’s stochastic reactions numerically for the three models. In the simplifying cases for the ratio of rate constants in the binary and three-state models, we show that the probability distribution of the time to arrestin binding is accurately predicted. To simulate SPRs, we then integrate the differential equations for the downstream reactions using a standard model of the rod outer segment that includes longitudinal diffusion of cGMP and Ca2+. Results Our simulations of SPRs in the conventional model of graded shut-off of R* conform closely to the simulations in a recent study. However, the gain factor required to account for the observed mean SPR amplitude is higher than can be accounted for from biochemical experiments. In addition, a substantial minority of the simulated SPRs exhibit features that have not been reported in published experiments. Our simulations of SPRs using the model of binary R* shut-off appear to conform closely to experimental results for wild type (WT) mouse rods, and the required gain factor conforms to

  18. [Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols].

    PubMed

    Nabiev, I R; Efremov, R G; Chumanov, G D

    1986-01-01

    Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.