Search

Multi-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested the efficacy of several commonly used detergents and membrane-mimicking nanodiscs with respect to solubilization, spectral properties, thermal stability and oligomeric profile of two membrane proteins from the eubacterial rhodopsin family, green proteorhodopsin (PR) and Gloeobacter violaceus rhodopsin (GR). Good solubilization was observed for the detergents TritonX-100 and dodecylphosphocholine (DPC), but DPC in particular strongly affected the thermal stability of PR and especially GR. The least deleterious effects were obtained with n-dodecyl-β-D-maltopyranoside (DDM) and octyl glucose neopentyl glycol (OGNG), which adequately stabilized the native oligomeric and monomeric state of PR and GR, respectively. The transition from the oligomeric to the monomeric state is accompanied by a small red-shift. Both GR and PR were rather unstable in SMA-nanodiscs, but the highest thermal stability was realized by the MSP-nanodisc environment. The size of the MSP-nanodisc was too small to fit the PR hexamer, but large enough to contain the PR monomer and GR trimer. This permitted the comparison of the photocycle of trimeric GR in a membrane-mimicking (MSP-nanodisc) and a detergent (DDM) environment. The ultrarapid early phase of the photocycle (femto- to picosecond lifetimes) showed very similar kinetics in either environment, but the slower part, initiated with proton transfer and generation of the M intermediate, proceeded faster in the nanodisc environment. The implications of our results for the biophysical characterization of PR and GR are discussed.

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

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications., The proton pump archaerhodopsin‐3 (AR3) is widely used in optogenetics as a silencer of neural activity or as a fluorescent indicator of transmembrane potential. In this research, we utilize analogs of its native chromophore all‐trans retinal to generate variants of AR3 and a single mutant. One analog from this study generated redshifted AR3 pigments which could be activated by near‐infrared light. Furthermore, these pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared. We anticipate that these analog AR3 pigments have potential for exploiting near‐infrared light in several optogenetic and bioenergy‐based applications.

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

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon-based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO2 , water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. 'Direct conversion' of CO2 and H2O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale-up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism, and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past two years and these will be discussed: (1) increasing the carbon partitioning to > 50%; (2) aligning product formation with the growth of the cells; and (3) expanding the PAR region for oxygenic photosynthesis.

To fill the “green absorption gap”, a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Contains fulltext : 172525.pdf (Publisher’s version ) (Closed access) Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10(5) molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes ( sll1541 , slr1648 , slr0091 , slr1192 , and slr0574 ) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo- carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541 , is an indispensable enzyme for retinal synthesis in Synechocystis , presumably via asymmetric cleavage of β- apo -carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648 , competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192 , causes an increase (relative to the level in wild-type Synechocystis ) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13 C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo -proteorhodopsin with exogenous retinal analogues, as demonstrated here for all- trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal. IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of β-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.

Contains fulltext : 174417.pdf (Publisher’s version ) (Closed access) Proteorhodopsins are light-driven proton pumps that occur widespread in Nature, where they function predominantly in environments with high incident irradiance. Their maximal absorbance is usually in the blue range, but can be extended into the (far)red range of the electromagnetic spectrum. Because they can be expressed heterologously, they may be exploited in studies aimed at increasing the efficiency of photosynthesis. Here we report further studies toward this goal, by comparing the expression of two different bacterial rhodopsins (Proteorhodopsin and Gloeobacter rhodopsin) in the model cyanobacterium Synechocystis sp. PCC6803. In particular, we investigated the pigments bound by the respective apo-opsins, and the oligomeric state of the corresponding holo-rhodopsins, both in Escherichia coli and in the cyanobacterial membranes. We conclude that the two proton-pumping rhodopsins are predominantly present in an oligomeric state (hexamers for Proteorhodopsin and trimers for Gloeobacter rhodopsin). Furthermore, Gloeobacter rhodopsin is able to bind an antenna carotenoid (in addition to retinal) and has the highest pumping rate at given light intensity. However, its lower expression level will decrease its physiological effectiveness. It remains to be established which of these two bacterial rhodopsins is best in stimulating the growth rate of its cyanobacterial host.

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

The aim of this paper is to identify and investigate an endophytic fungus (strain 28) that was isolated from Houttuynia cordata Thunb, a famous and widely-used Traditional Chinese Medicine. Based on morphological methods and a phylogenetic analysis of ITS sequences, this strain was identified as Chaetomium globosum. An antifungal activity bioassay demonstrated that the crude ethyl acetate (EtOAc) extracts of strain 28 had a wide antifungal spectrum and strong antimicrobial activity, particularly against Exserohilum turcicum (Pass.) Leonard et Suggs, Botrytis cinerea persoon and Botrytis cinerea Pers. ex Fr. Furthermore, the fermentation conditions, extraction method and the heat stability of antifungal substances from strain 28 were also studied. The results showed that optimal antifungal activity can be obtained with the following parameters: using potato dextrose broth (PDB) as the base culture medium, fermentation for 4–8 d (initial pH: 7.5), followed by extraction with EtOAc. The extract was stable at temperatures up to 80 °C. This is the first report on the isolation of endophytic C. globosum from H. cordata to identify potential alternative biocontrol agents that could provide new opportunities for practical applications involving H. cordata.

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

The major biological active ingredients of Bulbus Fritillariae cirrhosae (BFC) are steroidal alkaloids, such as peimisine, imperialine-3β-D-glucoside, and peimine. The bulbus of Fritillaria unibracteata var. wabuensis (FUW) was officially recorded in the National Pharmacopoeia of China (2010 edition) as one of the sources of BFC because of its positive therapeutic effects and few side effects. The endophytic fungus strain 6WBY3 was isolated from the fresh bulbus of FUW that had been cultivated for six years. Based on morphological methods and the phylogenetic analysis of internal transcribed spacer (ITS) sequences, this strain was identified as Fusarium redolens. Using color reaction analysis, high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD), and mass spectrometry (MS), it was demonstrated that F. redolens 6WBY3 could produce peimisine and imperialine-3β-D-glucoside, similar to its host plant. The yields of peimisine and imperialine-3β-D-glucoside were 16.0 μg·l(-1) and 18.8 μg·l(-1), respectively, in one week of culture. These results indicate that F. redolens 6WBY3 is a promising candidate for the large scale production of peimisine and imperialine-3β-D-glucoside. In addition, the results from the strain 6WBY3 lay the foundation for further study into the mechanism of Fritillaria alkaloids biosynthesis in fungi.

Steroidal alkaloids, as the major biologically active components in Bulbus Fritillariae, possess a variety of toxicological and pharmacological effects on humans. The objective of this work was to determine whether endophytic fungi isolated from fresh bulbs of Fritillaria unibracteata var. wabensis can produce one or more alkaloids like its host plant. Four classical reagents including Wagner's, iodine-potassium iodide, Mayer's and improved Dragendorff's were used for primary screening. Then thin-layer chromatography (TLC) and high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) were employed to identify the fermentation products of the selected strains. The results showed that extract from one stain (WBS007) has positive reactions in process of primary screening. A further TLC scan and HPLC-ELSD showed that strain WBS007 had two components with the same TLC relative front (Rf) value and HPLC retention time (RT) as authentic peimisine and peiminine. In addition, strain WBS007 was identified as Fusarium sp. based on phylogenetic analysis of ITS sequences. Thus, strain WBS007 produced the bioactive ingredient peimisine and peiminine, as does its host plant, and could be used for the production of peimisine and peiminine by fermentation.

This study presents the first experimental results of confining miscible magnetic fluids in a rotating Hele-Shaw cell. Variations in the prominence of labyrinthine instabilities are observed under a range of experimental conditions, with different magnetic field strengths, gap depths, and rotation speeds. These instabilities are characterized by two modified Peclect numbers, namely, Pem (the ratio of the characteristic magnetic advection rate and the diffusion rate) and Pec (the ratio of characteristic rotation advection and the diffusion rate). The magnetic effect is characterized by dipolar repulsion, which triggers a distinctive fingering pattern differing from the progressive diffusion pattern that occurs without magnetic fields or rotation. Under the same rotation speed, the magnetoviscous effect will hinder the growth rate of the magnetic drops at the later stage. However, both the rotation effect and the gap depth greatly enhance the growth rate of the magnetic drops, as these conditions help to in...

OBJECTIVE To study testing methods of seed quality, and provide a basis for establishing seed testing specification of Cyathula officinalis. METHOD Referring to the Specifications for Agricultural Seed Testing, the optimal testing methods of seed quality of C. officinalis were screened. RESULT AND CONCLUSION The testing method for C. officinalis seed quality has been initially established. At least 8 g seeds should be sampled and passed through 20-mesh sieve for purity analysis. The phenotypic observation and size measurement were used for authenticity testing. The seeds were inoculated directly on PDA medium, cultured 5 days on 28 degrees C for seed health testing. The weight of 1 000 seeds was determined by using the 500-seed method. The water content of the seeds was determined under the higher temperature (133 +/- 2) degrees C for 3 hours. The seeds were dipped into 0.1% TTC solution 3 hours for determining viability. The seeds were cultured on pleated paper at 25 degrees C for 2-9 days for germination testing.

This survey study explored the effects of different types of interpersonal conflict on counterproductive work behavior (CWB). We distinguished interpersonal conflict into four types (task outcome conflict, task process conflict, non-task organizational conflict, relationship conflict). We also testified the moderating effects of negative affectivity(NA). Using data of 447 participants, hierarchical regression analyses revealed that only the non-task organizational conflict had significant predictive power on all kinds of CWBs. NA were found to moderate the relationship between task outcome conflict and production deviance, non-task organizational conflict and production deviance, and also between general interpersonal conflict and production deviance. The implications of these findings are discussed.