Diversity and functional analysis of proteorhodopsin in marine Flavobacteria

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c. 124 H(+) PR(-1) min(-1) ) in recently isolated marine Flavobacteria. Among 75 isolates, 38 possessed the PR gene. Illumination of cell suspensions from all eight tested strains in five genera triggered marked pH drops. The action spectrum of proton pump activity closely matched the spectral distribution of the sea surface green light field. Addition of hydroxylamine to a solubilized membrane fraction shifted the spectrum to a form characteristic of PR photobleached into retinal oxime, indicating that PRs in flavobacterial cell membranes transform the photon dose in incident radiation into energy in the form of membrane potential. Our results revealed that PR-mediated proton transport can create the sufficient membrane potential for the ATP synthesis in native flavobacterial cells.     

Carbon nanodots with a controlled N structure by a solvothermal method for generation of reactive oxygen species under visible light

Carbon nanodots can function as photosensitizers that have the ability to generate reactive oxygen species such as singlet oxygen, hydroxy (OH) radicals, and superoxide ions. However, most of these can only be generated upon ultraviolet light excitation. Additionally, the mechanism of reactive oxygen species generation by carbon nanodots remains unclear. The development of carbon nanodots that can photosensitize under visible light irradiation is desirable for applications such as photodynamic therapy and pollutant decomposition under visible light. Here, we report novel carbon nanodot-based photosensitizers that generate reactive oxygen species under visible light; they were synthesized using a solvothermal method with two solvents (formamide and water) and amidol as the carbon source. Carbon nanodots from the solvothermal synthesis in formamide showed blue fluorescence, while those obtained in water showed green fluorescence. The photo-excited blue-fluorescent carbon nanodots produced OH radicals, superoxide ions, and singlet oxygen, and therefore could function as both type I and type II photosensitizers. In addition, photo-excited green-fluorescent carbon nanodots generated only singlet oxygen, therefore functioning as type II photosensitizers. It is proposed that the two photosensitizers have different origins of reactive oxygen species generation: the enrichment of graphitic N for blue-fluorescent carbon nanodots and molecular fluorophores for green-fluorescent carbon nanodots.     

Multiple functions of photosystem II

The most important function of photosystem II (PSII) is its action as a water-plastoquinone oxido-reductase. At the expense of light energy, water is split, and oxygen and plastoquinol are formed. In addition to this most important activity, PSII has additional functions, especially in the regulation of (light) energy distribution. The downregulation of PSII during photoinhibition is a protection measure. PSII is an anthropogenic target for many herbicides. There is a unique action of bicarbonate on PSII. Decrease in the activity of PSII is the first effect in a plant under stress; this decreased activity can be most easily measured with fluorescence. PSII is a sensor for stress, and induces regulatory processes with different time scales: photochemical quenching, formation of a proton gradient, state transitions, downregulation by photoinhibition and gene expression.     

Photoactivated perylenequinone toxins in fungal pathogenesis of plants

Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.     

Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O₂ consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O₂ consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.     

Neustonic Marine Craspedomonadales (Choanofiagellates) from Washington and California

SYNOPSIS. Neustonic choanoflagellates can be found in marine tide pools in the San Juan Islands, Washington, and on the Monterey Peninsula, California. Several marine photo-synthetic Chrysophyceae (in the Pedinellaceae), which also occur in these regions, have a basic structure so similar to choanoflagellates that this family is placed in the Craspedomonadales. In pointing out this relationship, the derivation of the Craspedomonadales from pigmented Chrysophyceae is strongly indicated.
In addition to the naked choanoflagellates, which are placed in the Codonosigaceae, these organisms produce loricae of two different types: 1) loricae possibly of cellulose and without visible structure in the light microscope (Salpingoecaceae), 2) loricae composed of silica strands, sometimes forming a mesh with large open spaces (Acanthoecaceae). Members of the latter family seem to be confined to a marine environment and are a prominent part of this investigation. Examination of several species with the electron microscope has revealed interesting details of lorica morphology that are not visible with the light microscope.
Several new combinations of taxa are proposed in addition to new taxa, including 4 new species of Salpingoeca , 3 new species of Diploeca and 4 new species of Pleurasiga. Three new genera are described, Ellisiella gen. nov., Acanthoecopsis gen. nov., and Sportelloeca gen. nov.     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.     

Uncoupling of oxidative phosphorylation. 2. Alternative mechanisms: intrinsic uncoupling or decoupling?

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), oleic acid, and chloroform is further investigated by measuring in the presence of a certain concentration of each type of uncoupler (i) the mitochondrial P/O and respiratory control ratios upon progressive inhibition of the redox pumps and (ii) delta mu H and the rate of either electron transfer or adenosine 5'-triphosphate (ATP) hydrolysis in static head upon progressive inhibition of either the redox or the adenosine triphosphatase (ATPase) proton pumps. Chloroform exhibits in all the experiments a behavior very different from that of FCCP and oleic acid. For example, upon addition of antimycin to chloroform-supplemented mitochondria, the respiratory control ratio remains unchanged and the P/O ratio slightly increases (in a certain range of inhibition) instead of decreasing as expected for an increased membrane conductance (and as indeed measured in the presence of either FCCP or oleic acid). From the kinetic model of chemiosmotic free energy coupling described by Pietrobon and Caplan [Pietrobon, D., & Caplan, S.R. (1986) Biochemistry 25, 7690-7696] all the results can be simulated by making the assumptions that (i) chloroform acts specifically at the level of the proton pumps and intrinsically uncouples electron transfer and ATP hydrolysis/synthesis from proton translocation and (ii) FCCP and oleic acid have a mixed behavior and act both as protonophores and as intrinsic uncouplers of the redox pumps (but not of the ATPases). The consistency of the results with the alternative hypothesis that the three agents interfere either with localized energy coupling sites or with a direct interaction between proton pumps is discussed.     

Comparative genomics reveals new functional insights in uncultured MAST species

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.Subject terms: Genomics, Microbiology     

Role of fungi in marine ecosystems

Marine fungi are an ecological rather than a taxonomic group and comprise an estimated 1500 species, excluding those that form lichens. They occur in most marine habitats and generally have a pantropical or pantemperate distribution. Marine fungi are major decomposers of woody and herbaceous substrates in marine ecosystems. Their importance lies in their ability to aggressively degrade lignocellulose. They may be important in the degradation of dead animals and animal parts. Marine fungi are important pathogens of plants and animals and also form symbiotic relationships with other organisms. The effect of disturbances on marine fungi is poorly investigated. Keystone marine species may exist, especially in mutualistic symbioses. However, as many saprophytes appear to carry out the same function simultaneously, they may be functionally redundant. The need for a concerted effort to investigate the biodiversity and role of marine fungi globally and on as many substrata as possible is presented.     

Effects of visible light and other environmental factors on the production of oxygen radicals by hamster embryos

Previous studies have demonstrated that developing hamster embryos are very sensitive to visible light. In order to elucidate why visible light exerts a toxic effect on hamster embryos, we examined the effect of visible light on the production of hydrogen peroxide (H(2)O(2)) within individual embryos, using a fluorimetric method. In addition, we examined the H(2)O(2) generating capacity of other factors which are known to be related to the in vitro developmental capacity of hamster embryos. One-cell hamster embryos were cultured with 2',7'-dichlorodihydrofluorescin diacetate, and the fluorescence emissions of the H(2)O(2)-dependent oxidative product in the embryos were measured using an Olympus microscopic photometry system. When embryos were exposed to visible light (14,000 lux) for a specified period (0, 0.5, 1, 2 or 3 min) prior to measurement, the fluorescence emissions from embryos increased with the time of exposure to visible light. An exposure of even 0.5 min resulted in a significant increase in hydrogen peroxide. This increase was more rapid in embryos cultured under 20% O(2) than in those cultured under 5% O(2), and the response was quicker than that observed in mouse embryos. The fluorescence emissions from embryos cultured under 5% O(2) were significantly (P<0.001) lower than those from embryos cultured under 20% O(2) in TLP medium. However, the effects of different oxygen tensions on fluorescence emissions were medium-dependent, and were not significant in embryos cultured in HECM-1 medium. The addition of L-cysteine to or elimination of phenol red from the media decreased the fluorescence emissions from embryos (P<0.001), but glucose and phosphate did not affect them. These results suggest that the toxic effect of visible light on the in vitro development of hamster embryos might be due to increased generation of reactive oxygen species, induced by the visible light. This could be one of the explanations for the strict conditions required for overcoming the in vitro developmental block. It is also suggested that the promotive effects of low oxygen culture and L-cysteine on embryo development seem to be derived from their ability to reduce reactive oxygen species.     

Factors affecting the distribution of lignicolous marine fungi in Hong Kong

An investigation into the effect of environmental factors on the general distribution and occurrence of lignicolous marine fungi using submerged blocks of pine (Pinus massoniana Lamb.) and teak (Tectona grandis L.) was carried out for 18 months in the coastal waters of Hong Kong. Five test sites, with environmental conditions varying from estuarine to oceanic, and from polluted to non-polluted, were selected. During each collection, salinity, temperature, pH, dissolved oxygen, biochemical oxygen demand, nitrate-nitrogen, inorganic phosphate-phosphorus and light transmission were measured. A total of 51 species of fungi were recorded among which only 28 were either obligate or facultative marine forms. Neither the general distribution pattern nor the distribution of the more frequent fungi could be solely accounted for by differences in salinity at the test sites and it is suggested that other ecological factors such as heavy sediments in the waters, low pH, and the presence of an abundant source of inocula may be important.     

Resistance to photodamage in evergreen conifers

Conifers in the temperate zone are subject to extremes in climatic conditions, such as low temperatures and water deficits. Under such conditions chloroplast antenna pigments can absorb more light energy than can be utilized in photosynthesis. If the plant is unable to dissipate this excess energy by combined radiationless decay (heat), fluorescence emission and carotenoid quenching, photodamage can result. Potentially damaging conditions exist during winter when low temperatures often occur simultaneously with intense light levels and desiccation. Photodamage is caused when regulatory controls on the production of toxic oxygen species produced in the chjoroplast and the capacity of scavenging systems to dispose of them are exceeded. By becoming cold-hardened and dormant, conifer species can resist all but the most severe effects of the winter climates to which they are adapted. Mechanisms which protect chloroplasts from photodamage appear to be among the essential adaptations enabling temperate conifers to resist the effects of drought and low winter temperatures, especially when light levels are high.     

Speculations on the evolution of ion transport mechanisms.

Primate cells evolved a plasma membrane to restrict the loss of important molecules. The osmotic problems that then arose were solved in one of several ways. Of major importance was the evolution of specific ion pumps, to actively extrude those salts whose inward diffusion would have led to swelling and lysis. In addition, these pumps allowed the cell to store energy in the form of ion gradients across the membrane. Thus, even in the earliest stages, the evolution of ion transport systems coincided with the development of mechanisms which catalyzes the energy transformations. It is postulated that an "ATP"-driven proton pump was one of the first ion transport systems. Such a proton pump would extrude hydrogen ions from the cell, establishing both a transmembrane pH gradient (alkaline inside) and a membrane potential (negative inside). This difference in electrochemical potential for protons (the proton-motive force) could then drive a variety of essential membrane functions, such as the active transport of ions and nutrients. A second major advance was the evolution of an ion transport system that converted light energy into a form which could be used by the cell. The modern model for this is the "purple membrane" of Halobacterium halobium, which catalyzes the extrusion of protons after the capture of light. The protonmotive force generated by such a light-driven proton pump could then power net synthesis of ATP by a reversal of the ATP-driven proton pump. A third important evolutionary step associated with ion transport was the development of a system to harness energy released by biological oxidations. Again, the solution of this problem was to conserve energy as a protonmotive force by coupling the activity of a respiratory chain to the extrusion of protons. Finally, with the development of animal cells a more careful regulation of internal and external pH was required. Thus, an ATP-driven Na+-K+ pump replaced the proton-translocating ATPase as the major ion pump found in plasma membranes.     

The photoactivated toxin cercosporin as a tool in fungal photobiology

Cercospora species are a highly successful group of fungi which pathogenize diverse species of economically important plants. Many Cercospora species produce a unique photoactivated and photoinduced polyketide toxin, cercosporin, which has been implicated as a pathogenicity factor. Illuminated cercosporin interacts with molecular oxygen to produce highly toxic singlet oxygen. Although nearly all organisms tested, including plants, mice and most fungi, are susceptible to cercosporin-mediated cell damage, Cercospora species are resistant. In general, little is known about how organisms protect themselves against singlet oxygen. Studies on how Cercospora species avoid autotoxicity are proving to be a valuable model in understanding this process in other systems. Furthermore, advances are being made in the understanding of how light regulates gene expression and cercosporin synthesis in Cercospora species. These studies are helping to elucidate mechanisms of gene regulation and light signal transduction for an environmental signal important in numerous fungal developmental processes, including secondary metabolite production.     

Studies on the tropical marine fungi of Brunei

Driftwood, mangrove roots and branches, and seaweeds were examined for marine fungi from beaches, rocky shorelines, an artificial lake and mangrove stands. Ninety-five species, some new to science were recorded. This is the largest number of marine fungi reported from a single study. Our knowledge of the geographic distributions of these fungi has been extended. New and rare species are illustrated at the light microscope and SEM levels.     

Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells

Low energy visible light (LEVL) irradiation has been shown to exert some beneficial effects on various cell cultures. For example, it increases the fertilizing capability of sperm cells, promotes cell proliferation, induces sprouting of neurons, and more. To learn about the mechanism of photobiostimulation, we studied the relationship between increased intracellular calcium ([Ca2+]i) and reactive oxygen species production following LEVL illumination of cardiomyocytes. We found that visible light causes the production of O2. and H2O2 and that exogenously added H2O2 (12 microm) can mimic the effect of LEVL (3.6 J/cm2) to induce a slow and transient increase in [Ca2+]i. This [Ca2+]i elevation can be reduced by verapamil, a voltage-dependent calcium channel inhibitor. The kinetics of [Ca2+]i elevation and morphologic damage following light or addition of H2O2 were found to be dose-dependent. For example, LEVL, 3.6 J/cm2, which induced a transient increase in [Ca2+]i, did not cause any cell damage, whereas visible light at 12 J/cm2 induced a linear increase in [Ca2+]i and damaged the cells. The linear increase in [Ca2+]i resulting from high energy doses of light could be attenuated into a non-linear small rise in [Ca2+]i by the presence of extracellular catalase during illumination. We suggest that the different kinetics of [Ca2+]i elevation following various light irradiation or H2O2 treatment represents correspondingly different adaptation levels to oxidative stress. The adaptive response of the cells to LEVL represented by the transient increase in [Ca2+]i can explain LEVL beneficial effects.     

Proton pumps: mechanism of action and applications

Recent progress in understanding the molecular structures and mechanisms of action of proton pumps has paved the way to their novel applications in biotechnology. Proton pumps, bacteriorhodopsin and ATP synthases in particular, are capable of continuous, renewable conversion of light to chemical, mechanical or electrical energy, which can be used in macro- or nano-scale devices. The capability of protein systems incorporated into liposomes to generate ATP, which can be used to drive chemical reactions and to act as molecular motors has been already demonstrated. Other possible applications of such biochemical devices include targeted drug delivery and biocatalytic reactors. All these devices might prove superior to their inorganic alternatives.