E. K. Rodicheva

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Isolation of luminescence system from the luminescent fungus Neonothopanus nambi

56 This study is devoted to the problem of isolation of the lighttemitting system able to luminesce in vitro from the fungus Neonothopanus nimbi. The study was performed with the mycelium of the luminous higher fungus N. nambi, inhabiting the tropp ical forests of South Vietnam. [1] The fungus culture was kindly provided for experiments by Vietnamese researcher Dao Thi Van (private collection of strains To obtain fungal biomass, the mycelium was cultured in Petri plates in a liquid nutrient medium by the techh nology described earlier [2]. The grown mycelium was taken from the Petri dish and washed with deionized (DI) water (MilliiQ system, Millipore, United States) to remove the residual components of the nutrient medium and exometaboliltes. After washing, the remaining water was removed from the mycelial biom ass with filter paper. The isolation of the luminescent system of N. nambi mycelium included the following steps, which were carried out at 0–4°C. Mycelium washed with DI water was ground in the cold with scissors and transferred to a beaker placed in an ice bath. The bioo mass was poured with cold 0.1 M phosphate buffer (pH 7.0) supplemented with 0.1–1.0% BSA (Serva, Germany) in the ratio 1 : 5 (wet biomass weight : buffer volume). The biomass was destroyed with a Volna ultrasonic disintegrator (Russia). Sonication was perr formed at a power of 200 W three times for 5 s at 11min intervals. The homogenate was transferred into chilled tubes and centrifuged at 48 000 g for 30–60 min in an Avanti ® JE centrifuge (BeckmannCoulter, United States). The pellet was discarded, and the supernatant was either used immediately for study (in this case, it was stored at 4°C) or immediately frozen at –20°C and stored at this temperature. The luminescence of the supernatants was meaa sured using a BLM 8801 luminometer (Nauka Special Engineering and Design Department, Krasnoyarsk, Russia) calibrated using the Hastings–Weber radioacc tive standard [3] (one luminescent unit was 10 8 phoo tons per 1 s). The signals were recorded using an LKB 2210 recorder (LKB, Sweden). It was found that supernatants isolated from the mycelium of the luminous fungus N. nambi by the method described above emitted long luminescence (Fig. 1). This fact allowed us to conclude that a selff sufficient luminescent system that ensures luminess cence in vitro was isolated from this fungal species. After filtering the supernatant through a membrane with an …

Chemiluminescent emission of tissues of fruit bodies of higher fungi

105 Among the dozens of thousands of species of higher fungi known to date, more than 80 species possess bioluminescence—the ability to emit light that is visii ble with the naked eye. This ability was found in the to note that the luminescent species phylogenetically coexist with the nonluminescent ones. Sometimes even one taxonomically defined species includes both luminescent and nonluminescent forms [2, 3]. Such a mosaic distribution of bioluminescence suggests that the ability to luminesce has occurred in the kingdom of fungi repeatedly and independently and that its evolutionary basis is a fundamental bioo chemical process, a small deviation in which (even in one or two stages of the metabolic chain) gives rise to luminescence. However, such a process remains unree vealed as yet. The absence of intermediate forms makes it highly difficult to elucidate the evolutionary pathway of the emergence of bioluminescence in the kingdom of fungi. Although the notions on the mechanism of light emission by fungi are far from complete [3, 4], it is obviously distinct from the understood emission mechanisms in animals and bacteria. Weak chemiluu minescence is characteristic of animal tissues [5]. It is known that the major source of luminescence in animals is lipid peroxidation [6] and that chemilumii nescence in plants is related to the photosynthesis system [7]. We studied the emission of nonbioluminescent higher fungi. Studies were performed with different species of higher fungi growing in forests of the Eastt ern Siberian region of Russia (Krasnoyarsk Krai). The objects of the study were 150 samples of fungi collected in forests in vicinities of Krasnoyarsk in summer 2011. The collections were representatives of five orders, 15 families, 21 genera, and 13 species. As many as 136 samples of the collected material were identified to the genus level and 35 samples to the species level (table). We measured luminescence of fungal fragments taken from different parts of the fruit body. Measuree ments were performed with the Glomax 20/20 lumii nometer (Promega, United States), which was calii brated using the Hastings–Weber radioactive standard [8] (2.7 × 10 3 quanta/s was taken as one unit of lumii nescence). The emission of each sample was recorded for 10 s. Signals exceeding the background level by at least 5 times were taken as reliable. Then, each sample was air dried to a constant weight to calculate the spee cific luminosity per unit mass. The table shows the …

Analysis of river water by bioluminescent biotests.

The bacterial bioluminescence has high sensitivity to the action of various inhibitors of biological activity. The lyophilized luminous bacteria Photobacterium phosphoreum (Microbiosensor B17 677F) and luminous strain Escherichia coli (Microbiosensor EC) from the Culture Collection IBSO were used to create bioluminescent biotests. They have been applied in ecological monitoring to determine the overall toxicity of the Yenisei and Angara Rivers and some water sources of Altai Territory. As a rule the heaviest pollution of water in studied rivers was registered near cities and settlements. The luminous bacteria biotests are simple and convenient in work, standardized and quantitative, have rapid response to actions of different substances and high sensitivity to environmental pollutants. It takes less than 30 min to do the biotest (the other biotests take 48--96 h).

Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDCM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40–70% of the cell dry mass weight) were determined. The capacity for synthesizing two-and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.

Biotesting of effluent and river water by lyophilized luminous bacteria biotest

Waters of the Yenisei River, certain rivers and lakes of the Altai Territory, and effluents of some industrial factories in Krasnoyarsk were studied by luminous bacteria biotest Microbiosensor B17 677F. The lyophilized luminous bacteria Photobacterium phosphoreum from the IBSO collection were used to design this biotest. The bioluminescent test is based on bioluminescence quenching resulting from the action of water samples on luminous bacteria. The test results indicated locations and zones of impaired water quality. The heaviest pollution of water in the Yenisei River was recorded in the zones 0–116 km downstream from Krasnoyarsk (Krasnoyarsk and satellite towns). The effluents of most factories were found to be toxic. Underground and surface waters of some areas of the Altai Territory had different toxicity levels; there were deviations from the norm in most water samples taken from the different lakes and rivers. The data from this study show that the luminous bacteria biotest is simple and convenient, and that the results obtained are within acceptable levels of accuracy for the evaluation of the toxicity of effluent and river water. It takes no more than 30 min to do the biotest. It can be used in ecological monitoring like the Microtox toxicity test.

A database on natural and transgenic luminous microorganisms: BiolumBase

The database BiolumBase was designed for the collection and systematization of available information on microorganisms containing bioluminescent systems; it includes two sections: natural and transgenic luminous microorganisms. By now, logic schemes of these sections have been developed, classification of the objects has been performed, ways of presentation of characteristics and structure of fields for input of information have been elaborated, and the necessary program modules have been developed. The database is filled on the basis of published data and our own experimental results; subsequent linkage of the database to the Internet is envisaged. Users will be able to obtain not only catalogues of strains but also information concerning the properties and functions of the known species of luminous bacteria, the structure, regulatory mechanisms, and application of bioluminescent systems and genetically engineered constructions with lux genes, as well as to find references and to search strains by using any set of attributes. The database will provide information that is of interest for the development of microbial ecology and biotechnology, in particular, for the prediction of biological hazard from the application of transgenic strains.

Components of the luminescent system of the luminous fungus Neonothopanus nambi

65 Earlier, we reported that a luminescent system with continuous luminescence in vitro was isolated from the mycelium of the luminous fungus Neonothopanus nambi [1]. The results of this study led us to the conn clusion that the isolated luminescent system is a fairly stable complex that includes protein and nonprotein components required for the light emission reaction. The aim of this work was to separate the protein and nonprotein components of the lighttemitting syss tem of the fungus Neonothopanus nambi and to study some of their properties. The study was performed with the mycelium of the higher luminous fungus N. nambi, inhabiting tropical forests of Southern Vietnam [2]. The culture of fungus for experiments was kindly provided by Dao Thi Van (BIOOLUMI Co., Ltd., Vietnam). The film mycelium biomass was obtained by culturing the fungus in Petri dishes on a liquid nutrient medium by the technology developed by us earlier [3]. Grown mycelium was taken from the Petri dishes and incubated in distilled water to remove the residual components of the nutrii ent medium and exometabolites. To obtain the extracts containing the luminescent reaction substrate, the fungal biomass was incubated in water for 8 h. To obtain the extracts containing the protein components of the luminescent system (primarily the enzymes involved in the light emission reaction), the mycelial biomass was incubated in water for 16 h. The luminescent reaction substrate was extracted from the mycelium biomass as follows. Mycelium incubated in distilled water was placed in a heattresiss tant glass, in which distilled water was added in a 1 : 1 ratio (wet biomass weight : water volume). Thus pree pared sample was placed in a microwave oven and heated to water boiling. Thereafter, the sample was rapidly cooled in an ice bath. The liquid portion of the sample was collected, transferred into chilled tubes, and centrifuged at 30000 g for 20 min at 4°C in an Avanti® JJE centrifuge (BeckmannCoulter, United States). The pellet was discarded, and the supernatant was collected and frozen at –20°C and stored at this temperature until use. The protein components (including the enzymes involved in the light emission reaction) were extracted from the N. nambi mycelium at 0–4°C. Fungal biom ass incubated in distilled water frozen at –80°C (ILS kelvinator, Nuaire Inc., Korea), after which the biomass was thawed and repeatedly washed with diss tilled water. The washed mycelium was ground and transferred …

Effect of ionizing radiation on the luminescence of mycelium of luminous fungus Neonothopanus nambi

30 Currently, the luminescent systems and the mechh anisms of luminescence of many living organisms are well studied: the enzymes (luciferases) catalyzing the lighttemission reactions and their substrates (luciferins) of these organisms were isolated and charr acterized [1]. However, this problem for higher lumii nous fungi remains unsolved, and the molecular orgaa nization of their luminescent system is still poorly understood. First of all, it remains unclear which enzyme (or enzyme complex) performs the function of luciferase in fungi and what is the structure of luciferin, the substrate of lighttemitting reaction. In the early 1990s, it was assumed that the mechanism of fungal luminescence involve reactive oxygen species (ROS) and enzymes with oxidase function [1, 2]. The results of our recent studies of the luminous fungus Neonothopanus nambi also indicate that ROS and oxidases are involved in the mechanism of its luminescence and indicate the relationship of the fungal luminescent system with membrane structures [3–5]. On the basis of these data, it was assumed that the foll lowing membraneebound enzyme systems can be involved in the lighttemitting reaction: oxidases (including peroxidases) of the ligninndegrading comm plex, cytochrome PP450 system, and enzymes of the mitochondrial respiratory chain. These enzyme syss tems can produce ROS, and two of them (ligninolytic oxidase complex and cytochrome PP450 system) can catalyze the oxidation of organic substrates (including luciferin) with the involvement of ROS. Thus, the study of the relationship between the forr mation and transformation of reactive oxygen radicals and the luminescence of higher fungi is important for understanding the mechanisms of luminescence. It is well known [6, 7] that the production of ROS in bioo logical objects can be stimulated by exposure to physs ical, chemical, and biological factors. In particular, the activation of ROS generation under the influence of ionizing radiation and, as a result, the stimulation of superweak chemiluminescence of plant and animal cells was shown [8, 9]. However, in available literature we found no similar papers describing the stimulation of luminescence of luminous fungi. In the present study, we investigated the effect of ionizing radiation on the luminescence of the fungus N. nambi. Experiments were performed with the mycelium of N. nambi inhabiting the tropical forests of South Viett nam [10]. The culture of the fungus for research was kindly provided by Vietnamese researcher Dao Thi Van (private collection of strains of the BIOOLUMI Co., Ltd. company, Vietnam). Samples of luminous …