BIOLUMBASE--the database of natural and transgenic bioluminescent organisms.

The Institute of Biophysics SB RAS hosts and maintains a specialized collection of luminous bacteria (CCIBSO 836) containing over 700 strains isolated in various regions of the world's oceans. The culture collection is a source of lux genes and biologically active substances. The wide application of bioluminescence in medicine and ecology has given importance to analysing information on the structure and functioning of bioluminescence systems in natural and transgenic microorganisms, as well as on their features that are closely interrelated with bioluminescence. The aims of our BIOLUMBASE database are: gathering information on microorganisms with lux genes, their analysis and free access, and distribution of this data throughout the global network. The database includes two sections, natural and transgenic luminous microorganisms, and is updated by our own experimental results, the published literature and internet resources. For the future, a publicly available internet site for BIOLUMBASE is planned. This will list the strains and provide comprehensive information on the properties and functions of luminous bacteria, the mechanisms of regulation of bioluminescence systems, constructs with lux genes, and applications of bioluminescence in microbiology, ecology, medicine and biotechnology. It is noteworthy that this database will also be useful for evaluation of biological hazards of transgenic strains. Users will be able to carry out bibliographic and strain searches starting from any feature of interest.     

Effect of surface-active substances on bioluminescence intensity of bacteria

The study of sensitivity of luminous bacteria isolated from the Black and Azov seas to surfactants from various classes was carried out. It was shown that cationic surfactants had a strong inhibition effect on bacterial luminescence in contrast to anionic and in particular nonionic surfactants. To increase the luminous bacteria sensitivity to the action of OP-10 (nonionic surfactant) and ABS (anionic surfactant), which are widely used in industry, several approaches have been developed. They include modulation of bacterial sensitivity by the additives of cationic substances, use of luminous bacteria at a logarithmic stage of growth, realization of biotesting at low pH = 5.5. The use of these approaches allows to lower effective concentrations of OP-10 and ABS, which caused a decrease of bioluminescence by 50%, 3-200 times and opens perspectives for the use of the bioluminescent method to study these surfactants toxicity on the principle of biosensorics.     

Bioluminescence characteristics of Lake Shira water

Bioluminescence bioassays based on luminous bacteria (Photobacterium phosphopreum) and coupled enzyme system NADH-FMN-oxidoreductase-luciferase were adapted for monitoring the saline-water conditions of Lake Shira (Khakasia, Siberia). The differences in bioluminescence responses have been found to be related to the salt composition and the oxidation-reduction properties of water. Bioluminescent kinetics parameters, which are mostly sensitive to pollution under conditions of saline water, have been observed. The enzymatic system in the presence of 1,4-benzoquinone are shown to be more sensitive to redox characteristics of the salt water than this in the absence of 1,4-benzoquinone. 1,4-benzoquinone should be applied for the preparation of a model solution for the monitoring of redox properties of the salt water. Using this technique, the results of bioluminescence analysis are used to construct a heterogeneity map that characterizes the spatial and temporal water quality of lake Shira. A partial map was based on the bioluminescence characteristics of water samples taken along the shoreline, sampling stations in the different places and in different depths of the lake. It has been demonstrated that the bioluminescence assay measurements must be done within two hours after the sampling time.     

The photobioluminometer,an instrument for the study of ecological factors affecting photosynthesis

An instrument for measurement of bioluminescence and photosynthesis is described. It may be used to detect chemicals toxic to luminous bacteria or to measure photosynthetic oxygen production of algae in mixed culture with bacteria. The latter technique has been used to study the effect on algal photosynthesis of environmental factors such as light quality, temperature, and salinity, and to study the factors affecting chlorophyll synthesis. The technique is ideal for rapid detection of photosynthesis-inhibiting herbicides and other toxic substances in water.     

Long-term preservation of active luminous bacteria by lyophilization

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCI) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4°C. Luminous bacteria were resuscitated affer six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.     

发光蚯蚓的发光体系研究进展

发光蚯蚓在世界范围内广泛分布。大多数发光蚯蚓的发光体系包含于蚯蚓体腔液内充满颗粒的细胞内。早期对不同种发光蚯蚓的生理学及生物化学方面的对比研究表明大多数发光蚯蚓的发光体系是类似的,但最近对线蚓科的两个种的研究发现它们不仅发光源的定位特殊,而且发光反应所需要的成分也明显不同于其他种类。本文对发光蚯蚓的发光器官和发光体系的研究现状及其进展进行了综述,并将有代表性的发光蚯蚓的发光体系进行了对比总结。     

Identification of Antifungal Compounds Produced by Lactobacillus casei AST18

Lactobacillus casei AST18 was screened as an antifungal lactic acid bacteria which we have reported before. In this research, the antifungal properties of cell-free culture filtrate (CCF) from L. casei AST18 were detected, and the antifungal compounds of CCF were prepared by ultrafiltration, and semi-preparative HPLC, and then determined by GC-MS. CCF was sensitive to pH and heat treatment but it was not affected by the treatment of trypsin and pepsin. Through the treatment of ultrafiltration and semi-preparative HPLC there were two parts of CCF which showed antifungal activities: part 1 and part 4. Lactic acid was identified as the main antifungal compound in part 1. In part 4, three small molecular substances were detected with GC-MS. The three potential antifungal substances were cyclo-(Leu-Pro), 2,6-diphenyl-piperidine, and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;1',2'-d]pyrazine. The antifungal activity of L. casei AST18 was a synergistic effect of lactic acid and cyclopeptides.     

Bioluminescence of Marine Dinoflagellates : I. An underwater photometer for day and night measurements

Portable light-baffled underwater photometers have been designed for the measurement of dinoflagellate bioluminescence by day and night. Maximal light emission is obtained by mechanical stimulation in a defined volume. The pump which stimulates the dinoflagellates also constantly replenishes the sample volume so that continuous measurements are possible. Evidence for both diurnal variation and vertical migration is presented. Using luminous bacteria for calibration a single dinoflagellate has been found to emit of the order of 10¹⁰ light quanta per flash. The technique suggests that large scale mapping of bioluminescence is feasible.     

The Luminescent Systems of Pony Fishes

The anatomy of bioluminescent organs and mode of light production in 18 species of pony fish have been investigated using fresh and preserved material. The luminescent systems are similarly arranged in all. Basically, the system consists of a light organ located at the distal end of the esophagus, and a series of abdominal accessory structures positioned in tandem for controlling light intensity and for directing and dispersing the light. Light is produced by numerous symbiotic luminous bacteria in the light organ. A simple classification of the luminescent systems is proposed. The light organs of Leiognathus elongatus and L. rivulatus show marked sexual dimorphism. The bacteria present in the light organs of many pony fishes are easily culturable, but not those from L. elongatus. Electron micrographs of the light organs of L. elongatus and L. rivulatus show the presence of numerous rod-shaped bacteria measuring approximately 0.8 µ x 2.4 µ and 0.8 µ x 7.3 µ, respectively. It is concluded that the light organ of L. elongatus contains another example of a type of non-culturable luminous bacteria that have been found elsewhere. Such bacteria appear to require from the host some special factor for growth and luminescence.     

Contribution by symbiotically luminous fishes to the occurrence and bioluminescence of luminous bacteria in seawater

Seawater samples from a variety of locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. When the symbiotically luminous fishCleidopus gloriamaris was placed in a sterile aquarium, plate counts of water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light measurements of water samples from a sterile aquarium containingPhotoblepharon palpebratus, another symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern of increasing light which rapidly decreased upon removal of the fish. These experiments suggest that symbiotically luminous fishes release brightly luminous bacteria from light organs into their environment and may be a source of planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples from environments with populations of symbiotically luminous fish may show detectable levels of light.     

Bioluminescence characteristics of a tropical terrestrial fungus (Basidiomycetes).

Freshly collected samples of luminous mycelium of a terrestrial fungus from Panama were investigated for their bioluminescence characteristics. Taxonomic identification of fungal species could not be determined because of the lack of fruiting bodies. Fluorescence excited by 380 nm illumination had an emission spectrum with a main peak at 480 nm and additional chlorophyll peaks related to the wood substrate. Bioluminescence appeared as a continuous glow that did not show any diel variation. The light production was sensitive to temperature and decreased with temperatures higher or lower than ambient. Bioluminescence intensity was sensitive to hydration, increasing by a factor of 400 immediately after exposure to water and increasing by a factor of 1 million after several hours. This increase may have occurred through dilution of superoxide dismutase, which is a suppressive factor of bioluminescence in fungus tissue. The mycelium typically transports nutritive substances back to the fruiting body. The function of luminescent mycelium may be to increase the intensity of light from the fungus and more effectively attract nocturnal insects and other animals that serve as disseminating vectors for fungal spores.     

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.     

萤火虫(鞘翅目:萤科)两性交流中的闪光信号

对国内外萤火虫两性交流闪光信号的研究进行了综述,萤火虫发光器因种而异,多数发出黄绿色萤光,闪光信号的频率、光谱、强度及其时空分布的闪光模式包含着两性交流信息。萤火虫闪光交流系统有两种分类方法,其一是萤火虫具两个类型的闪光信号交流系统,及系统和系统,前者多在旧大陆,后者多在新大陆;其二是萤火虫具6个类型闪光信号交流系统,即HP,LL,LC,PR,CR和LB型,其中PR型与系统相对应,HP型与系统对应。萤火虫两性交流闪光信号常因时间和空间上的差异及外界物体的干扰使两性闪光交流的效率受到影响。萤火虫两性交流的闪光信号起源于鞘翅目的幼虫阶段,并起警戒天敌的作用,经过两性选择成为成虫两性交流的一种途径,进而成为新大陆的一些萤火虫间捕食猎物和逃避天敌的生存策略。     

Microtiter plate tests for segregation of bioluminescent bacteria

It has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self‐organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux‐gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three‐phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch‐like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller–Segel–Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two‐state) bacteria. The modified KSF model successfully simulated the observed switch‐like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three‐phase contact line region. Copyright © 2015 John Wiley & Sons, Ltd.     

Bacterial bioluminescence as a bioassay for mycotoxins.

The use of bacterial bioluminescence as a toxicological assay for mycotoxins was tested with rubratoxin B, zearalenone, penicillic acid, citrinin, ochratoxin A, PR-toxin, aflatoxin B1, and patulin. The concentrations of mycotoxins causing 50% light reduction (EC50) in Photobacterium phosphoreum were determined immediately and at 5 h after reconstitution of the bacteria from a freeze-dried state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of the above mycotoxins on bioluminescence were determined after 5, 10, 15, and 20 min of incubation with the bacterial suspensions. The concentration of rubratoxin B necessary to elicit an EC50 increased with time, whereas the concentration of citrinin, penicillic acid, patulin, and PR-toxin necessary decreased with time. There was very little change in the concentration of zearalenone, aflatoxin B1, and ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence assay was most sensitive to patulin and least sensitive to rubratoxin B.     

Immobilization ofPhotobacterium phosphoreum for monitoring of toxic substances

A new sensing system based on the immobilization of luminescent bacteria,Photobacterium phosphoreum, was proposed for continuous real-time monitoring of pollutants. The response curves demonstrate thatPhotobacterium phosphoreum immobilized on the strontium alginate was very sensitive to seven reference chemicals used. The significant inhibitory concentrations for bioluminescence emission were 5 ppm for Pb(NO₃)₂, NiCl₂, CdCl₂, 50 ppm for NaAsO₂, 0.1 ppm for HgCl₂, 0.5 ppm for pentachlorophenol and less than 5 ppm for SDS, respectively. The alginate mixed-cells (AMC) retained their luminescence during experimental period (29 days) under storage condition of ? 80°C. The variables affecting performance of continuous flow through monitoring (CFTM) were optimized in order to ensure stability and efficiency. The flow through cell with strontium-alginate immobilized luminescent bacteria was tested with salicylate and 4-nitrophenol and a rapid response of luminescence was recorded by time drivemode in bioluminescence spectrometer after exposure to both toxicants.     

A novel continuous toxicity test system using a luminously modified freshwater bacterium

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.     

Continuous culture of photobacterium

The design and performance characteristics of a small volume (20 ml) continuous culture device for the cultivation of luminous bacteria are described. This simply constructed device can be used to supply luminescent bacteria with constant properties for either laboratory use or the assay of environmental pollutants. Furthermore, bacteria can be deployed to make sensitive (<1 nM) oxygen measurements. The culture device may be configured, alone or in combination, as a chemostat, turbidostat or a "bioluminostat" where bacterial bioluminescence becomes the controlling variable. Over extended periods (>1 week) it was possible to maintain steady-state luminescence within 5% of a pre-set value, although occasionally a non-bioluminescent "mutant" became dominant; in this case light emission was irreversibly lost. A secondary chamber provided additional flexibility and easy manipulation of the cultivated bacteria for testing. The continuous culture system is also suitable for the growth of recombinant microorganisms that either constitutively express luciferase, or do so in response to stress promoter activity. The non-standard control methodologies reported may have important research and industrial applications, for example in providing immediate process control or as an inferential method to optimize biomass: product yield ratios.     

The use of multiple-strain algal sensor chips for the detection and identification of volatile organic compounds

Although biosensors detecting a great variety of toxicants have been developed during the last decades, the simultaneous detection and identification of several targets by one biosensor is not possible in the majority of the biosensor systems. In our study we proved the concept of the detection and identification of two different volatile toxic compounds with a non-selective biochip-based algal biosensor. For that purpose we produced array plate biochips to utilise three membrane-immobilised algal strains of genus Klebsormidium and Chlorella in one biosensor system. A novel IMAGING-PAM chlorophyll fluorometer was applied to measure the impact of volatile organic compounds (VOC) on photosynthesis of chip-immobilized algae in terms of quantum efficiency of electron transport (DeltaF/F'm). Formaldehyde (FA) vapour was detectable with statistical significance in concentrations relevant to human health from 10 ppb to 10 ppm. The biosensor response recorded within minutes was concentration-dependent and reversible. Moreover, vapours of formaldehyde (0.05-1 ppm) and methanol (MeOH) (200-1000 ppm) were significantly identified by the compound-specific response rate as a quotient of the biosensor responses of the respective algal strains. Using the IMAGING-PAM chlorophyll fluorometer, data sampling proved to be highly efficient. Based on our results we conclude that the principle of the algal sensor chip (ASC) suggests further research on the detection and identification of VOCs and other toxic substances in gaseous environment with that biochip system.     

3种拟除虫菊酯类农药对草地贪夜蛾的毒力测定及田间应用效果评价

评价使用氟氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯喷雾和喇叭口点施处理对草地贪夜蛾的活性与防效,为草地贪夜蛾综合防控提供技术支持。采用喷雾法测定了3种农药原药对草地贪夜蛾3龄幼虫毒力;采用喷雾和喇叭口点施2种方式田间施用5.7%氟氯氰菊酯乳油、25 g/L溴氰菊酯乳油、5.0%高效氯氟氰菊酯乳油,药后第1天、第3天、第7天调查挂牌标记玉米上草地贪夜蛾活虫数,计算防治效果。3种拟除虫菊酯农药对草地贪夜蛾3龄幼虫LC 50值大小顺序依次为氟氯氰菊酯(29.80 mg/L)<高效氯氟氰菊酯(42.39 mg/L)<溴氰菊酯(49.88 mg/L);3种拟除虫菊酯类农药在54.00 g a.i./ha剂量下均能明显降低草地贪夜蛾田间虫口数量,同等剂量防效氟氯氰菊酯乳油>高效氯氟氰菊酯乳油>溴氰菊酯微乳剂。由于草地贪夜蛾低龄幼虫和高龄幼虫取食部位有差异,喷雾法防治低龄幼虫的效果好,喇叭口点施防治高龄幼虫的效果好。拟除虫菊酯类农药对草地贪夜蛾具有较好的防治效果,喇叭口点施方式节省药剂、提高对高龄幼虫的防效,在防治草地贪夜蛾等害虫方面具有广阔的应用前景。