387 research outputs found

    Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination

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    Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5’-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FM

    The Role of Bacterial Symbionts and Bioluminescence in the Pyrosome, Pyrosoma atlanticum

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    The pelagic tunicate, Pyrosoma atlanticum, is known for its brilliant bioluminescence, but the mechanism causing this bioluminescence has not been fully characterized. This study identifies the bacterial bioluminescent symbionts of P. atlanticum collected in the northern Gulf of Mexico using various methods such as electron microscopy, light microscopy, and molecular genetics. The bacteria are localized within a specific pyrosome light organ. Bioluminescent symbiotic bacteria of Vibrionaceae composed \u3e50% of taxa in tunicate samples (n=13), which was shown by utilizing current molecular genetics methodologies. While searching for bacterial lux genes in 2 tunicate samples, we also serendipitously generated a draft tunicate mitochondrial genome which was used for P. atlanticum pyrosome identification. Furthermore, a total of 396K MiSeq16S rRNA reads provided pyrosome microbiome profiles to determine bacterial symbiont taxonomy. After comparing with the Silva rRNA database, a 99% sequence identity matched a Photobacterium sp. R33-like bacterium (referred to as Photobacterium-Pa1) as the most abundant bacteria within P. atlanticum samples. Specifically-designed 16S rRNA V4 probes for fluorescence in situ hybridization (FISH) verified the Photobacterium-Pa1 location around the periphery of each pyrosome luminous organ. Scanning and transmission electron microscopy (SEM, TEM respectively) confirmed a rod-like bacterial presence which also appears intracellular in the light organs. This intracellular bacterial localization may represent a bacteriocyte formation reminiscent of other invertebrates

    Luminescent microbial bioassays and microalgal biosensors as tools for environmental toxicity evaluation

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    This chapter deals with toxicity bioassays and biosensors based on luminescent microorganisms that report on global toxicity of a sample in such a way that luminescence is reduced or inhibited in the presence of toxic compounds that impair metabolism. Both natural as well as recombinant microorganisms are considered. A detailed description of their main characteristics and environmental applications is reported. A few examples of bioassays for detecting oxidative stress (both bioluminescent and fluorescent bioreporters) are also mentioned and discussed as reactive oxygen species (ROS) formation and subsequent oxidative stress if the antioxidant defenses of the cells are surpassed is one of the main mechanisms of toxicity for most pollutants. There is also a section dedicated to microalgal–based biosensors given their ecological relevance as primary producers, their easiness of culture and immobilization in different matrices, ability to acclimate to low nutrients conditions and ubiquity in aquatic environments. The most used toxicity endpoints for this type of biosensors are the alteration of photosynthetic activity (optical and amperometric biosensors) and the inhibition of enzymes such as alkaline phosphatase (APA) or sterases (mostly conductometric biosensors). The main information is shown in tables that include the microorganisms, their main characteristics (reporter gene systems, transducer types for biosensors) and their main environmental applications as well as relevant references. Although some of these bioassays have already been standardized by different international organizations, there are still many which are also promising tools for environmental global toxicity evaluation and should be fully validated and standardized for regulatory purposesThis research was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grant CTM2016-74927-C2-2-

    Whole-Cell Fluorescent Biosensors for Bioavailability and Biodegradation of Polychlorinated Biphenyls

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    Whole-cell microbial biosensors are one of the newest molecular tools used in environmental monitoring. Such biosensors are constructed through fusing a reporter gene such as lux, gfp or lacZ, to a responsive promoter. There have been many reports of the applications of biosensors, particularly their use in assaying pollutant toxicity and bioavailability. This paper reviews the basic concepts behind the construction of whole-cell microbial biosensors for pollutant monitoring, and describes the applications of two such biosensors for detecting the bioavailability and biodegradation of Polychlorinated Biphenyls (PCBs)

    Development of a Fully Automated Flow Injection Analyzer Implementing Bioluminescent Biosensors for Water Toxicity Assessment

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    This paper describes the development of an automated Flow Injection analyzer for water toxicity assessment. The analyzer is validated by assessing the toxicity of heavy metal (Pb2+, Hg2+ and Cu2+) solutions. One hundred μL of a Vibrio fischeri suspension are injected in a carrier solution containing different heavy metal concentrations. Biosensor cells are mixed with the toxic carrier solution in the mixing coil on the way to the detector. Response registered is % inhibition of biosensor bioluminescence due to heavy metal toxicity in comparison to that resulting by injecting the Vibrio fischeri suspension in deionised water. Carrier solutions of mercury showed higher toxicity than the other heavy metals, whereas all metals show concentration related levels of toxicity. The biosensor’s response to carrier solutions of different pHs was tested. Vibrio fischeri’s bioluminescence is promoted in the pH 5–10 range. Experiments indicate that the whole cell biosensor, as applied in the automated fluidic system, responds to various toxic solutions

    Environmental Biosensors: A Microbiological View

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    In this mini-review, the potential of using microorganisms to design biosensors for detecting environmental pollutants is analyzed and discussed. A distinction is made between a classical biosensor (CB) and a whole-cell biosensor (WCB), emphasizing their structural components and the possibility of using whole microorganisms as their bioreceptor elements. The advantages and disadvantages of using prokaryotic microorganisms as opposed to eukaryotic microorganisms are described. Likewise, the advantages of using protozoa ciliates) over other eukaryotic microorganisms are also shown. We analyze the current bibliography on biosensors built on microorganisms as bioreceptors of pollutant molecules, such as inorganic (metal (loid)s) or organic (xenobiotics). New trends, such as the prokaryotic riboswitches, microbial two-component systems where the pollutant can be simultaneously detected and bioremediated, along with advances in synthetic biology, are shown as promising tools in the design of environmental biosensors

    Development of a bioassay reagent using Photobacterium phosphoreum as a test for the detection of aquatic toxicants

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    Lyophilized cells of Photobacterium phosphoreum, rehydrated in 2% (w/v) NaCl in 0.022M KH₂PO₄ at pH 7.0, were used for developing an assay to test the acute toxicity of organic and inorganic compounds. The standardized assay gave good reproducibility of results with 11 organic and four inorganic compounds. Results were compared with reported data obtained with other test organisms and are within their sensitivity ranges. Environmental screening of wastes from oil and petrochemical industries is discussed.Centro de Investigaciones del Medioambient

    Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria

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    Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations

    Development of an on-line biosensor for optimization and control of continuous bioreactor process

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    During the operation of any bioreactor process two of the most crucial elements are the microbial biomass products and their metabolic state of growth. One of the major inhibiting factors in the development and implementation of effective bioreactor control regimes has been the lack of suitable on-line sensors for measuring such parameters and variables. The aim of the work is to develop and evaluate an on-line biosensor for a continuous bioreactor based on the light emission capabilities of the recombinant host cell culture. The cells have been genetically engineered to include the luz gene operon, originally isolated from bioluminescent marine bacteria, such as Vibrio fischeri. The lux gene codes for products that lead to the bioluminescence phenomenon. The focus of this work is to derive an on-line growth and biomass estimation scheme based on the intensity of emitted light. Experimental data is presented, and it appears that bioluminescence level corresponds to the relative dilution rate which is equal to the growth rate at steady state. It was found that higher dilution rate leads to higher bioluminescence level. It confirmed the batch experimental results that bioluminescence is a reflection of the relative rate of growth rather than the absolute amount of biomass
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