Assessment of rapid bioassays for detecting cyanobacterial toxicity

Simple and easy-to-use bioassays with Artemia salina (brine shrimp) larvae, luminescent bacteria and Pseudomonas putida were evaluated for the detection of toxicity due to cyanobacterial hepato-and neurotoxins. The hepatotoxins and a neurotoxin, anatoxin-a, were extracted from laboratory-grown cultures and natural bloom samples by the solid phase fractionation method and dissolved in diluent for different bioassays. The toxin concentration of cyanobacterial extracts was determined with HPLC. The Artemia biotest appeared to be quite sensitive to cyanobacterial hepatotoxins, with LC 50 values of 3–17 mg l^-1. The Artemia test was also shown to be of value for the detection of toxicity caused by anatoxin-a. The fractionated extract of anatoxin-a was not lethal to Artemia but it disturbed the ability of the larvae to move forwards. Filtered cyanobacterial cultures with anatoxin-a, on the other hand, caused mortality of Artemia larvae at concentrations of 2–14 mg l^-1. With the solid phase fractionation of cyanobacterial samples, no non-specific toxicity due to compounds other than hepato- and neurotoxins was observed. In the luminescent bacteria test, the inhibition of luminescence did not correlate with the abundance of hepatotoxins or anatoxin-a. The growth of Ps. putida was enhanced, rather than inhibited by cyanobacterial toxin fractions.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay

We have developed a sensitive bioluminescence assay for beta-galactosidase using a luminescent substrate, D-luciferin-O-beta-galactopyranoside (LuGal). The detection limit for beta-galactosidase was 3 x 10-20 mol per assay, which was approximately 50-fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10-min enzyme assay using LuGal were comparable to those made after a 22-24-h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms.     

UV and arsenate toxicity: a specific and sensitive yeast bioluminescence assay

We describe a Saccharomyces cerevisiae bioluminescence assay for UV and arsenate in which bacterial luciferase genes are regulated by the promoter of the yeast gene, UFO1. UFO1 encodes the F-box subunit of the Skp1–Cdc53–F-box protein ubiquitin ligase complex and is induced by DNA damage and by arsenate. We engineered the UFO1 promoter into an existing yeast bioreporter that employs human genes for detection of steroid hormone-disrupting compounds in water bodies. Our analysis indicates that use of an endogenous yeast promoter in different mutant backgrounds allows discrimination between different environmental signals. The UFO1-engineered yeast give a robust bioluminescence response to UVB and can be used for evaluating UV protective sunscreens. They are also effective in detecting extremely low concentrations of arsenate, particularly in pdr5Δ mutants that lack a mechanism to extrude toxic chemicals; however, they do not respond to cadmium or mercury. Combined use of endogenous yeast promoter elements and mutants of stress response pathways may facilitate development of high-specificity yeast bioreporters able to discriminate between closely related chemicals present together in the environment.     

A rapid fluorescence-based assay for detecting soluble methane monooxygenase

A fluorescence-based assay was developed to estimate soluble methane monooxygenase (sMMO) activity in solution. Whole cells of Methylosinus trichosporium OB3b expressing sMMO were used to oxidize various compounds to screen for fluorescent products. Of the 12 compounds tested, only coumarin yielded a fluorescent product. The UV absorbance spectrum of the product matches that of 7-hydroxycoumarin, and this identification was confirmed by 13C-NMR spectroscopy. The dependence of the fluorescent reaction on sMMO activity was investigated by pre-incubation with acetylene, a known inhibitor of sMMO activity. Apparent kinetic parameters for whole cells were determined to be Km(app)=262 microM and Vmax(app)=821 nmol 7-hydroxycoumarin min(-1) mg protein(-1). The rate of coumarin oxidation by sMMO correlates well with those of trichloroethylene degradation and naphthalene oxidation. Advantages of the fluorescence-based coumarin oxidation assay over the naphthalene oxidation assay include a more stable product, direct detection of the product without additional reagents, and greater speed and convenience.     

A new rapid and sensitive bioluminescence assay for antibiotics that inhibit protein synthesis

N aveh , A., P otasman , I., B assan , H. & U litzur , S. 1984. A new rapid and sensitive bioluminescence assay for antibiotics that inhibit protein synthesis. Journal of Applied Bacteriology 56 , 457–463.
A new sensitive, rapid and simple bioluminescence assay for antibiotics inhibiting protein synthesis is described. In this assay the ability of the tested antibiotic to inhibit the de novo synthesis of the enzymes participating in the bacterial luminescence system is determined by means of a dark variant of a luminous bacterium that undergoes prompt induction of the luminescence system with certain DNA-intercalating agents. Upon induction, the in vivo luminescence of the dark variant is increased more than 50-fold within 30 min. Antibiotics that block the de novo synthesis of protein limit the development of luminescence at a level that was found to be a function of the antibiotic concentration. The minimum detectable concentration of antibiotics in the bioluminescence test, after 45–60 min of incubation, was 0.1 μg ml for streptomycin, gentamicin, kanamycin, lincomycin and chlorampheni-col and 0.3 μg/ml for neomycin, clindamycin and spectinomycin. The new bioluminescence test has been used to assay these antibiotics in serum.     

Quantitative detection of crystalline lysine supplementation in poultry feeds using a rapid bacterial bioluminescence assay

Lysine is an essential amino acid for both humans and animals; and it is usually the first or second limiting amino acid in most formulated diets. In order to estimate the lysine content in feeds and feed sources, rapid amino acid bioassays have been developed. The objective of this work is to assess a rapid assay for lysine supplementation in chicken feeds, using a luminescent Escherichia coli lysine-auxotrophic strain, to avoid prior thermal sterilization. An E. coli lysine auxotroph carrying a plasmid with lux genes was used as the test organism. The lysine assay was conducted using depleted auxotrophic cells in lysine samples. Luminescence was measured with a Dynex MLX luminometer after addition of the aldehyde substrate. Growth response (monitored as optical density at 600 nm) and light emission response of the assay E. coli strain were monitored to generate standard curves. Bioluminescent analysis of feed samples indicated that the method works well in the presence of a complex feed matrix. Comparison of both optical density and luminescent-based methods indicated that, when the assay takes place under optimal conditions, both methodologies correlated well ( r(2)=0.99). Except for the 0.64% lysine-supplemented feed, estimates for lysine based on the bacterial assay were over 80% (82-97%) of the theoretical values. Animal data showed that the bacterial bioluminescent method correlated well with the chick bioassay when diets with different levels of lysine supplementation were assayed for lysine bioavailability ( r(2)=0.97). Luminescent methodology coupled with a bacterial growth assay is a promising technique to assess lysine availability in supplemented animal feeds.     

一种快速检测微囊藻毒素mcyG基因的新技术——环介导恒温扩增

利用环介导恒温扩增(LAMP)技术,以微囊藻毒素(Microcystins,MCs)合成基因簇中的mcyG基因为靶序列,设计了1 套LAMP引物,建立了LAMP反应体系并进行灵敏度和特异性实验。结果表明mcyG基因的最低检测限为:24 cfu/mL,远低于常规PCR(Polymerase Chain Reaction)。整个检测过程仅需40 min,且可直接目测结果。特异性实验中, 13 株淡水常见水华蓝藻分属:色球藻属(Chroococcus)、念珠藻属(Nostoc)、鱼腥藻属(Anabaena)、束丝藻属(Aphanizomenon)、微囊藻属(Microcystis),其中10 株呈阳性反应, 3 株为阴性。在野外样品检测中,来自太湖与黄庆苗池塘的水样PCR检测显示阴性反应,而LAMP检测均呈阳性反应,提示此两处水样中可能含有产毒微囊藻,显示出了LAMP检测方法良好的野外检测和预警能力。综合上述,LAMP检测方法能够快速检测产微囊藻毒素的关键基因,且结果可视化。该方法简便、快捷、不依赖特殊检测设备,极具推广前景。     

A sensitive bioluminescence assay for myo-inositol

A specific, sensitive bioluminescence assay for myo-inositol is described employing myo-inositol dehydrogenase linked to a commercial NADH bioluminescence kit. Optimum conditions providing a linear response over 4 orders of magnitude are presented with reproducibility of 6% CV and a sensitivity of 1 pmol. The assay is suitable for the measurement of inositol at low concentrations or in small volumes.     

A new,rapid and sensitive bioluminescence assay for drug screening on Leishmania

We validated a new method, based on luciferine/luciferase bioluminescence, for drug screening on promastigotes of different Leishmania species. Results obtained with this new, rapid, reproducible, and reliable method are in good accordance with results obtained by the conventional MTT assay. This bioluminescence assay has a lower detection limit.     

Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.     

Appraising freeze-drying for storage of bacteria and their ready access in a rapid toxicity assessment assay

Direct toxicity assessment (DTA) techniques seek to measure the impact of toxic chemicals on biological materials resident in the environment. This study features the use of freeze-dried bacterial cells in combination with a rapid DTA analyser, SciTOX^?. The effects of three factors—cryoprotectant type, bacterial strain, and storage temperature—were tested in order to validate the shelf life of the freeze-dried cells. Three freeze-dried Gram-negative bacterial strains, Acinetobacter calcoaceticus, Escherichia coli and Pseudomonas putida, were tested by using the bacteria in the SciTox^? DTA assay and recording their responses to two standard toxicants: 2,4-dicholorophenol and 3,5-dichlorophenol. Each freeze-dried strain of bacteria was prepared in two forms—either pre-treatment with polyethylene glycol (PEG) or with sucrose/Tween 80—prior to storing at either 4 or ?20 °C for three different storage periods (1, 2 or 3 months). While the sucrose/Tween 80 pre-treated freeze-dried cells exhibited better cell viability, we concluded that PEG was a more suitable cryoprotectant for the bacteria used in the DTA assay because of EC₅₀ parity with fresh cell and zero-time freeze-dried cell assays. The results showed that freeze-dried cells, with appropriate materials and conditions, can give reproducible DTA results for up to 3 months. The availability of a biocomponent that can be activated by simple rehydration makes the deployment of this technology much easier for an end user.     

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

Background  

Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy). mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.     

Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins

A rapid and sensitive method for determining protein concentrations using fluorescamine has been characterized for use in the analysis of intact lipoproteins. It was shown that there is no interference with the assay due to the presence of lipid-associated turbidity or primary amine content. The assay was shown to be sensitive to as little as 0.3 microgram of lipoprotein and to yield similar results when compared to the Lowry method.     

Sensitive and rapid detection of microcystin synthetase E Gene (mcyE) by loop-mediated isothermal amplification: A new assay for detecting the potential microcystin-producing Microcystis in the aquatic ecosystem

In order to develop a new molecular technique that has the potential to assist with monitoring and management of water bodies for potential microcystin producing cyanobacterial species that occur in mixed populations in many regions of the world, we designed a new loop-mediated isothermal amplification (LAMP) assay based on microcystin biosynthesis genes. Four sets of primers were designed to recognize six distinct sequences on target the mcyE gene that encodes a protein (McyE) being responsible to catalyze the addition of d-glutamate to Adda. One set (MCYE2) was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for mcyE detection were determined. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 40 min at an isothermal temperature of 61 °C. For the sensitivity of LAMP, the detection limit was 8.5 pg/μl (approximately 17 pg) DNA. The eleven microcystin producing and four non-toxic cyanobacterial strains were selected for testing of specificity. The results of the amplification were positive with all microcystin-producing strains tested and not with four non-toxic strains, which showed that the primers had good levels of specificity. For testing the application of LAMP assay in the aquatic ecosystem, seven environmental samples from ponds and lakes in Ningbo City were also analyzed using the LAMP targeting the mcyE gene as well as an ELISA assay. Compared with these results of ELISA assay, LAMP assay is satisfied. All of these validated LAMP method being fast, simple and low in cost is a potentially valuable means for potential toxic of cyanobacterial blooms detection, especially for routine monitoring purposes in future.     

Inhibition of several protein phosphatases by a non-covalently interacting microcystin and a novel cyanobacterial peptide, nostocyclin

Microcystins produced by cyanobacterial 'blooms' in reservoirs and lakes pose significant public health problems because they are highly toxic due to potent inhibition of protein serine/threonine phosphatases in the PPP family. A dehydrobutyrine (Dhb)-containing microcystin variant [Asp3, ADMAdda5, Dhb7]microcystin-HtyR isolated from Nostoc sp. was found to potently inhibit PP1, PP2A, PPP4 and PPP5 with IC50 values similar to those of microcystin-LR. However, in contrast to microcystin-LR, which forms a covalent bond with a cysteine residue in these protein phosphatases, Asp,ADMAdda,Dhb-microcystin-HtyR did not form any covalent interaction with PP2A. Since the LD50 for Asp,ADMAdda,Dhb-microcystin-HtyR was 100 microg kg(-1) compared to 50 microg kg(-1) for microcystin-LR, the data indicate that the non-covalent inhibition of protein phosphatases accounts for most of the harmful effects of microcystins in vivo. A 3-amino-6-hydroxy-2-piperidone containing cyclic peptide, nostocyclin, also isolated from Nostoc sp., was non-toxic and exhibited more than 500-fold less inhibitory potency towards PP1, PP2A, PPP4 and PPP5, consistent with the conclusion that potent inhibition of one or more these protein phosphatases underlies the toxicity of microcystins, both lacking and containing Dhb.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

The presence of the cyanobacterial toxin microcystin in black band disease of corals

Black band disease (BBD) is a migrating, cyanobacterial dominated, sulfide-rich microbial mat that moves across coral colonies lysing coral tissue. While it is known that BBD sulfate-reducing bacteria contribute to BBD pathogenicity by production of sulfide, additional mechanisms of toxicity may be involved. Using HPLC/MS, the cyanotoxin microcystin was detected in 22 field samples of BBD collected from five coral species on nine reefs of the wider Caribbean (Florida Keys and Bahamas). Two cyanobacterial cultures isolated from BBD, Geitlerinema and Leptolyngbya sp. contained microcystin based on HPLC/MS, with toxic activity confirmed using the protein phosphatase inhibition assay. The gene mcyA from the microcystin synthesis complex was detected in two field samples and from both BBD cyanobacterial cultures. Microcystin was not detected in six BBD samples from a different area of the Caribbean (St Croix, USVI) and the Philippines, suggesting regional specificity for BBD microcystin. This is the first report of the presence of microcystin in a coral disease.     

Direct colorimetric assay of microcystin using protein phosphatase

A new direct colorimetric assay of microcystin in water and algal samples is proposed consisting of two procedures as follows: 1) the elimination of phosphorus in the sample and concentration of microcystin using a C₁₈ cartridge, 2) the detection of the released phosphorus by the ascorbic acid method and determination of protein phosphatase (PP) inhibition by microcystin. The optimum amounts of phosphorylase α and PP-1 in 50 μL concentrated sample were 50 μg/50 μL buffer and 1.0 unit/50 μL buffer, respectively, for the best assay. The pH for the maximum activity of PP-1 was 8. The minimum detectable concentration for this method was about 0.02 μg/L, which is sufficient to meet the proposed guideline level of 1 μg microcystin/L in drinking water. Consequently, it would seem that the proposed direct colorimetric assay using PP is a rapid, easy, and convenient method for the detection of microcystin in water and algal samples.