Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman&#39;s rank correlation, &rho;&nbsp;&gt;&nbsp;0.66, P&nbsp;&lt;&nbsp;0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC&ndash;MS). Positive correlations were observed also for single lakes.</p

Characterization of enzymatic activity of MlrB and MlrC proteins involved in bacterial degradation of cyanotoxins microcystins

Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed

Non-competitive ELISA with broad specificity for microcystins and nodularins

Simple and cost-effective methods with sufficient sensitivities for preliminary screening of cyanobacterial toxins are in high demand for assessing water quality and safety. We have recently developed a highly sensitive and rapid time-resolved fluorometry based noncompetitive immunoassay for detection of microcystins and nodularins. The assay is based on a synthetic broad-specific anti-immunocomplexantibody SA51D1 capable of recognizing the immunocomplex formed by a generic anti-Adda monoclonal antibody (mAb) bound to either microcystins or nodularins. Using the same antibody pair, here we describe a very simple and cost-efficient non-competitive ELISA test for microcystins and nodularins based on conventional alkaline phosphatase (AP) activity measurement. The recombinant SA51D1 single-chain fragment of antibody variable domain (scFv) was produced as a fusion with bacterial alkaline phosphatase in Escherichia coli. After one step affinity purification through His-tag, the scFv-AP fusion protein could directly be used in the assay. For the assay, toxin standard/sample, biotinylated anti-Adda mAb and the scFv-AP were incubated together for one hour on streptavidin-coated microtiter wells, washed and AP activity was then measured by incubating (1 h at 37°C) with chromogenic substrate para-nitrophenylphosphate (pNPP). The assay was capable of detecting all the eleven tested toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LA -LY, -LF -LW, -WR, and nodularin-R) below WHO guide line value of 1 μg L–1. The detection limit (based on blank+3SD response) for microcystin-LR was 0.2 μg L–1. The assay was verified using spiked (0.25-4 μg L–1 of microcystin-LR) tap, river and lake water samples with recoveries from 64 to 101%. The assay showed good correlation (r2>0.9) with four reference methods for its performance in detecting extracted intracellular microcystin/nodularin from 17 natural surface water samples. The described easy-to-perform assay has a high potential to be used in resource-poor settings as quantitative measurements can be obtained using a simple ELISA reader or easy-to-interpret qualitative results by visual readout. Based on the non-competitive format, the assay does not need any chemical toxin conjugates and offers robustness as compared to the currently available competitive format assays.</p

Cyanobacteria and loess-an underestimated interaction

Background: Biocrusts are important functional units in dryland ecosystems. Regarded as ecosystem engineers, cyanobacteria in biocrusts contribute several major physico-chemical and biological processes. However, the role of cyanobacteria in the process of loess formation has been underestimated. Recently, their contribution to sediment development was presented in the BLOCDUST model of loess formation. Scope: This perspective paper features the environmental impact of cyanobacteria and biocrusts with a focus on processes involved in the formation of loess sediments. We propose that the formation of loess can be mediated by cyanobacteria, including initial trapping, and the accumulation and preservation of loess-forming particles. Moreover, the initial structure may be further altered by weak mineral weathering, dissolution and mineral re-precipitation due to cyanobacterial metabolic processes. Possible negative aspects of environmental impact related to the potential toxicity of cyanobacterial biocrusts are also discussed. We highlight specific biotic-abiotic interactions between biocrusts and loess (e.g. exudation of organic polymers, carbonate dissolution and re-precipitation, and dust-dependent metabolic activities of cyanobacteria) which are essential for the formation of stabilized loess and propose the term “synergosis” to comprise these interactions. Conclusion: The role of cyanobacteria in loess formation has only recently been recognized and the possible biogenic nature of loessification is underestimated as compared to their eolian nature. Mineral weathering and mineral precipitation processes as well as mineral dust flux between litho- and atmosphere mediated by cyanobacteria and biocrusts require more attention due to their significant contribution to ecosystem properties

Cyanotoxin production in seven Ethiopian Rift Valley Lakes

We hypothesized that unusual deaths and illnesses in wild and domestic animals in lake areas of the Rift Valley south of Addis Ababa were caused by toxic cyanobacteria. In the first cyanotoxic analyses conducted in samples from Ethiopia, we found lakes Chamo, Abaya, Awassa, Chitu, Langano, Ziway, and Koka all had concentrations of microcystins (MC) ranging from trace to hazardous, whereas only traces less than limits of detection (LOD) of cylindrospermopsin (CYN) were found. In the December 2006 dry season we sampled the lakes for analyses of MC, CYN, species structures, and calculations of cyanobacteria biomass. We used the Uterm&ouml;hl technique to analyse cyanobacterial biomass and monitored MC toxins using HPLC-DAD, LC-ESI-MS-MRM, and ELISA-test and CYN with HPLC-DAD and ELISA. The various toxicity tests coincided well. In 4 of the lakes (Chamo, Langano, Ziway, and Koka), the inter-lake range of total MC concentration was 1.3&ndash;48 &mu;g L-1; in 3 (Abaya, Awassa, and Chitu), we found only traces of MC. Microcystis aeruginosa was the dominant species, with Microcystis panniformis, Anabaena spiroides, and Cylindrospermopsis spp. as subdominants. The MC concentration, especially in Lake Koka, exceeded levels for serious health hazards for humans, cattle, and wildlife

Assessment of Common Cyanotoxins in Cyanobacteria of Biological Loess Crusts

Cyanotoxins are a diverse group of bioactive compounds produced by cyanobacteria that have adverse effects on human and animal health. While the phenomenon of cyanotoxin production in aquatic environments is well studied, research on cyanotoxins in terrestrial environments, where cyanobacteria abundantly occur in biocrusts, is still in its infancy. Here, we investigated the potential cyanotoxin production in cyanobacteria-dominated biological loess crusts (BLCs) from three different regions (China, Iran, and Serbia) and in cyanobacterial cultures isolated from the BLCs. The presence of cyanotoxins microcystins, cylindrospermopsin, saxitoxins, and beta-N-methylamino-L-alanine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, while the presence of cyanotoxin-encoding genes (mcyE, cyrJ, sxtA, sxtG, sxtS, and anaC) was investigated by polymerase chain reaction (PCR) method. We could not detect any of the targeted cyanotoxins in the biocrusts or the cyanobacterial cultures, nor could we amplify any cyanotoxin-encoding genes in the cyanobacterial strains. The results are discussed in terms of the biological role of cyanotoxins, the application of cyanobacteria in land restoration programs, and the use of cyanotoxins as biosignatures of cyanobacterial populations in loess research. The article highlights the need to extend the field of research on cyanobacteria and cyanotoxin production to terrestrial environments

A collaborative evaluation of LC-MS/MS based methods for BMAA analysis: soluble bound BMAA found to be an important fraction.

Exposure to β-Ν-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery ( < 10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis