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Rapid Characterization of Microcystin-Producing Cyanobacteria in Freshwater Lakes by TSA-FISH (Tyramid Signal Amplification-Fluorescent In Situ Hybridization)

Luc Brient, Nihel Ben Gamra, Marine Periot, Marie Roumagnac, Perrine Zeller, Myriam Bormans, Annick Méjean, Olivier Ploux and Isabelle C. Biegala
Front. Environ. Sci., 25 July 2017 | https://doi.org/10.3389/fenvs.2017.00043

Abstract

Microcystin (MC) is a common and widespread toxin which represents a health hazard to humans and animals. MC toxin concentrations are monitored by various direct or proxy techniques (HPLC, LC-MS/MS, ELISA, PPIA), however, these techniques do not discriminate producing species from non-producing ones. In order to simultaneously provide the identity and activity of cyanotoxin producing species in freshwater lakes, we applied simple, and fully detailed, whole cell fluorescent in situ hybridization enhanced by tyramid signal amplification (TSA-FISH). DNA oligonucleotide probes MICR3 and MCYA were targeting 16S rRNA and mcyA-mRNA, respectively. The mcyA gene is coding for the MC synthetase enzyme involved in MC synthesis. Controls were acquired with the general eubacterial 16S rRNA probe EUB338, for TSA-FISH assay, and standard HPLC and LC-MS/MS as standard methods for the measurements of MC concentration. Results obtained from monoclonal strains and natural samples demonstrated a specific identification of Microcystis species and were able to discriminate MC producing from non-producing ones. In addition, the MCYA probe allowed the specific detection of MC-synthetase mRNA within Planktothrix isothrix (Oscillatoriale) filaments. Two kinds of mcyA-mRNA labeling were observed in these cells, spots like and plasmid like, which illustrates the well-known plasticity of microbial genome to adapt to environmental stresses. We demonstrated that a simple TSA-FISH assay allows acquiring rapidly dual information of the presence and abundance of potentially toxic species, while identifying species actively producing MC-synthetase mRNA, a proxy of MC toxin. This technique has the potential to be developed into an effective environmental monitoring tool. In addition, detail visualization of cellular mRNAs is powerful for the acquisition of ecological and biomolecular studies of toxic cyanobacteria.
Introduction

Many freshwater cyanobacteria produce blooms which may be toxic to fauna, flora, and humans (Chorus and Bartram, 1999) and raises the importance of making rapid diagnosis of toxin content and/or the toxic potential of these blooms. General phycocyanin probes can be easily deployed in a natural environment to rapidly detect total cyanobacteria concentrations (Brient et al., 2008), while toxin concentrations are measured by standard HPLC or mass spectrometry based techniques (Lawton and Edwards, 2008). Although these latter methods are time consuming, they remained references for the calibration of rapid biochemical (ELISA and PPIA) assays. Potentially toxic species are often identified by biomolecular (PCR) assays (Carmichael and An, 1999 ; Pearson and Neilan, 2008 ; Humpage et al., 2012) but they involve DNA extraction which destroys cells and colony organization. Identification of cyanotoxin producing species requires additional time consuming microscopic isolation, purification, and cultivation. Thus a rapid assay, which simultaneously provides the identity and the activity of cyanotoxin producing species, still remains a challenge.

The mcyA gene has been frequently used in many molecular based approaches, as it is involved in the ribosomal production of microcystine (MC) synthetase, responsible for MC production, through non-ribosomal pathways (Tillett et al., 2000). Many studies have shown a transcriptional regulation of mcyA gene in accordance with changes in toxin concentrations, although discrepancies have been described since early studies which suggested a post-transcriptional regulation of mcyA gene expression (Schawbe et al., 1988 ; Meissner et al., 1996). Very recently, a whole cell Tyramid Signal Amplification-Fluorescent in situ Hybridization (TSA-FISH) assay was developed to target mcyA mRNA transcription as a proxy of MC-synthetase production (Zeller et al., 2016). This assay could verify an up-regulation of mcyA-mRNA synthesis by light, and show similar spot-like fluorescent labeling as the rpob-mRNA probe, where the rpob gene is involved in RNA polymerase synthesis (Gaget et al., 2011). That study demonstrated the potential interest to distinguish toxic from non-toxic organisms, by combining the precision of molecular based technique with species identification, as cellular integrity was not destroyed. However, direct measurement of MC toxin by standard HPLC methods was not provided, and it’s in situ application remained to be demonstrated.

The aim of the present study was to demonstrate that (i) mcyA-mRNA TSA-FISH assay could label MC producing laboratory strains, as well as cells from the natural environment and (ii) identification of MC producing species could be easily acquired simultaneously by the use of specific identity MICR3-16S rDNA probe ; when (iii) toxin concentrations were verified by standard HPLC and/or LC-MS/MS protocols.

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