Abstract :
[en] ‘Blooms’, an increasing worldwide phenomenon, are adversely affecting surface water resource, including reservoirs and lakes, used for drinking water supplies, recreation, crop, irrigation and fisheries. These amenities are affected by recurrent mass proliferations of cyanobacteria. The latter are responsible for the production of a wide range of bioactive compounds, including potent toxins (cyanotoxins). These comprise neurotoxins, cytotoxins, inflammatory agents, and hepatotoxins.
Microcystins (MCs), hepatotoxins and tumour promoters are the most documented of the cyanotoxins. The microcystin synthetase gene cluster (mcy) involved in MC biosynthesis consists of a succession of non-ribosomal peptide synthase (NRPS) and polyketide synthetase (PKS) genes. The main producers of MCs are Anabaena, Microcystis, and Planktothrix. However, it is not possible to distinguish a toxic from a non-toxic strain on the basis of their morphology.
In the present study, molecular tools were used, optimized and developed to (i) characterize the 16S rRNA gene diversity of planktonic cyanobacteria, (ii) to detect the cyanobacteria responsible for the production of MCs, (iii) to identify the MCs producing taxa, and (iv) to determine the environmental factors that influence the dynamic of toxic and non-toxic genotypes in Belgian freshwaters.
Eighty-nine strains were isolated and their 16S rRNA genes sequenced. The 16S rRNA gene diversity was studied in 32 samples by denaturing gradient gel electrophoresis (DGGE). In order to evaluate the contribution of this work to the study of the molecular diversity of cyanobacteria in Belgian waterbodies, 114 (strains and DGGE) sequences obtained during this PhD thesis were compared to Belgian sequences obtained by others. As a result, 14 previously undiscribed operational taxonomic units (OTUs) were found in the present study.
For polymerase chain reaction (PCR) detection of the mcyA/B/E genes, the DNA from a total of 162 environmental samples was extracted. The three genes were found together in 64.2% of the samples, whereas the mcyB alone was detected in 95.1% of the samples.
In order to identify the mcyE-carriers present in the freshwaters, a restriction fragment length polymorphism (RFLP) was performed on the mcyE gene. The presence of potentially toxic Microcystis was observed in most of the cases.To bypass the constraint of bacterial cultivation, a combination of whole genome amplification (WGA) and enzyme-linked immunoassay (ELISA) was tested on individual colonies of members of two cyanobacteria, Microcystis and Woronichinia, directly from the natural environment. Sequences of 3 different housekeeping genes (ftsZ, gltX, and recA), of 3 mcy genes, and the Internal Transcribed Spacer (ITS) were analyzed for 11 colonies of Microcystis. MCs were detected and quantified by ELISA in 7 of the 11 Microcystis colonies tested, in agreement with the detection of mcy genes. Sequence types (ST) based on the concatenated sequences of housekeeping genes from cyanobacterial colonies from Belgian water bodies appeared to be endemic when compared to those of strains described in the literature. One colony belonged to a yet undiscovered lineage. A similar protocol was used for 6 colonies of the genus Woronichinia, a taxon that is very difficult to cultivate in the laboratory. The 16S rRNA analysis confirmed the colony identification based on morphology. In addition, we obtained for the first time new genetic data for this genus, such as the rpoC1 gene sequences and the sequences and secondary structures of the ITS. The first discovery of NRPS and PKS DNA sequences in Woronichinia colonies highlights the need for further study of this widely occurring genus, to better assess its ability to produce MCs and/or related metabolites.
For the first time, in this study, we were able to simultaneously monitor one toxic and one non-toxic genotype of M. aeruginosa using real time qPCR technology during a monitoring of 2 years. Both toxic and non-toxic genotypes dynamics appeared influenced by the photoperiod. In addition, the dynamic of the toxic genotype was influenced of light intensity.
The results obtained during this PhD research show the need to characterize toxic cyanobacteria in freshwaters, as well as the conditions that influence MCs concentration dynamics. We showed that factors controlling the dynamics of toxic and non-toxic genotypes are complex. Nevertheless, detection tools can be developed to better understand these widely occurring phenomena. Therefore, efforts should go on in this field with collaborations between the scientists and the authorities.
