Assessing genetic diversity and identification of Microcystis aeruginosa strains through AFLP and peRRFLP analyses.

[摘要] English: There are 150 cyanobacterial genera and approximately 2 000 species known in the world.More than 40 of these have toxin producing strains. Cyanobacteria, commonly known asblue-green algae, are often present in small numbers together with a diverse assemblageof other photosynthetic algae that naturally occur in surface water worldwide. However,under conditions of warm temperatures, minimal water movement and elevatedconcentrations of phosphorus in a water body, cyanobacteria may frequently becomedominant and form thick scums of floating algal cells. These dense aggregations of floatingcells, termed 'blooms', presents a number of water quality problems; most often offensiveodours and tastes, and sometimes biotoxins that can be divided into alkaloid neurotoxinsand cyclic peptide hepatotoxins, commonly from the genus Microcystis and released inwaterbodies. The neurotoxins act chiefly at neuromuscular junctions and cause rapid deathbecause of respiratory paralysis. The hepatotoxins act on the hepatocyte cytoskeleton andcause intrahepatic haemorrhage and centrilobular necrosis. Clinically the hepatotoxin mostoften causes peracute or acute death, or subacute poisoning with signs such as icterus andhepatogenous photosensitivity.Currently cyanobacterial taxonomy does not provide an unequivocal system for theidentification of toxigenic and bloom-forming genus Microcystis. The ambiguities that existin the cyanobacterial taxonomy are due to the expressed variability, minor morphologicaland developmental characteristics that are used for identification. In this studygeographically unrelated axenic strains of Microcystis aeruginosa were obtained from thePasteur Institute, France (PCC); the National Institute for Environmental Studies, Japan(NIES); the Institute of Freshwater Ecology, UK (CCAP); the Pflanzen PhysiologischesInstitut, Universitat Gottingen, Germany (SAG) and the University of the Free State, SouthAfrica (UV) culture collections. Nonaxenic strains were collected from Hartbeespoort,Rietvlei and Roodeplaat Dams in South Africa. After screening 20 primer combinations ona subset of strains eight IRDye700�?labeled EcoR1 primer pairs were selected foramplified fragment length polymorphism (AFLP) analysis to determine the geneticrelationship of these geographically unrelated strains. A total of 909 bands were amplifiedfrom the eight primer combinations, of which 665 were informative, 207 non-informativeand 37 monomorphic, with an average of 83.12 polymorphic bands per primer combination.The genetic relationship among all the Microcystis aeruginosa strains based on thecombination of data obtained with the eight primer combinations was analysed employingthe Unweighted Pair Group Method using Arithmetic Means (UPGMA) algorithm andpresented as a dendrogram. In the dendrogram, the strains from Rietvlei (UP01) andHartbeespoort Dams (UP04) grouped together and were thus genetically closer to eachother, than to the strain from the Rhoodeplaat Dam (UP03). The Japanese strains (NIES88,NIES89, NIES90, NIES99, NIES299) also grouped separate from the other strains, withNIES90 and NIES299, genetically closest to each other. Interestingly, Microcystisaeruginosa strain PC7806 that originated from The Netherlands, also grouped within thisgroup. Microcystis aeruginosa strains CCAP1450/1 (UK), UV027 (South Africa) andPC7813 grouped together, and are genetically closer to the UP-strains, than any of theother strains. In the present study, AFLP analysis proved useful for the identification ofgenetic diversity and analysis of population structure within Microcystis aeruginosa.In order to link the identification of strains with toxicity, the utility of the mcyB genesequence for identification of strains was tested. Based on conserved motifs present inknown sequences of mcyB four primer pairs were designed. Using the primer pairs Tax3P/2M, Tax 1P/1M, Tax 7P/3M and Tax 10P/4M, the mcyB gene from PCC7813 andUV027 were sequenced, resulting in fragments of 2174 and 2170 base pairs in size,respectively. The obtained sequences were analyzed using nucleotide BLASTN annotationof the Basic Local Alignment Search Tool (BLAST). The sequence alignment indicatedhigh homology to other published sequences in GenBank (AY034601 for pee7813 andAY034602 for UV027; e-value = 0.0). Upon further analysis of the sequences it wasobvious that there are several base differences between the sequences of the two strains,which led us to investigate the potential of using differences in restriction sites, and thusinsertions/deletions (indels) in nucleotide sequence to discriminate between the other M.aeruginosa strains, as well as using the mcyB gene to discern between M. aeruginosa andM. wesenbergii in raw water samples. A vast number of restriction sites were identifiedwith differences followed by restriction digest of the specific polymerase chain reaction(PCR) mcyB gene fragment. This work demonstrates that PCR assays provide a usefulindicator of toxicity as well as the identification of taxonomical characteristics betweenlaboratory cultures and environmental isolates.A number of questions arise from the present study and future research thereforeneeds to address the following issues:�?Are there more than one Microeystis aeruginosa strain / population present at agiven time in a specific water reservoir? Do these populations change through theseason? What role does the individual populations play in a cyanobacterial bloom?Thus, the dynamics and structure of populations need to be clarified.�?Which mcy gene in the cluster is mostly responsible for toxin production? Does theexpression of the genes correlate with gene structure/sequence? What role doesthe environment play in determining the level of expression, and thus toxinproduction?

[发布机构] University of the Free State