Molecular characterization of bloom-forming Aphanizomenon strains isolated from Vela Lake (Western Central Portugal)

The investigation on microcystin topics is increasing due to the related ecological and public health risks. Recent investigation confirms a gap in establishing global patterns relating a particular environment to the bloom occurrence of a species and the production of certain microcystin variants. All the results concerning the environmental effects on the microcystin synthesis of one species must be checked in the light of genome diversity. Thus, the poisoning risks of a bloom depend on the strain causing toxicity. To be more effective, specific water treatment methods are required for blooms of different microcystin producing species (such as colonial and filamentous cyanobacteria found in stratified and unstratified water bodies, respectively). With the increasing number of new microcystin variants discovered, the development of new rapid, inexpensive and sensitive enough monitoring methods to promptly screen simultaneously a great diversity of toxins and also check their toxic effects is becoming necessary.

The heterocytous cyanobacteria form a monophyletic group according to 16S rRNA gene sequence data. Within this group, phylogenetic and morphological studies have shown that genera such as Anabaena and Aphanizomenon are intermixed. Moreover, the phylogeny of the genus Trichormus, which was recently separated from Anabaena, has not been investigated. The aim was to study the taxonomy of the genera Anabaena, Aphanizomenon, Nostoc and Trichormus belonging to the family Nostocaceae (subsection IV.I) by morphological and phylogenetic analyses of 16S rRNA gene, rpoB and rbcLX sequences. New strains were isolated to avoid identification problems caused by morphological changes of strains during cultivation. Morphological and phylogenetic data showed that benthic and planktic Anabaena strains were intermixed. In addition, the present study confirmed that Anabaena and Aphanizomenon strains were not monophyletic, as previously demonstrated. The evolutionary distances between the strains indicated that the planktic Anabaena and Aphanizomenon strains as well as five benthic Anabaena strains in cluster 1 could be assigned to a single genus. On the basis of the 16S rRNA, rpoB and rbcLX gene sequences, the Anabaena/Aphanizomenon strains (cluster 1) were divided into nine supported subclusters which could also be separated morphologically, and which therefore might represent different species. Trichormus strains were morphologically and phylogenetically heterogeneous and did not form a monophyletic cluster. These Trichormus strains, which were representatives of three distinct species, might actually belong to three genera according to the evolutionary distances. Nostoc strains were also heterogeneous and seemed to form a monophyletic cluster, which may contain more than one genus. It was found that certain morphological features were stable and could be used to separate different phylogenetic clusters. For example, the width and the length of akinetes were useful features for classification of the Anabaena/Aphanizomenon strains in cluster 1. This morphological and phylogenetic study with fresh isolates showed that the current classification of these anabaenoid genera needs to be revised.

Heterocyst differentiation in filamentous cyanobacteria provides an excellent prokaryotic model for studying multicellular behaviour and pattern formation. In Anabaena sp. strain PCC 7120, for example, 5-10% of the cells along each filament are induced, when deprived of combined nitrogen, to differentiate into heterocysts. Heterocysts are specialized in the fixation of N(2) under oxic conditions and are semi-regularly spaced among vegetative cells. This developmental programme leads to spatial separation of oxygen-sensitive nitrogen fixation (by heterocysts) and oxygen-producing photosynthesis (by vegetative cells). The interdependence between these two cell types ensures filament growth under conditions of combined-nitrogen limitation. Multiple signals have recently been identified as necessary for the initiation of heterocyst differentiation, the formation of the heterocyst pattern and pattern maintenance. The Krebs cycle metabolite 2-oxoglutarate (2-OG) serves as a signal of nitrogen deprivation. Accumulation of a non-metabolizable analogue of 2-OG triggers the complex developmental process of heterocyst differentiation. Once heterocyst development has been initiated, interactions among the various components involved in heterocyst differentiation determine the developmental fate of each cell. The free calcium concentration is crucial to heterocyst differentiation. Lateral diffusion of the PatS peptide or a derivative of it from a developing cell may inhibit the differentiation of neighbouring cells. HetR, a protease showing DNA-binding activity, is crucial to heterocyst differentiation and appears to be the central processor of various early signals involved in the developmental process. How the various signalling pathways are integrated and used to control heterocyst differentiation processes is a challenging question that still remains to be elucidated.