Nitrogen fixation in a non-heterocystous cyanobacterial mat from a mountain river.

In situ nitrogen fixation was investigated in a cyanobacterial mat growing on the bed of rocks of the Muga River, Spain. The filamentous non-heterocystous cyanobacterium Schizothrix dominated the mat, showing nitrogenase activity in the light at similar rates to those found in nearby heterocystous Rivularia colonies. N2 fixation in the light was significantly increased by an inhibitor of PSII and oxygen evolution, DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea), and anaerobic conditions. However, no nitrogenase activity was found in the dark. Addition of fructose as a respiratory substrate induced nitrogenase activity in samples incubated under aerobic conditions in the dark but not in anaerobic conditions. Microelectrode oxygen profiles showed internal microaerobic microzones where nitrogen fixation might concentrate. Analyses of the 16S rRNA gene revealed only the presence of sequences belonging to filamentous non-heterocystous cyanobacteria. nifH gene diversity showed that the major phylotypes also belonged to this group. One of the three strains isolated from the Schizothrix mat was capable of fixing N2 and growing in the absence of combined N. This was consistent with the nifH gene analysis. These results suggest a relevant contribution of non-heterocystous cyanobacteria to nitrogen fixation in these mats.

Polyphasic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama river (Spain): morphological, molecular, and ecological approaches(1).

The occurrence and environmental factors responsible for the distribution of benthic cyanobacteria in running waters remain largely unexplored in comparison with those of other aquatic ecosystems. In this study, combined data of ecological characteristics, molecular analysis (based on 16S rRNA gene), and direct microscopic inspection of environmental samples were analyzed in parallel with the morphological characterization of the isolated strains to investigate benthic cyanobacterial diversity in the Guadarrama river (Spain). A total of 17 species were identified that belonged to the genera Aphanocapsa, Pleurocapsa, Chroococcus, Chamaesiphon, Cyanobium, Pseudan-abaena, Leptolyngbya, Phormidium, Nostoc, and Tolypothrix. Phenotypic features were associated with the results of 16S rRNA gene sequencing, complementing existing morphological and genetic databases. A decrease in the cyanobacterial diversity was observed along a pollution gradient in the river. Water quality differed among the sampling sites, and variation in nutrient content was the principal difference among locations. These characteristics were closely associated with an upstream-downstream eutrophic gradient. Canonical correspondence analysis distinguished three groups of species with respect to the eutrophication gradient. The first group (Tolypothrix cf. tenuis, Nostoc punctiforme, Nostoc piscinale, Chamaesiphon investiens, Chroococcus minor, Leptolyngbya nostocorum, and Leptolyngbya tenuis) was characteristic of waters with low levels of nutrients. The second group (Cyanobium sp., Chamaesiphon polymorphus, Leptolyngbya boryana, Phormidium autumnale, Phormidium sp., and Aphanocapsa cf. rivularis) was characteristic of polluted waters, its members appearing mainly in great abundance under eutrophic-hypertrophic conditions. The third group of species (Pseudanabaena catenata, Aphanocapsa muscicola, and Nostoc carneum) was present at upstream and downstream sites.

Phenotypic variability and phylogenetic relationships of the genera Tolypothrix and Calothrix (Nostocales, Cyanobacteria) from running water.

The taxonomy of heterocystous cyanobacteria belonging to the genera Calothrix and Tolypothrix has long been a matter of debate, but their phylogenetic relationships are still not well understood. Our aim was to compare the phylogeny and morphology of members of these genera, which exhibit basal-apical polarity. A phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with the morphological characterization of new isolates and environmental samples. Strains isolated from several rivers and streams showed a high degree of tapering when they were cultured in a nutrient-rich medium. However, clear differences were apparent when they were transferred to a nutrient-poor medium. Some strains showed a low degree of tapering and other morphological features corresponding to the genus Tolypothrix, such as false branching, whereas others maintained the morphological characteristics of the genus Calothrix. Phylogenetic analysis was congruent with the phenotypic characterization, in which the strains and environmental samples of the Tolypothrix and Calothrix morphotypes could be clearly separated. Isolates with a low degree of tapering and natural samples of Tolypothrix distorta were grouped in the same cluster, but strains of the genus Calothrix fell into well separated clades. Results from this study showed that representatives of the genus Tolypothrix share most morphological and developmental properties and a high degree of 16S rRNA gene sequence similarity. However, although similar and sometimes overlapping morphologies may occur in isolates of the genus Calothrix, these morphotypes may be distinguished on the basis of their clear genetic divergence.

Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers.

The diversity within the genus Nostoc is still controversial and more studies are needed to clarify its heterogeneity. Macroscopic species have been extensively studied and discussed; however, the microscopic forms of the genus, especially those from running waters, are poorly known and likely represented by many more species than currently described. Nostoc isolates from biofilms of two Spanish calcareous rivers were characterized comparing the morphology and life cycle in two culture media with different levels of nutrients and also comparing the 16S rRNA gene sequences. The results showed that trichome shape and cellular dimensions varied considerably depending on the culture media used, whereas the characteristics expressed in the course of the life cycle remained stable for each strain independent of the culture conditions. Molecular phylogenetic analysis confirmed the distinction between the studied strains established on morphological grounds. A balanced approach to the evaluation of diversity of Nostoc in the service of autecological studies requires both genotypic information and the evaluation of stable traits. The results of this study show that 16S rRNA gene sequence similarity serves as an important criterion for characterizing Nostoc strains and is consistent with stable attributes, such as the life cycle.

Genetic and morphological characterization of Rivularia and Calothrix (Nostocales, Cyanobacteria) from running water.

In this study, a polyphasic approach was adopted to investigate natural freshwater (river and stream) samples of Rivularia colonies and isolated strains of cyanobacteria with a high degree of trichome tapering (genera Rivularia and Calothrix). Analysis of the phycocyanin (PC) operon and the intervening intergenic spacer (cpcBA-IGS) and 16S rRNA gene sequences were used for genetic characterization. In addition, a molecular fingerprinting method, temperature-gradient gel electrophoresis, which allows sequence-dependent separation of PCR products, was used to assess genotypic diversity in environmental samples and isolated strains. The results showed a high variability of the PC-IGS among the genotypes that was not associated with the morphologies observed. This study underlines the importance of choosing a low-nutrient-content culture medium, especially one with a low phosphorus concentration, for studying typical morphological features of Rivularia for taxonomic purposes. Molecular fingerprinting methods and morphological analyses confirmed the diversity in Rivularia colonial structure and trichome features corresponding to genetic diversity within a single colony. Phylogenetic analysis of cpcBA-IGS was largely consistent with that obtained from 16S rRNA gene sequence analysis and confirmed the high level of divergence between genotypes. The sequences of Rivularia and Calothrix from this study and database sequences showed great heterogeneity and were clearly not monophyletic. The results of this genetic and morphological study of field samples and fresh isolates indicated that the current classification of these genera needs to be revised.