Variability in the anatoxin gene clusters of Cuspidothrix issatschenkoi from Germany, New Zealand, China and Japan.

Anatoxin-a and homoanatoxin-a are neurotoxic cyanotoxins produced by benthic and planktonic cyanobacteria worldwide. These toxins are produced by the cyanobacterial genera Dolichospermum, Cuspidothrix, Phormidium, Oscillatoria, Tychonema and Cylindrospermum. In the present study the ana gene clusters (anaA-anaG; c. 21.1 kilobases) of two anatoxin producing Cuspidothrix issatschenkoi strains from Germany: (NIVA-CYA 711) and New Zealand (CAWBG02) were sequenced and compared with the ana gene clusters of two C. issatschenkoi strains from Japan (RM-6 and LBRI48) and one from China (CHABD3). All five ana gene clusters are characterized by the same gene order for anaA-anaG. Similarities were highest (99.56-99.57%) between German (NIVA-CYA 711), New Zealand (CAWBG02) and Chinese (CHABD3) strains. Similarities were lower (91.40-91.67%) when compared to the Japanese strains (RM-6 and LBRI48). Collectively, 2,037 variable sites (328 single nucleotide polymorphisms and 9 insertions/deletions, comprising 1,709 nucleotides) were found in the ana gene clusters of the German, New Zealand and Japanese strains compared to the Chinese strain (CHABD3). The ana gene clusters of the German (NIVA-CYA 711), New Zealand (CAWBG02) and Japanese (RM-6 and LBRI48) strains were characterized by 83, 84, 255 and 231 SNP's compared to the Chinese strain (CHABD3), respectively. The anaE and anaF genes showed the highest variability in all five strains and are recommended as the best genetic markers for further phylogenetic studies of the ana gene cluster from C. issatschenkoi.

Influence of cyanobacteria, mixotrophic flagellates, and virioplankton size fraction on transcription of microcystin synthesis genes in the toxic cyanobacterium Microcystis aeruginosa.

Toxic cyanobacteria such as Microcystis aeruginosa are a worldwide concern in freshwater reservoirs. Problems associated with their mass occurrence are predicted to increase in the future due to global warming. The hepatotoxic secondary metabolite microcystin is of particular concern in this context. This study aimed to determine whether co-occurring microorganisms influence the expression of microcystin biosynthesis genes. To this end, we performed cocultivation experiments and measured mcyB and mcyD transcripts in M. aeruginosa using RT-qPCR. We utilized representatives from three different plankton groups: the picocyanobacterium Synechococcus elongatus, the unicellular flagellate grazer Ochromonas danica, and virioplankton from two different lakes. The presence of S. elongatus significantly increased mcyB and mcyD transcription in M. aeruginosa. Cocultivation with the mixotrophic chrysophyte O. danica did not increase the transcription of mcyB and mcyD; in fact, mcyD transcripts decreased significantly. The virioplankton size fraction of environmental water samples induced a significant increase in mcyB and mcyD transcription when obtained from lakes with cyanobacterial blooms. Our results show that co-occurring microorganisms influence the expression of microcystin biosynthesis genes in M. aeruginosa.

Influence of temperature, mixing, and addition of microcystin-LR on microcystin gene expression in Microcystis aeruginosa.

Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change-related factors. In this study, a new RT-qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin-LR (10 µg L-1 or 60 µg L-1 ) significantly altered mcyB and mcyD expression. The expression levels of mcyB and mcyD were correlated but not identical.

Temporal Dynamics of the Microbial Community Composition with a Focus on Toxic Cyanobacteria and Toxin Presence during Harmful Algal Blooms in Two South German Lakes.

Bacterioplankton plays an essential role in aquatic ecosystems, and cyanobacteria are an influential part of the microbiome in many water bodies. In freshwaters used for recreational activities or drinking water, toxic cyanobacteria cause concerns due to the risk of intoxication with cyanotoxins, such as microcystins. In this study, we aimed to unmask relationships between toxicity, cyanobacterial community composition, and environmental factors. At the same time, we assessed the correlation of a genetic marker with microcystin concentration and aimed to identify the main microcystin producer. We used Illumina MiSeq sequencing to study the bacterioplankton in two recreational lakes in South Germany. We quantified a microcystin biosynthesis gene (mcyB) using qPCR and linked this information with microcystin concentration to assess toxicity. Microcystin biosynthesis gene (mcyE)-clone libraries were used to determine the origin of microcystin biosynthesis genes. Bloom toxicity did not alter the bacterial community composition, which was highly dynamic at the lowest taxonomic level for some phyla such as Cyanobacteria. At the OTU level, we found distinctly different degrees of temporal variation between major bacteria phyla. Cyanobacteria and Bacteroidetes showed drastic temporal changes in their community compositions, while the composition of Actinobacteria remained rather stable in both lakes. The bacterial community composition of Alpha- and Beta-proteobacteria remained stable over time in Lake Klostersee, but it showed temporal variations in Lake Bergknappweiher. The presence of potential microcystin degraders and potential algicidal bacteria amongst prevalent Bacteroidetes and Alphaproteobacteria implied a role of those co-occurring heterotrophic bacteria in cyanobacterial bloom dynamics. Comparison of both lakes studied revealed a large shared microbiome, which was shaped toward the lake specific community composition by environmental factors. Microcystin variants detected were microcystin-LR, -RR, and -YR. The maximum microcystin concentrations measured was 6.7 µg/L, a value still acceptable for recreational waters but not drinking water. Microcystin concentration correlated positively with total phosphorus and mcyB copy number. We identified low abundant Microcystis sp. as the only microcystin producer in both lakes. Therefore, risk assessment efforts need to take into account the fact that non-dominant species may cause toxicity of the blooms observed.