Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis

Abstract

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.

Impacts of microbial assemblage and environmental conditions on the distribution of anatoxin-a producing cyanobacteria within a river network

“…The anaG gene was ~1670 base pairs shorter than in the three reference sequences. This missing region contained the ~300 base pair methyltransferase domain, proposed to encode the formation of either anatoxin-a or homoanatoxin-a [35, 67]. All samples also lacked the anaK gene, which is proposed to encode the formation of dihydroanatoxin-a [67].…”

Fatal Neurotoxicosis in Dogs Associated with Tychoplanktic, Anatoxin-a Producing Tychonema sp. in Mesotrophic Lake Tegel, Berlin

“…In addition to ATX, Italian strains of T. bourrellyi produce minor amounts of homoanatoxin-a [ 35 ], which has not been detected in Lake Tegel to date. However, there were indications of the presence of dihydroanatoxin-a in the Fontinalis / Tychonema samples from Lake Tegel, which is interesting, as only recently dihydroanatoxin-a has been shown to be not only an ATX degradation product, but also an ATX analogue synthesised by Cylindrospermum stagnale [ 26 , 36 ].…”

Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats

“…A significant factor impeding advancements in knowledge on the ecological function of anatoxins and the ability to predict its concentrations in Microcoleus -dominated samples is the coexistence of multiple strains with varying anatoxin quota (concentration of anatoxin per cell; [ 1 , 7 ]). Some strains do not carry the gene cluster ( anaA–anaK ; [ 8 ]) responsible for anatoxin synthesis, while others carry the gene cluster but produce different quantities of each anatoxin variant [ 1 , 7 , 9 ]. The co-existence of toxic and non-toxic strains suggests that the benefits of producing anatoxins do not always outweigh the costs associated with anatoxin production.…”