Characterisation of Cyanobacterial Strains Originating from the Baltic Sea with Emphasis on Nodularia and its Toxin, Nodularin

Abstract

In the Baltic Sea, Nodularia has been confirmed as the only toxin-producing cyanobacterium so far, but the role of environmental factors on nodularin production is not known yet. Hepatotoxic Nodularia blooms have been observed in almost all parts of the Baltic Sea. In these blooms, the concentration of nodularin often rises high enough to cause a health risk for animals through adverse effects on the liver. In addition to Nodularia, the blooms are dominated also by Aphanizomenon, which is not known to be toxic. These two genera are capable of forming blooms in the nitrogen-depleted water mass of the Baltic Sea in late summer due to their ability to fix nitrogen. However, they differ from each other in physiology. For example, Nodularia is absent from surface waters during most of the year, while Aphanizomenon is found all year round. In order to understand toxin production in Nodularia under different environmental conditions, we studied the effect of several growth factors on intracellular and extracellular concentrations of nodularin of two batch-cultures of Nodularia using high performance liquid chromatography (I, II). The non-toxic Nodularia strain was cultured under the same growth conditions as the toxic strains in order to reveal physiological differences between toxic and non-toxic strains (I). The growth and nitrogen fixation rates of Nodularia and Aphanizomenon under different growth conditions were studied using batch-culture experiments in order to obtain information on the co-dominance of these two cyanobacterial genera in late summer blooms (II). The three-dimensional structure of nodularin in water was determined by nuclear magnetic resonance spectroscopy and molecular dynamics simulations in order to understand the inhibition of protein phosphatases by nodularin, the mechanism underlying its hepatotoxicity (III). The taxonomy of Nodularia from different geographical origins and with different toxin production abilities was studied using several molecular methods based on the 16S rRNA gene and whole genome (IV). In addition, the morphology of strains was examined using light microscopy (IV). Nodularin concentrations under different growth conditions were studied using non-axenic (I) and axenic (II) Nodularia strains. Toxin concentrations in cells and in growth media were generally highest under conditions that promoted growth. Intracellular nodularin concentrations of the axenic Nodularia strain studied increased with increases in temperature, phosphate concentration, and irradiance (II). They decreased at low and high salinities and high inorganic nitrogen concentrations. The associated bacteria of non-axenic cultures had no effect on nodularin concentration. According to our studies, growth at different temperature, light, salinity, and phosphorus conditions as well as growth stage may have an effect on the release of nodularin from cells into the growth medium (I, II). When comparing the growth responses of the toxic strains and the non-toxic strain it was shown that the non-toxic strain grew poorer than the toxic ones under all conditions except at the lowest temperature and phosphate concentration tested (I). Nitrogen fixation of Aphanizomenon and Nodularia was often, but not always, highest under conditions which promoted the growth and lowest in cultures with poor growth. Differences in growth and nitrogen fixation rates of Nodularia and Aphanizomenon were observed (II). Aphanizomenon preferred lower irradiances (test range 2-155 μmol ms), temperatures (7-28oC), and salinities (0-30‰) than Nodularia. The different responses of Nodularia and Aphanizomenon may explain the different vertical, horizontal and temporal distribution of the two genera in the Baltic Sea. The preference of Aphanizomenon for low light and that of Nodularia for high light mirrored their vertical distribution patterns in the field; Aphanizomenon is more homogeneously distributed in the water column than Nodularia, which usually forms scum on the water surface. The ability of Aphanizomenon to grow at low temperatures shown in this study may explain why it is abundant in the water mass during most of the year. Nodularia showed a capacity to tolerate much higher temperatures than it experiences in its natural environment. The growth and nitrogen fixation rates of Nodularia were highest in the same salinity range (5 to 20‰) in which the genus forms mass occurrences in the Baltic Sea and other brackish waters. The incapability of Aphanizomenon to tolerate salinities higher than 10‰ suggests that salinity is an important factor restricting the distribution of this genus. The different salinity optima of the two genera is also seen in their different horizontal distribution patterns in the Baltic Sea: with increasing salinity from freshwater in the north to approximately 15‰ salinity in the southern Baltic Proper, the abundance of Aphanizomenon decreases while the abundance of Nodularia increases. High phosphorus and low nitrogen concentrations have been linked to mass occurrences of Aphanizomenon and Nodularia in the Baltic Sea. Similarly, in these laboratory studies, high phosphorus and low nitrogen concentrations increased the growth of Nodularia and Aphanizomenon. Furthermore, the growth was increased with the presence of accompanying bacteria. The solution conformation of nodularin was remarkably similar to the three-dimensional structure of microcystin-LR, which implies that nodularin inhibits protein phosphatases in the same way as microcystin-LR (III). Both toxins had a saddle-shaped backbone conformation, but microcystin-LR was more buckled than nodularin. In particular, the backbone fold in the conserved region of MeAsp-Arg-Adda-Glu was almost identical between nodularin and microcystin-LR. The molecular dynamics simulations, nevertheless, reveal a certain degree of sway for the trans peptide bonds. The proximal part of the Adda’s side-chain was also very similar. The remote parts of Adda and Arg were not structurally defined and they were also mobile in both peptides. No groups of Nodularia strains could be recognised on the basis of cell size whereas toxin production separated the strains into two groups. In this study, nodularin production of Nodularia strains was consistent with the genotypic analysis. Therefore, this character may be useful when identifying Nodularia strains. The toxic Nodularia strains were separated from non-toxic strains by RFLP of the 16S rRNA gene, 16S rRNA gene sequencing, REPand ERIC-PCR, and ribotyping (IV). All strains were closely related despite their different abilities to produce toxin or geographical origins. The profiles of REPand ERIC-sequences indicated high genetic homogeneity among toxic Nodularia strains from the Baltic Sea. These strains were found to be different from the toxic Nodularia strains from Australia and France by 16S rRNA-based methods and by REPand ERIC-PCR. Our results indicated that two closely related Nodularia genotypes are found in the Baltic Sea. One genotype consists of only nontoxic strains. 16S rRNA gene sequencing showed that these strains were identical to the proposed type strain of Nodularia spumigena PCC 73104/1, which is not a typical N. spumigena strain according to morphological taxonomy. All the genetic markers separate the proposed type strain, and other non-toxic strains, from toxic strains. The toxic Baltic Sea strains form another genotype, which most closely fits the descriptions of Nodularia baltica and N. spumigena whereas the morphological characters of non-toxic Nodularia strains fit most closely to the description of Nodularia sphaerocarpa.