Appearance of Planktothrix rubescens bloom with [D-Asp3, Mdha7]MC-RR in gravel pit pond of a shallow lake-dominated area

Abstract

Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing genera in temperate lakes. The reddish color of cyanobacterial blooms viewed in a gravel pit pond with the appearance of a dense 3 cm thick layer (biovolume: 28.4 mm(3) L(-1)) was an unexpected observation in the shallow lake-dominated alluvial region of the Carpathian Basin. [D-Asp(3), Mdha(7)]MC-RR was identified from the blooms sample by MALDI-TOF and NMR. Concentrations of [D-Asp(3), Mdha(7)]MC-RR were measured by capillary electrophoresis to compare the microcystin content of the field samples and the isolated, laboratory-maintained P. rubescens strain. In analyzing the MC gene cluster of the isolated P. rubescens strain, a deletion in the spacer region between mcyE and mcyG and an insertion were located in the spacer region between mcyT and mcyD. The insertion elements were sequenced and partly identified. Although some invasive tropical cyanobacterial species have been given a great deal of attention in many recent studies, our results draw attention to the spread of the alpine organism P. rubescens as a MC-producing, bloom-forming species.

Figure 1

(a) Location of Kocka pond in Hungary, indicated by a filled circle; (b) The Planktothrix rubescens bloom in the gravel pit pond; and (c) a microscopic observation of Planktothrix rubescens trichomes from the pond.

Figure 2

(A) Maximum likelihood trees showing the phylogenetic position of BGSD-500 based on the 16S rRNA gene and (B) the phycocyanin operon. In the case of the 16S rRNA gene, 1329 nt positions were involved in the analysis that was performed with the HKY + G substitution model, while for the construction of the cpcBA-IGS tree, 464 nt were used and the Kimura 2-parameter model was applied. Type strains of Planktothrix species according to Suda et al. [29] are marked with superscript T. Arthrospira platensis PCC 7345 was used as an outgroup in both phylogenetic analyses. Bootstrap values lower than 70 are not shown (based on 500 replicates).

Figure 3

DEAE-52 chromatography and Blue-Green Sinapis Test of [d-Asp³, Mdha⁷]MC–RR from Planktothrix rubescens. Absorbance at 239 nm (-○-); hypocotyl length of three-day-old mustard seedlings (-●-), gradient between 0 and 0.2 M NaCl in 5 mM Tris-HCl buffer (---).

Figure 4

Chemical structure of the identified cyanobacterial heptapeptide [d-Asp³, Mdha⁷]MC–RR.

Figure 5

Effect of crude P. rubescens-dominated bloom-sample extract (-●-) and the isolated P. rubescens BGSD-500 (--) on the growth of Sinapis alba etiolated seedlings (Blue-Green-Sinapis-Test).

Figure 6

Capillary electrophoresis of P. rubescens-dominated bloom-sample extract (A) and the isolated P. rubescens BGSD-500 (B). Peak of [d-Asp³, Mdha⁷]MC–RR is indicated by black arrow. (separation conditions: capillary: 64.5 cm, 50 µm i.d., buffer electrolyte: 25 mM borate and 75 mM SDS, pH 9.3, applied voltage: +25 kV, detection: UV absorption at 238 nm).

Figure 7

Localization of the detected and partly identified 1194 nt length insertion element in the spacer region between mcyT and mcyD of mcy gene cluster of Planktothrix.