Microcystin-Bound Protein Patterns in Different Cultures of Microcystis aeruginosa and Field Samples

Abstract

Micocystin (MC) exists in Microcystis cells in two different forms, free and protein-bound. We examined the dynamic change in extracellular free MCs, intracellular free MCs and protein-bound MCs in both batch cultures and semi-continuous cultures, using high performance liquid chromatography and Western blot. The results showed that the free MC per cell remained constant, while the quantity of protein-bound MCs increased with the growth of Microcystis cells in both kinds of culture. Significant changes in the dominant MC-bound proteins occurred in the late exponential growth phase of batch cultures, while the dominant MC-bound proteins in semi-continuous cultures remained the same. In field samples collected at different months in Lake Taihu, the dominant MC-bound proteins were shown to be similar, but the amount of protein-bound MC varied and correlated with the intracellular MC content. We identified MC-bound proteins by two-dimensional electrophoresis immunoblots and mass spectrometry. The 60 kDa chaperonin GroEL was a prominent MC-bound protein. Three essential glycolytic enzymes and ATP synthase alpha subunit were also major targets of MC-binding, which might contribute to sustained growth in semi-continuous culture. Our results indicate that protein-bound MC may be important for sustaining growth and adaptation of Microcystis sp.

Non‐canonical localization of RubisCO under high‐light conditions in the toxic cyanobacterium Microcystis aeruginosa PCC7806

“…In order to assess how far the distinct subcellular localization of RubisCO in the MC‐producing WT and the mutant relate to the binding of MC to RubisCO, we performed diurnal cultivation experiments (16 h light of 60 μmol photons m −2 s −1 , 8 h darkness) with the WT strain at high cell density (OD 750 : 0.6) and low cell density (OD 750 : 0.3). As seen in a previous study, MC‐binding to proteins was only detected in the high cell density cultures but not in the low cell density cultures (Wei et al ., ) (Fig. B and D).…”

“…One may therefore speculate that the phenotypic heterogeneity observed for RubisCO reflects the multicellular lifestyle of this colonial cyanobacterium, while the low‐density single‐celled state may more closely resemble planktonic unicellular cyanobacteria such as Synechococcus and Synechocystis sp. MC may be one of the specialized molecules that differentiate low‐density cultures from high‐density cultures as the toxin specifically associates with protein complexes and is increasingly secreted as a signal at higher cell density (Wei et al ., ). Phenotypic heterogeneity of multicellular cyanobacteria with regard to carbon fixation activities was already reported in several field studies utilizing the high‐resolution NanoSIMS technology for the measurement of 13 C fixation rates.…”

“…Two independent proteomic studies have previously identified a striking number of Calvin-Benson cycle enzymes as predominant MC-binding partners (Zilliges Wei et al, 2016). Hence, we considered the possibility that the high-molecular-mass complexes comprising MC and RbcS represent Calvin-Benson cycle enzyme super complexes that are known from chloroplasts of eukaryotic algae and plants, which were also postulated for cyanobacteria (Suss et al, 1993;Agarwal et al, 2009).…”

“…Furthermore, M. aeruginosa PCC7806 shows higher photosynthesis rates and growth under high‐light conditions compared with the MC‐deficient mutant (Meissner et al ., ). Binding of MC to proteins was also observed in field colonies of Microcystis , while in the laboratory it is only observed at higher cell densities (Wei et al ., ).…”

“…Two independent studies have revealed that the MC-producing strain Microcystis aeruginosa PCC7806 can grow better under C i limitation than the non-toxic ΔmcyB mutant which vice versa outcompetes the wild-type (WT) strain at high CO 2 levels (Jahnichen et al, 2007;Van de Waal et al, 2011). Moreover, the toxin was shown to bind to a number of proteins in Microcystis, among which the large (RbcL) and small (RbcS) subunits of RubisCO and a striking number of Calvin-Benson cycle enzymes such as phosphoribulokinase (PRK), phosphoglycerate kinase, fructose-bisphosphate aldolase and glyceraldehyde-3 phosphate dehydrogenase were identified as predominant binding partners (Zilliges et al, 2011;Wei et al, 2016). MC-bound RubisCO, in turn, is significantly more stable against protease degradation (Zilliges et al, 2011).…”