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Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria
(2018)
Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine beta-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.
Cyanobacteria produce about 40 percent of the world’s primary biomass, but also a variety of often toxic peptides such as microcystin. Mass developments, so called blooms, can pose a real threat to the drinking water supply in many parts of the world. This study aimed at characterizing the biological function of microcystin production in one of the most common bloom-forming cyanobacterium Microcystis aeruginosa.
In a first attempt, the effect of elevated light intensity on microcystin production and its binding to cellular proteins was studied. Therefore, conventional microcystin quantification techniques were combined with protein-biochemical methods. RubisCO, the key enzyme for primary carbon fixation was a major microcystin interaction partner. High light exposition strongly stimulated microcystin-protein interactions. Up to 60 percent of the total cellular microcystin was detected bound to proteins, i.e. inaccessible for standard quantification procedures. Underestimation of total microcystin contents when neglecting the protein fraction was also demonstrated in field samples. Finally, an immuno-fluorescence based method was developed to identify microcystin producing cyanobacteria in mixed populations.
The high light induced microcystin interaction with proteins suggested an impact of the secondary metabolite on the primary metabolism of Microcystis by e.g. modulating the activity of enzymes. For addressing that question, a comprehensive GC/MS-based approach was conducted to compare the accumulation of metabolites in the wild-type of Microcystis aeruginosa PCC 7806 and the microcystin deficient ΔmcyB mutant. From all 501 detected non-redundant metabolites 85 (17 percent) accumulated significantly different in either of both genotypes upon high light exposition. Accumulation of compatible solutes in the ΔmcyB mutant suggests a role of microcystin in fine-tuning the metabolic flow to prevent stress related to excess light, high oxygen concentration and carbon limitation.
Co-analysis of the widely used model cyanobacterium Synechocystis PCC 6803 revealed profound metabolic differences between species of cyanobacteria. Whereas Microcystis channeled more resources towards carbohydrate synthesis, Synechocystis invested more in amino acids. These findings were supported by electron microscopy of high light treated cells and the quantification of storage compounds. While Microcystis accumulated mainly glycogen to about 8.5 percent of its fresh weight within three hours, Synechocystis produced higher amounts of cyanophycin. The results showed that the characterization of species-specific metabolic features should gain more attention with regard to the biotechnological use of cyanobacteria.
The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.
The impact of environmental stimuli on the production of the widespread cyanobacterial hepatotoxin microcystin (MC) is under debate. Whereas transcriptional studies of the biosynthetic genes suggest a clear influence of light conditions on toxin production the data for the metabolite itself are inconsistent and highly strain-specific. Here, we have reassessed the MC content by using two immunological detection techniques that allow a parallel quantification of MC in the methanolic extracts and the residual pellet fraction that contains high molecular weight proteins. Our results show a significant proportion of MC in the protein bound fraction in strains of Microcystis and Planktothrix and of the related toxin nodularin (NOD) in Nodularia. Moreover, we could show a very strong increase of MC after high light illumination in the protein fraction contributing to a significant overall increase in MC production under these conditions that is not seen in extracts analysed by LC-MS and ELISA. The fact that a considerable portion of MC is neglected with current analysis techniques was also confirmed for selected field samples. Immunofluorescence studies suggest strain-specific differences in the amount of MC conjugate formation.
Microcystis is a freshwater cyanobacterium frequently forming nuisance blooms in the summer months. The genus belongs to the predominant producers of the potent hepatotoxin microcystin. The success of Microcystis and its remarkable resistance to high light conditions are not well understood. Here, we have compared the metabolic response of Microcystis aeruginosaPCC7806, its microcystin-deficient mcyB mutant (Mut) and the cyanobacterial model organism SynechocystisPCC6803 to high light exposure of 250molphotonsm(-2)s(-1) using GC/MS-based metabolomics. Microcystis wild type and Mut show pronounced differences in their metabolic reprogramming upon high light. Seventeen percent of the detected metabolites showed significant differences between the two genotypes after high light exposure. Whereas the microcystin-producing wild type shows a faster accumulation of glycolate upon high light illumination, loss of microcystin leads to an accumulation of general stress markers such as trehalose and sucrose. The study further uncovers differences in the high light adaptation of the bloom-forming cyanobacterium Microcystis and the model cyanobacterium Synechocystis. Most notably, Microcystis invests more into carbon reserves such as glycogen after high light exposure. Our data shed new light on the lifestyle of bloom-forming cyanobacteria, the role of the widespread toxin microcystin and the metabolic diversity of cyanobacteria.
Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant Delta mcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.