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Harmful cyanobacteria producing toxic microcystins are a major concern in water quality management. In recent years, hydrogen peroxide (H2O2) has been successfully applied to suppress cyanobacterial blooms in lakes. Physiological studies, however, indicate that microcystin protects cyanobacteria against oxidative stress, suggesting that H2O2 addition might provide a selective advantage for microcystin-producing (toxic) strains. This study compares the response of a toxic Microcystis strain, its non-toxic mutant, and a naturally non-toxic Microcystis strain to H2O2 addition representative of lake treatments. All three strains initially ceased growth upon H2O2 addition. Contrary to expectation, the non-toxic strain and non-toxic mutant rapidly degraded the added H2O2 and subsequently recovered, whereas the toxic strain did not degrade H2O2 and did not recover. Experimental catalase addition enabled recovery of the toxic strain, demonstrating that rapid H2O2 degradation is indeed essential for cyanobacterial survival. Interestingly, prior to H2O2 addition, gene expression of a thioredoxin and peroxiredoxin was much lower in the toxic strain than in its non-toxic mutant. Thioredoxin and peroxiredoxin are both involved in H2O2 degradation, and microcystin may potentially suppress their activity. These results show that microcystin-producing strains are less prepared for high levels of oxidative stress, and are therefore hit harder by H2O2 addition than non-toxic strains.
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.
Mycosporine-like amino acids (MAAs) are UV-absorbing metabolites found in cyanobacteria. While their protective role from UV in Microcystis has been studied in a laboratory setting, a full understanding of the ecology of MAA-producing versus non-MAA-producing Microcystis in natural environments is lacking. This study presents a new tool for quantifying MAA-producing Microcystis and applies it to obtain insight into the dynamics of MAA-producing and non-MAA-producing Microcystis in Lake Erie. This study first developed a sensitive, specific TaqMan real-time PCR assay that targets MAA synthetase gene C (mysC) of Microcystis (quantitative range: 1.7 × 101 to 1.7 × 107 copies/assay). Using this assay, Microcystis was quantified with a MAA-producing genotype (mysC+) in water samples (n = 96) collected during March-November 2013 from 21 Lake Erie sites (undetectable − 8.4 × 106 copies/ml). The mysC+ genotype comprised 0.3–37.8% of the Microcystis population in Lake Erie during the study period. The proportion of the mysC+ genotype during high solar UV irradiation periods (mean = 18.8%) was significantly higher than that during lower UV periods (mean = 9.7%). Among the MAAs, shinorine (major) and porphyra (minor) were detected with HPLC-PDA-MS/MS from the Microcystis isolates and water samples. However, no significant difference in the MAA concentrations existed between higher and lower solar UV periods when the MAA concentrations were normalized with Microcystis mysC abundance. Collectively, this study’s findings suggest that the MAA-producing Microcystis are present in Lake Erie, and they may be ecologically advantageous under high UV conditions, but not to the point that they exclusively predominate over the non-MAA-producers.
Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.
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 cyanobacterial genus Microcystis is known to produce an elaborate array of structurally unique and biologically active natural products, including hazardous cyanotoxins. Cytotoxic aeruginoguanidines represent a yet unexplored family of peptides featuring a trisubstituted benzene unit and farnesylated arginine derivatives. In this study, we aimed at assigning these compounds to a biosynthetic gene cluster by utilizing biosynthetic attributes deduced from public genomes of Microcystis and the sporadic distribution of the metabolite in axenic strains of the Pasteur Culture Collection of Cyanobacteria. By integrating genome mining with untargeted metabolomics using liquid chromatography with mass spectrometry, we linked aeruginoguanidine (AGD) to a nonribosomal peptide synthetase gene cluster and coassigned a significantly smaller product to this pathway, microguanidine (MGD), previously only reported from two Microcystis blooms. Further, a new intermediate class of compounds named microguanidine amides was uncovered, thereby further enlarging this compound family. The comparison of structurally divergent AGDs and MGDs reveals an outstanding versatility of this biosynthetic pathway and provides insights into the assembly of the two compound subfamilies. Strikingly, aeruginoguanidines and microguanidines were found to be as widespread as the hepatotoxic microcystins, but the occurrence of both toxin families appeared to be mutually exclusive.
Nodularia spumigena is a filamentous diazotrophic cyanobacterium that dominates the annual late summer cyanobacterial blooms in the Baltic Sea. But N. spumigena also is common in brackish water bodies worldwide, suggesting special adaptation allowing it to thrive at moderate salinities. A draft genome analysis of N. spumigena sp. CCY9414 yielded a single scaffold of 5,462,271 nucleotides in length on which genes for 5,294 proteins were annotated. A subsequent strand-specific transcriptome analysis identified more than 6,000 putative transcriptional start sites (TSS). Orphan TSSs located in intergenic regions led us to predict 764 non-coding RNAs, among them 70 copies of a possible retrotransposon and several potential RNA regulators, some of which are also present in other N2-fixing cyanobacteria. Approximately 4% of the total coding capacity is devoted to the production of secondary metabolites, among them the potent hepatotoxin nodularin, the linear spumigin and the cyclic nodulapeptin. The transcriptional complexity associated with genes involved in nitrogen fixation and heterocyst differentiation is considerably smaller compared to other Nostocales. In contrast, sophisticated systems exist for the uptake and assimilation of iron and phosphorus compounds, for the synthesis of compatible solutes, and for the formation of gas vesicles, required for the active control of buoyancy. Hence, the annotation and interpretation of this sequence provides a vast array of clues into the genomic underpinnings of the physiology of this cyanobacterium and indicates in particular a competitive edge of N. spumigena in nutrient-limited brackish water ecosystems.
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.
Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium Microcystis via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene mdnA encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in Microcystis aeruginosa strain NIES843 and heterologously produced variants.
Over the past 15 years, the genetic basis for production of many cyanobacterial bioactive compounds has been described. This knowledge has enabled investigations into the environmental factors that regulate the production of these toxins at the molecular level. Such molecular or systems level studies are also likely to reveal the physiological role of the toxin and contribute to effective water resource management. This review focuses on the environmental regulation of some of the most relevant cyanotoxins, namely the microcystins, nodularin, cylindrospermopsin, saxitoxins, anatoxins and jamaicamides.