Refine
Has Fulltext
- no (16)
Document Type
- Article (16) (remove)
Language
- English (16)
Is part of the Bibliography
- yes (16)
Keywords
- Cyanobacteria (3)
- Microcystis aeruginosa (3)
- Microcystis (2)
- biosynthesis (2)
- cyanobacteria (2)
- lake monitoring (2)
- microcystin (2)
- natural products (2)
- Aphanizomenon (1)
- Biosynthesis (1)
Institute
Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.
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.
Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.
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.
Cyanobacteria are important primary producers in temperate freshwater ecosystems. However, studies on the seasonal and spatial distribution of cyanobacteria in deep lakes based on high-throughput DNA sequencing are still rare. In this study, we combined monthly water sampling and monitoring in 2019, amplicon sequence variants analysis (ASVs; a proxy for different species) and quantitative PCR targeting overall cyanobacteria abundance to describe the seasonal and spatial dynamics of cyanobacteria in the deep hard-water oligo-mesotrophic Lake Tiefer See, NE Germany. We observed significant seasonal variation in the cyanobacterial community composition (p < 0.05) in the epi- and metalimnion layers, but not in the hypolimnion. In winter-when the water column is mixed-picocyanobacteria (Synechococcus and Cyanobium) were dominant. With the onset of stratification in late spring, we observed potential niche specialization and coexistence among the cyanobacteria taxa driven mainly by light and nutrient dynamics. Specifically, ASVs assigned to picocyanobacteria and the genus Planktothrix were the main contributors to the formation of deep chlorophyll maxima along a light gradient. While Synechococcus and different Cyanobium ASVs were abundant in the epilimnion up to the base of the euphotic zone from spring to fall, Planktothrix mainly occurred in the metalimnetic layer below the euphotic zone where also overall cyanobacteria abundance was highest in summer. Our data revealed two potentially psychrotolerant (cold-adapted) Cyanobium species that appear to cope well under conditions of lower hypolimnetic water temperature and light as well as increasing sediment-released phosphate in the deeper waters in summer. The potential cold-adapted Cyanobium species were also dominant throughout the water column in fall and winter. Furthermore, Snowella and Microcystis-related ASVs were abundant in the water column during the onset of fall turnover. Altogether, these findings suggest previously unascertained and considerable spatiotemporal changes in the community of cyanobacteria on the species level especially within the genus Cyanobium in deep hard-water temperate lakes.
Sedimentary ancient DNA-based studies have been used to probe centuries of climate and environmental changes and how they affected cyanobacterial assemblages in temperate lakes. Due to cyanobacteria containing potential bloom-forming and toxin-producing taxa, their approximate reconstruction from sediments is crucial, especially in lakes lacking long-term monitoring data. To extend the resolution of sediment record interpretation, we used high-throughput sequencing, amplicon sequence variant (ASV) analysis, and quantitative PCR to compare pelagic cyanobacterial composition to that in sediment traps (collected monthly) and surface sediments in Lake Tiefer See. Cyanobacterial composition, species richness, and evenness was not significantly different among the pelagic depths, sediment traps and surface sediments (p > 0.05), indicating that the cyanobacteria in the sediments reflected the cyanobacterial assemblage in the water column. However, total cyanobacterial abundances (qPCR) decreased from the metalimnion down the water column. The aggregate-forming (Aphanizomenon) and colony-forming taxa (Snowella) showed pronounced sedimentation. In contrast, Planktothrix was only very poorly represented in sediment traps (meta- and hypolimnion) and surface sediments, despite its highest relative abundance at the thermocline (10 m water depth) during periods of lake stratification (May-October). We conclude that this skewed representation in taxonomic abundances reflects taphonomic processes, which should be considered in future DNA-based paleolimnological investigations.
The biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation through the carboxylic acid side chains of d-glutamate and -methyl d-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains, McyE-A and McyB-A(2), either in a didomain or a tridomain context with the cognate thiolation domain and the upstream condensation domain revealed that substrate activation of both domains strictly depended on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond-forming modules incorporating d-glutamate or d-aspartate. Our findings further contribute to the understanding of the multifaceted role of condensation domains in nonribosomal peptide synthetase assembly lines.
Liverwort Blasia pusilla L. recruits soil nitrogen-fixing cyanobacteria of genus Nostoc as symbiotic partners. In this work we compared Nostoc community composition inside the plants and in the soil around them from two distant locations in Northern Norway. STRR fingerprinting and 16S rDNA phylogeny reconstruction showed a remarkable local diversity among isolates assigned to several Nostoc clades. An extensive web of negative allelopathic interactions was recorded at an agricultural site, but not at the undisturbed natural site. The cell extracts of the cyanobacteria did not show antimicrobial activities, but four isolates were shown to be cytotoxic to human cells. The secondary metabolite profiles of the isolates were mapped by MALDI-TOF MS, and the most prominent ions were further analyzed by Q-TOF for MS/MS aided identification. Symbiotic isolates produced a great variety of small peptide-like substances, most of which lack any record in the databases. Among identified compounds we found microcystin and nodularin variants toxic to eukaryotic cells. Microcystin producing chemotypes were dominating as symbiotic recruits but not in the free-living community. In addition, we were able to identify several novel aeruginosins and banyaside-like compounds, as well as nostocyclopeptides and nosperin.
Microcystis is the most commonly found toxic cyanobacterial genus around the world and has a negative impact on the ecosystem. As a predominant producer of the potent hepatotoxin microcystin (MC), the genus causes outbreaks in freshwaters worldwide. Standard analytical methods that are used for the detection of microcystin variants can only measure the free form of microcystin in cells. Since microcystin was found as free and proteinbound forms in the cells, a significant proportion of microcystin is underestimated with analytical methods. The aim of the study was to measure protein-bound microcystins and determine the environmental factors that affect the binding of microcystin to proteins. Samples were taken at depths of surface, 1 m, 5 m, 10 m, 15 m, and 18 m in Kucukcekmece Lagoon to analyze depth profiles of two different microcystin forms from June to September 2012 at regular monthly intervals. Our findings suggest that the most important parameter affecting proteinbound microcystin at surface water is high light. Due to favorable environmental conditions such as temperature, light, and physicochemical parameters, the higher microcystin contents, both free and protein-bound MCs, were found in summer periods.
Deciphering chemical mediators regulating specialized metabolism in a symbiotic cyanobacterium
(2022)
Genomes of cyanobacteria feature a variety of cryptic biosynthetic pathways for complex natural products, but the peculiarities limiting the discovery and exploitation of the metabolic dark matter are not well understood. Here we describe the discovery of two cell density-dependent chemical mediators, nostoclide and nostovalerolactone, in the symbiotic model strain Nostoc punctiforme, and demonstrate their pronounced impact on the regulation of specialized metabolism. Through transcriptional, bioinformatic and labeling studies we assigned two adjacent biosynthetic gene clusters to the biosynthesis of the two polyketide mediators. Our findings provide insight into the orchestration of specialized metabolite production and give lessons for the genomic mining and high-titer production of cyanobacterial bioactive compounds.