Expression, purification, characterization of DNA binding activity and crystallization of a putative type II DNA Cytosine-5-methyltransferase from Microcystis aeruginosa.

DNA 5-methylcytosine modification plays an important role in the regulation of a variety of biological functions in both prokaryotic and eukaryotic organisms. Previous studies show that DNA Cytosine-5-methylation is predominantly associated with restriction-modification system in bacteria. IPF4390 is deduced to be a putative type II DNA Cytosine-5 methyltransferase from a fresh water cyanobacterium, Microcystis aeruginosa. Both its substrate sequence specificity and catalytic mechanism need to be revealed. In this study, the cloning, expression, purification, DNA binding assays and crystallization of IPF4390 are reported. Results of DNA binding assays demonstrate that IPF4390 can specifically recognize and bind two double-stranded DNAs containing GGNCC (N = A, T, C or G) sequences (HgiBI: 5'-ATAAGGACCAATA-3'; TdeIII: 5'-ATAAGGGCCAATA-3'). Therefore, IPF4390 is probably capable of blocking endonuclease cleavage once restriction sites containing these sequences. Moreover, the crystal of IPF4390 in the presence of TdeIII was obtained, and its X-ray diffraction data were collected and scaled to a maximum resolution of 2.46 Å.

Investigation of microcystin conformation and binding towards PPP1 by molecular dynamics simulation.

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [ß-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

Cell density-dependent regulation of microcystin synthetase genes (mcy) expression and microcystin-LR production in Microcystis aeruginosa that mimics quorum sensing.

As the secondary metabolites of cyanobacterial harmful algal blooms (Cyano-HABs), microcystins (MCs) were generated under various environmental and cellular conditions. The understanding of the causes of MCs generation is of great interest in the field of water treatment and environmental science. In this work, we studied how Microcystis aeruginosa (FACHB-905) cell densities affect the MCs synthetase genes (mcy) expression, microcystin-LR (MC-LR) and quorum sensing molecules (Acyl-homoserine lactones (AHLs)) production. An electrochemical sensor was developed here for sensitive and quantitative detection of MC-LR that cultured at different cell densities. The results showed that mcy expression and MC-LR concentration started to increase when the cell density reached ca. 22 × 106 cells/mL, and was significantly increased with increasing cell densities. Moreover, the up-regulation of AHLs with increasing cell densities revealed that MC-LR is quorum sensing-mediated. Our results undoubtedly confirmed that MC-LR was produced in a cell density-dependent way that mimics quorum sensing, and the minimum cell density (ca. 22 × 106 cells/mL) that was required to produce MC-LR was provided and offered a reference standard for the prevention and control of MCs pollution in the actual water environment.

Growth, physiological responses and microcystin-production/-release dynamics of Microcystis aeruginosa exposed to various luteolin doses.

By testing time-dependent IC50 of luteolin against Microcystis growth, this study revealed 6.5 mg/L as nearly IC50 value during prolonged stress until day 14, and explored chlorophyll-a (CLA) and phycobiliproteins (PBPs) contents, antioxidant responses and microcystin (MC)-production/-release dynamics at rising luteolin doses (0.5~2-fold IC50). Growth inhibition ratio (GIR) generally rose at rising luteolin dose, while at each dose GIR firstly increased and then leveled off or dropped. In early stage, CLA, allophycocyanin (APC), phycoerythrin (PE) and glutathione (GSH) contents, and superoxide dismutase (SOD) and catalase (CAT) activities, were increasingly stimulated at rising luteolin dose to enhance energy yield and antioxidant defense, but Microcystis was damaged more severely at rising dose, due to stress-repair imbalance. Such more severe damage in early stage, coupled with stronger PBPs-inhibition in mid-late stage, at rising dose could jointly account for rising GIR at rising dose. The CAT/GSH-stimulation persisting until late stage could alleviate cell damage in late stage, which explained for why GIR no longer increased in late stage at each luteolin dose. Besides, more MCs were produced and retained in cell to exert protective roles against luteolin-stress in early stage, but intracellular MCs decreased following inhibited MC-production by prolonged stress to decrease cell protectant. Extracellular MCs detection showed that less MCs amount existed in water phase than control along luteolin-stress, implying luteolin as eco-friendly algaecide with promising potential to remove MPM blooms and MC-risks. This is the first study to reveal the effect of various luteolin doses on MC-production/release and PBP-synthesis dynamics of Microcystis during prolonged stress. The findings shed novel views in anti-algal mechanisms of luteolin, and provided direct evidence for luteolin applied as safe agent to remediate Microcystis-dominant blooms.

Evaluating combined toxicity of binary heavy metals to the cyanobacterium Microcystis: A theoretical non-linear combined toxicity assessment method.

A theoretical non-linear combined toxicity assessment method is proposed and evaluated using Microcystis aeruginosa as the test organism. The combined toxicity of binary heavy metals was evaluated by comparing the actual inhibitory rates shown from the experiments with the theoretically calculated inhibitory rates. It was identified that the binary mixtures of Cu2++ Cd2+, Cu2++ Cr3+ and Zn2++ Cr3+ had the synergistic effects when the combined concentrations were low, but exhibited the antagonistic effects with the higher combined concentrations. Furthermore, the toxic effect of Pb2+ was not influenced by the addition of Cu2+ when combined concentration was low, but it was enhanced by Cu2+ at the high combined concentration. The binary mixtures of Zn2++ Cd2+, Pb2++ Cr3+, Pb2++ Cd2+, Pb2++ Zn2+, and Cr3++ Cd2+ always presented antagonistic effects, while the synergistic toxicity effect on M. aeruginosa was observed for the binary mixtures of Cu2++ Zn2+ regardless of combined concentration. The proposed assessment method was also validated by the antioxidant enzyme activity, which showed synergistic or antagonistic effects under different binary mixtures of heavy metals.

Expression and characterization of a thermostable citrate synthase from Microcystis aeruginosa PCC7806.

Citrate synthase (CS) is an important enzyme in energy conversion and material circulation, participating in many important biochemical processes. In the present study, CS from Microcystis aeruginosa PCC7806 (MaCS) was cloned and expressed in Escherichia coli Rosetta (DE3). The recombinant MaCS was purified and its enzymological properties were characterized. The results showed that MaCS formed dimers in native status. The optimum temperature and pH of MaCS was 30°C and 8.2, respectively. MaCS displayed relative high thermal stability. Treatment at 50°C for 20 min only decreased 11.30% activity of MaCS and the half-life of MaCS was approximately 35 min at 55°C. The kcat and Km of acetyl-CoA and oxaloacetic acid were 17.133 s-1 (kcat) and 11.62 µM (Km), 24.502 s-1 and 103.00 µM, respectively. MaCS activity was not drastically inhibited by monovalent ions and NADH but depressed by divalent ions and some small molecular compounds, especially Mg2+, Zn2+, Co2+ and DTT. Overall, these data contributed to further understanding of energy metabolism in cyanobacteria and also provided basic information for industrial application of CS.

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

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.

Synthesis and Activity of 1,2,3-Triazole Aminopyrimidines against Cyanobacteria as PDHc-E1 Competitive Inhibitors.

Cyanobacteria harmful algal blooms are of global concern, but all currently available algicides in the market are nonselective and have potential side effects on nontarget species. In the present work, two series of compounds (4 and 6) comprising 16 novel 1,2,3-triazole aminopyrimidines were rationally designed and synthesized as control agent for cyanobacteria. Our design focus was the inhibiting cyanobacteria by inhibition against pyruvate dehydrogenase complex E1 (PDHc-E1). Compounds 4 and 6 showed potent inhibition against Escherichia coli PDHc-E1 (IC50 = 4.13-23.76 µM) and also strong algicidal activities against Synechocystis sp. PCC 6803 (EC50 = 1.7-8.1 µM) and Microcystis sp. FACHB905 (EC50 = 2.1-11.8 µM). In particular, the algicidal activities of 6d against four algal species were not only higher than that of prometryn; they were also comparable to or higher than that of copper sulfate. The analogues 4c, 4d, 6d, and 6e displayed potent algicidal activities and inhibition of E. coli PDHc-E1 but exhibited negligible inhibition of porcine PDHc-E1. As revealed by molecular docking, site-directed mutagenesis, enzymatic assays, and an inhibition kinetic analysis, 4c and 6d inhibited PDHc-E1 in a competitive manner. Our results suggest that highly selective, effective algicides can be developed by rationally designing competitive PDHc-E1 inhibitors.

A Novel Chemoenzymatic Approach to Produce Cilengitide Using the Thioesterase Domain from Microcystis aeruginosa Microcystin Synthetase C.

Modern organic chemistry faces many difficulties in the reliable production of cyclopeptides, such as poor yields and insufficient regio- and stereoselectivity. Thioesterase (TE) shows impressive stereospecificity, region- and chemoselectivity during the cyclization of peptide substrates. The biocatalytic properties of TE provide high value for industrial applications. Herein, a novel chemoenzymatic method to synthesize cilengitide is described based on the cyclic activity of the TE domain from microcystin synthetase C (McyC) of Microcystis aeruginosa. In addition, a single active site mutation in the McyC TE was engineered to generate a more effective macrocyclization catalyst. Compared to the chemical approach to synthesize cilengitide, this novel enzyme-catalysed methodology exhibits a higher synthetic efficiency with an approximately 3.4-fold higher yield (49.2%).

Formation mechanism of the Microcystis aeruginosa bloom in the water with low dissolved phosphorus.

The utilization of phosphorus by algae in the low-phosphorus state has drawn wide concerns due to the high risk of forming algal blooms. The cyanobacteria Microcystis aeruginosa (M. aeruginosa) grew well under low-phosphorus condition by hydrolyzing dissolved organic phosphorus (DOP) to dissolved inorganic phosphorus (DIP) through alkaline phosphatase (AP). There was a negative correlation between DIP concentration and AP activity of algae. AP activity significantly increased at 0-3 d (p < 0.05), and reached the peak values of 43.06 and 49.11 King unit/gprot on day 5 for DIP (0.1 mg/L) and DOP (4.0 mg/L), respectively. The relative expression of phosphate transporter gene increased with decreasing phosphorus concentrations. The catalase activity under low-phosphorus condition increased significantly (p < 0.05) after one week, and was generally higher than 0.15 U/mgprot on day 14. Understanding the utilization efficiency and mechanism of DIP and DOP in the low-phosphorus state would help to inhibit the formation of algal blooms.

Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF.

L-amino acids represent the most common amino acid form, most notably as protein residues, whereas D-amino acids, despite their rare occurrence, play significant roles in many biological processes. Amino acid racemases are enzymes that catalyze the interconversion of L- and/or D-amino acids. McyF is a pyridoxal 5'-phosphate (PLP) independent amino acid racemase that produces the substrate D-aspartate for the biosynthesis of microcystin in the cyanobacterium Microcystis aeruginosa PCC7806. Here we report the crystal structures of McyF in complex with citrate, L-Asp and D-Asp at 2.35, 2.63 and 2.80 Å, respectively. Structural analyses indicate that McyF and homologs possess highly conserved residues involved in substrate binding and catalysis. In addition, residues Cys87 and Cys195 were clearly assigned to the key catalytic residues of "two bases" that deprotonate D-Asp and L-Asp in a reaction independent of PLP. Further site-directed mutagenesis combined with enzymatic assays revealed that Glu197 also participates in the catalytic reaction. In addition, activity assays proved that McyF could also catalyze the interconversion of L-MeAsp between D-MeAsp, the precursor of another microcystin isoform. These findings provide structural insights into the catalytic mechanism of aspartate racemase and microcystin biosynthesis.

Structural and functional insights into the role of a cupin superfamily isomerase in the biosynthesis of Choi moiety of aeruginosin.

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is a hallmark of aeruginosins, a class of cyanobacterial derived bioactive linear tetrapeptides that possess antithrombotic activity. The biosynthetic pathway of Choi has yet to be resolved. AerE is a cupin superfamily enzyme that was shown to be involved in the biosynthesis of Choi, but its exact role remains unclear. This study reports the functional characterization and structural analyses of AerE. Enzymatic observation reveals that AerE can dramatically accelerate 1,3-allylic isomerization of the non-aromatic decarboxylation product of prephenate, dihydro-4-hydroxyphenylpyruvate (H2HPP). This olefin isomerization reaction can occur non-enzymatically and is the second step of the biosynthetic pathway from prephenate to Choi. The results of comparative structural analysis and substrate analogue binding geometry analysis combined with the results of mutational studies suggest that AerE employs an induced fit strategy to bind and stabilize the substrate in a particular conformation that is possibly favorable for 1,3-allylic isomerization of H2HPP through coordinate bonds, hydrogen bonds, π-π conjugation interaction and hydrophobic interactions. All of these interactions are critical for the catalytic efficiency.

Phosphorus Influences the Interaction Between Toxigenic Microcystis and Chloramphenicol.

Microcystis growth and physiological responses to chloramphenicol (CAP)-stress were explored at different phosphorus (P) concentrations during 20-day exposure. Under CAP-stress, Microcystis exhibited (i) stronger total protein synthesis and antioxidant defenses at 5 mg/L P than 0.05-0.5 mg/L P in early test period (before day 8), and (ii) greater CAP-removal via biodegradation at 5 mg/L P in mid-late period. Due to above mechanisms, 5 mg/L P largely alleviated the inhibitory effect of CAP on Microcystis growth until test end, thus minimizing CAP toxicity to Microcystis, compared with 0.05-0.5 mg/L P. Moreover, microcystin-production and -release by Microcystis under CAP-stress were also P-dependent. These results suggested that under CAP-stress, although Microcystis growth was more inhibited at 0.05-0.5 mg/L P, higher microcystin-release and CAP residual at 0.05-0.5 mg/L P than at 5 mg/L P still caused eco-risks, which had important implication for risk assessment during Microcystis-dominated blooms and CAP pollution co-occurrence in different waters.

An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming.

The monitoring and control of toxic cyanobacterial strains, which can produce microcystins, is critical to protect human and ecological health. We herein reported an optical-biosensor-based quantification of the microcystin synthetase A (mcyA) gene so as to discriminate microcystin-producing strains from nonproducing strains. In this assay, the mcyA-specific ssDNA probes were designed in silico with an on-line tool and then synthesized to be covalently immobilized on an optical-fiber surface. Production of fluorescently modified target DNA fragment amplicons was accomplished through the use of Cy5-tagged deoxycytidine triphosphates (dCTPs) in the polymerase chain reaction (PCR) method, which resulted in copies with internally labeled multiple sites per DNA molecule and delivered great sensitivity. With a facile surface-based hybridization process, the PCR amplicons were captured on the optical-fiber surface and were induced by an evanescent-wave field into fluorescence emission. Under the optimum conditions, the detection limit was found to be 10 pM (S/N ratio = 3) and equaled 103 gene copies/mL. The assay was triumphantly demonstrated for PCR amplicons of mcyA detection and showed satisfactory stability and reproducibility. Moreover, the sensing system exhibited excellent selectivity with quantitative spike recoveries from 87 to 102% for M. aeruginosa species in the mixed samples. There results confirmed that the method would serve as an accurate, cost-effective, and rapid technique for in-field testing of toxic Microcystis sp. in water, giving early information for water quality monitoring against microcystin-producing cyanobacteria.

Physiological responses and toxin production of Microcystis aeruginosa in short-term exposure to solar UV radiation.

The aim of this study was to evaluate the effects of short-term (hours) exposure to solar UV radiation (UVR, 280-400 nm) on the physiology of Microcystis aeruginosa. Three solar radiation treatments were implemented: (i) PAR (PAR, 400-700 nm), (ii) TUVA (PAR + UVAR, 315-700 nm) and (iii) TUVR (PAR + UVAR + UVBR, 280-700 nm). Differential responses of antioxidant enzymes and the reactive oxygen species (ROS) production to UVR were observed. Antioxidant enzymes were more active at high UVR doses. However, different responses were observed depending on the exposure to UVAR or UVBR and the dose level. No effects were observed on the biomass, ROS production or increased activity of superoxide dismutase (SOD) and catalase (CAT) compared to the control when UVR + PAR doses were lower than 9875 kJ m-2. For intermediate doses, UVR + PAR doses between 9875 and 10 275 kJ m-2, oxidative stress increased while resistance was imparted through SOD and CAT in the cells exposed to UVAR. Despite the increased antioxidant activity, biomass decrease and photosynthesis inhibition were observed, but no effects were observed with added exposure to UVBR. At the highest doses (UVR + PAR higher than 10 275 kJ m-2), the solar UVR caused decreased photosynthesis and biomass with only activation of CAT by UVBR and SOD and CAT by UVAR. In addition, for such doses, a significant decrease of microcystins (MCs, measured as MC-LR equivalents) was observed as a consequence of UVAR. This study facilitates our understanding of the SOD and CAT protection according to UVAR and UVBR doses and cellular damage and reinforces the importance of UVR as an environmental stressor. In addition, our results support the hypothesized antioxidant function of MCs.

Design, Synthesis, and Potency of Pyruvate Dehydrogenase Complex E1 Inhibitors against Cyanobacteria.

Safe and effective algaecides are needed to control agriculturally and environmentally significant algal species. Four series (6, 10, 17, and 21) of 29 novel 4-aminopyrimidine derivatives were rationally designed and synthesized. A part of 10, 17, and 21 displayed potent inhibition of Escherichia coli pyruvate dehydrogenase complex E1 (E. coli PDHc-E1) (IC50 = 2.12-18.06 µM) and good inhibition of Synechocystis sp. PCC 6803 (EC50 = 0.7-7.1 µM) and Microcystis sp. FACH 905 (EC50 = 3.7-7.6 µM). The algaecidal activity of these compounds positively correlated with their inhibition of E. coli PDHc-E1. In particular, 21l and 10b exhibited potent algaecidal activity against PCC 6803 (EC50 = 0.7 and 0.8 µM, respectively), values that were 2-fold increased compared to that of copper sulfate (EC50 = 1.8 µM), and showed the best inhibition of cyanobacterium PDHc-E1 (IC50 = 5.10 and 6.06 µM, respectively). 17h and 21e, the best inhibitors of E. coli PDHc-E1, were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. These results revealed that the improved inhibition of novel inhibitors compared with that of the lead compound I was due to the formation of a new hydrogen bond with Leu264 at the active site of E. coli PDHc-E1. The results proved the great potential to obtain effective algaecides via the rational design of PDHc-E1 inhibitors.

Effects of Dihydroartemisinin and Artemether on the Growth, Chlorophyll Fluorescence, and Extracellular Alkaline Phosphatase Activity of the Cyanobacterium Microcystis aeruginosa.

Increased eutrophication in the recent years has resulted in considerable research focus on identification of methods for preventing cyanobacterial blooms that are rapid and efficient. The objectives of this study were to investigate the effects of dihydroartemisinin and artemether on the growth of Microcystis aeruginosa and to elucidate its mode of action. Variations in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular alkaline phosphatase activity (APA), and chlorophyll fluorescence parameters (Fv/Fm, ΦPSII, ETR, rapid light curves, fast chlorophyll fluorescence curves on fluorescence intensity, and relative variable fluorescence) were evaluated by lab-cultured experiments. Our results demonstrated that both dihydroartemisinin and artemether inhibited the growth of M.aeruginosa by impairing the photosynthetic center in photosystem II and reducing extracellular APA, with a higher sensitivity exhibited toward artemether. The inhibitory effects of dihydroartemisinin on M.aeruginosa increased with concentration, and the maximum growth inhibitory rate was 42.17% at 24 mg·L-1 after 120h exposure, whereas it was 55.72% at 6 mg·L-1 artemetherafter 120h exposure. Moreover, the chlorophyll fluorescence was significantly inhibited (p<0.05) after 120h exposure to 12 and 24 mg·L-1 dihydroartemisinin. Furthermore, after 120h exposure to 6 mg·L-1 artemether, Fv/Fm, ΦPSII, ETR and rETRmax showed a significant decrease (p<0.01) from initial values of 0.490, 0.516, 17.333, and 104.800, respectively, to 0. One-way analysis of variance showed that 6 mg·L-1 artemether and 24 mg·L-1 dihydroartemisinin had significant inhibitory effects on extracellular APA (p<0.01). The results of this study would be useful to further studies to validate the feasibility of dihydroartemisinin and artemether treatment to inhibit overall cyanobacterial growth in water bodies, before this can be put into practice.

Growth, toxin production, active oxygen species and catalase activity of Microcystis aeruginosa (Cyanophyceae) exposed to temperature stress.

Microcystis are known for their potential ability to synthesize toxins, mainly microcystins (MCs). In order to evaluate the effects of temperature on chlorophyll a (Chl a), growth, physiological responses and toxin production of a native Microcystis aeruginosa, we exposed the cells to low (23°C) and high (29°C) temperature in addition to a 26°C control treatment. Exponential growth rate was significantly higher at 29°C compared to 23°C and control, reaching 0.43, 0.32 and 0.33day(-)(1) respectively. In addition, there was a delay of the start of exponential growth at 23°C. However, the intracellular concentration of Chl a decreased significantly due to temperature change. A significant increase in intracellular ROS was observed in coincidence with the activation of enzymatic antioxidant catalase (CAT) during the first two days of exposure to 23° and 29°C in comparison to the control experiment, decreasing thereafter to nearly initial values. Five MCs were determined by LC-MS/MS analysis. In the experiments, the highest MC concentration, 205fg [Leu(1)] MC-LR.cell(-1) expressed as MC-LR equivalent was measured in the beginning of the experiment and subsequently declined to 160fg.cell(-1) on day 2 and 70fg.cell(-1) on day 4 in cells exposed to 29°C. The same trend was observed for all other MCs except for the least abundant MC-LR which showed a continuous increase during exposure time. Our results suggest a high ability of M. aeruginosa to perceive ROS and to rapidly initiate antioxidant defenses with a differential response on MC production.

Effects of iron on growth, antioxidant enzyme activity, bound extracellular polymeric substances and microcystin production of Microcystis aeruginosa FACHB-905.

Toxic cyanobacterial blooms have occurred in various water bodies during recent decades and made serious health hazards to plants, animals and humans. Iron is an important micronutrient for algal growth and recently, the concentration of which has increased remarkably in freshwaters. In this paper, the cyanobacterium Microcystis aeruginosa FACHB-905 was cultivated under non-iron (0µM), iron-limited (10µM) and iron-replete (100µM) conditions to investigate the effects of iron on growth, antioxidant enzyme activity, EPS and microcystin production. The results showed that algal cell density and chlorophyll-a content were maximal at the highest iron concentration. Antioxidant enzymes activity increased notably under all three conditions in the early stage of experiment, of which the SOD activity recovered soon from oxidative stress in 10µM group. The productions of some protein-like substances and humic acid-like substances of bound EPS were inhibited in iron-containing groups in the early stage of experiment while promoted after the adaptation period of Microcystis aeruginosa. Iron addition is a factor affecting the formation of cyanobacterial blooms through its impact on the content of LB-EPS and the composition of TB-EPS. The intracellular MC-LR concentration and the productivity potential of MC-LR were the lowest in 0µM group and highest in 10µM group. No obvious extracellular release of MC-LR was observed during the cultivation time. Therefore, iron addition can promote the physiological activities of M. aeruginosa, but a greater harm could be brought into environment under iron-limited (10µM) condition than under iron-replete (100µM) condition.

Biochemical Dissection of the Natural Diversification of Microcystin Provides Lessons for Synthetic Biology of NRPS.

The cyanobacterial hepatotoxin microcystin is assembled at a non-ribosomal peptide synthetase (NRPS) complex. The enormous structural diversity of this peptide, which is also found in closely related strains, is the result of frequent recombination events and point mutations. Here, we have compared the in vitro activation profiles of related monospecific and multispecific modules that either strictly incorporate leucine or arginine or incorporate chemically diverse amino acids in parallel into microcystin. By analyzing di- and tri-domain proteins we have dissected the role of adenylation and condensation domains for substrate specificity. We have further analyzed the role of subdomains and provide evidence for an extended gatekeeping function for the condensation domains of multispecific modules. By reproducing natural point mutations, we could convert a monospecific module into a multispecific module. Our findings may inspire novel synthetic biology approaches and demonstrate how recombination platforms of NRPSs have developed in nature.