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.

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.

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.

The art of destruction: revealing the proteolytic capacity of bacterial caspase homologs.

Caspases are proteases that initiate and execute programmed cell death in animal tissues, thereby facilitating multicellular development and survival. While caspases are unique to metazoans and specifically cleave substrates at aspartic acid residues, homologs are found in protozoa, plants, algae, fungi, bacteria and archaea, and show specificity for basic residues. In this issue of Molecular Microbiology, Klemencic and colleagues present the first biochemical characterization of a bacterial caspase homolog, classified as an orthocaspase. By expressing the gene MaOC1 from the cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli, the authors discovered specificity for substrates with arginine in the P1 position. The protein requires autocatalytic processing to become active and is dependent on an intact histidine-cysteine dyad. These results significantly extend our knowledge of the specificities of bacterial caspase homologs, which are known to be highly diverse in protein domain architectures and active site mutations. Although bacterial programmed cell death is one possible area of action, the function of most bacterial caspase homologs remains unexplored. Cyanobacteria represent the best studied group in terms of prokaryotic caspase-like proteins both genomically and experimentally, and thereby provide a suitable platform for further investigations into activation, regulation and physiological roles of orthocaspases.

Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa.

Caspases are a family of cysteine-dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase-like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the kcat /KM value for Z-RR-AMC substrate of the order 10(6) M(-1) s(-1). In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase-like proteins, we suggest to name these evolutionary primitive proteins orthocaspases.

An antialgal compound produced by Streptomyces jiujiangensis JXJ 0074(T).

Previous investigations suggested that Streptomyces jiujiangensis JXJ 0074(T) can secrete antialgal compounds. In this study, an antialgal compound was isolated from the cultured broth of S. jiujiangensis JXJ 0074(T) by using bioassay methods. Based on spectroscopic data, the active compound was identified as 2'-deoxyadenosine, which exhibited a greater antialgal activity against cyanobacteria than its analogues such as adenosine, guanosine, and 2'-deoxyguanosine. The antialgal activity of 2'-deoxyadenosine increased with the content and time. 2'-Deoxyadenosine severely damaged the vegetative cells of cyanobacteria, causing crumpling, collapse, expanding, perforation, breakage of filamentous cyanobacteria, and decrease of the chlorophyll. However, 2'-deoxyadenosine seemed to have less impact on the morphology of heterocysts of filamentous cyanobacteria. The superoxide dismutase (SOD) activity in the treated cells of M. aeruginosa FACHB-905 initially increased with 31.14 ± 2.00% higher than that of the control after 36 h and then decreased quickly. On the same time, there were rapid increases in superoxide anion radical (O2 (-)) and malondialdehyde (MDA) contents with 315.53 ± 12.81 and 84.72 ± 6.15% higher than these of the controls at 60 h, respectively. The intracellular microcystin-LR (MC-LR) content in the treated cells of M. aeruginosa FACHB-905 increased by 36.34 ± 7.35% 1 day later, followed by a rapid decrease with a rate of 90.50 ± 1.08% 8 days later, while the extracellular MC-LR content showed no significant difference with the control. Five days after adding 15 µg/ml of 2'-deoxyadenosine to the culture of M. aeruginosa FACHB-905, there was no 2'-deoxyadenosine detected by HPLC, suggesting that 2'-deoxyadenosine completely degraded. This study provides a new clue to screen natural-based antialgal compounds from nucleoside analogues.

Inhibitory mechanisms of Acacia mearnsii extracts on the growth of Microcystis aeruginosa.

Our previous work revealed that Acacia mearnsii extract can inhibit the growth of Microcystis aeruginosa, the common species forming toxic cyanobacterial blooms in eutrophic freshwater. In the present study, we demonstrated that this plant extract can significantly increase cell membrane permeability and Ca²âº/Mg²âº-ATPase activity on the membrane. Long-term exposure to concentrations of 20 ppm A. mearnsii extract led to algal cell membrane leakage or even lysis. Comparison of expression of three photosynthesis-related genes (rbcL, psaB and psbD) in M. aeruginosa with and without plant extract treatment revealed that their expression was remarkably reduced in the presence of the extract. Down-regulation of photosynthesis-related genes could indicate the inhibition of the photosynthetic process. Thus, our results suggested that both photosynthetic systems and membranes of M. aeruginosa are potentially damaged by A. mearnsii extract.

Structural and biochemical analyses of Microcystis aeruginosa O-acetylserine sulfhydrylases reveal a negative feedback regulation of cysteine biosynthesis.

O-acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis from O-acetylserine (OAS) and inorganic sulfide in plants and bacteria. Bioinformatics analyses combined with activity assays enabled us to annotate the two putative genes of Microcystis aeruginosa PCC 7806 to CysK1 and CysK2, which encode the two 75% sequence-identical OASS paralogs. Moreover, we solved the crystal structures of CysK1 at 2.30Ǻ and cystine-complexed CysK2 at 1.91Ǻ, revealing a quite similar overall structure that belongs to the family of fold-type II PLP-dependent enzymes. Structural comparison indicated a significant induced fit upon binding to the cystine, which occupies the binding site for the substrate OAS and blocks the product release tunnel. Subsequent enzymatic assays further confirmed that cystine is a competitive inhibitor of the substrate OAS. Moreover, multiple-sequence alignment revealed that the cystine-binding residues are highly conserved in all OASS proteins, suggesting that this auto-inhibition of cystine might be a universal mechanism of cysteine biosynthesis pathway.

Antioxidant responses and degradation of two antibiotic contaminants in Microcystis aeruginosa.

Cyanobacteria may interact with antibiotic contaminants in aquatic environments, but the interaction effects and mechanisms remain unclear. In the present study, aqueous culture of Microcystis aeruginosa was exposed to 50ng/l-1µg/l of spiramycin and amoxicillin for seven days. The influences of antibiotics on the antioxidant system of M. aeruginosa and the degradation of antibiotics by M. aeruginosa were investigated. The activities of superoxide dismutase (SOD) in spiramycin-treated M. aeruginosa were stimulated by up to 2.2 folds, while the activities of peroxidase (POD) and catalase (CAT) were inhibited by spiramycin at test concentrations of 500ng/l-1µg/l, with a decrease of up to 71% and 76% compared to the control, respectively. The activities of SOD, POD and CAT in M. aeruginosa were stimulated by amoxicillin during the whole exposure period, with respective increases of up to 60%, 30% and 120% relative to the control. At test concentrations of 500ng/l-1µg/l, the higher MDA contents in spiramycin-treated M. aeruginosa indicated a higher toxicity of spiramycin than amoxicillin, possibly due to the accumulation of hydrogen peroxide caused by the inhibited activities of POD and CAT under exposure to spiramycin. The increase of glutathione content, the stimulation of glutathione S-transferase activity and the degradation of each antibiotic were observed in M. aeruginosa during the 7-day exposure. At the end of exposure, 12.5%-32.9% of spiramycin and 30.5%-33.6% of amoxicillin could be degraded by M. aeruginosa from the culture medium, indicating the ability of M. aeruginosa to eliminate coexisting contaminants via detoxification.

Biochemical and genetic alterations in the freshwater neotropical fish Prochilodus lineatus after acute exposure to Microcystis aeruginosa

Microcystins are secondary metabolites produced by different species of cyanobacteria, such as Microcystis aeruginosa (MA). In this study, the biochemical and genetic effects of lyophilized MA were evaluated in the neotropical fish Prochilodus lineatus exposed to 1 or 2 mg L-1 lyophilized MA (treated group) or only water (control group) in static toxicity tests for 24 and 96 h. The gills and liver were used in the analysis of biotransformation enzymes and antioxidant defenses, blood and gill cells in genetic analysis and in brain and muscle it was determined the activity of acetylcholinesterase (AChE). The results showed the biotransformation pathway activation due to the increase in hepatic CYP1A and in branchial and hepatic glutathione S-transferase (GST). The antioxidant defense proved to be greatly affected by MA exposure leading to changes, both in gills and liver, in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and in the content of tripeptide glutathione (GSH). Lipid peroxidation was not detected, but damage to DNA molecule was observed in blood cells. In conclusion, it can be state the lyophilized MA is able to promote changes in the biochemical and genetic parameters of P. lineatus.

As microcistinas são metabólitos secundários produzidos por diferentes espécies de cianobactérias, como a Microcystis aeruginosa (MA). Neste estudo, os efeitos bioquímicos e genéticos de liofilizado de MA foram avaliados para juvenis da espécie de peixe Neotropical Prochilodus lineatus expostos a 1 ou 2 mg L-1 de liofilizado de MA (grupo tratado) ou apenas à água (grupo controle), em testes de toxicidade estáticos, durante 24 e 96 h. As brânquias e o fígado foram usados para as análises das enzimas de biotransformação e defesas antioxidantes, células do sangue e das brânquias para análises genéticas e no cérebro e músculo foi determinada a atividade da acetilcolinesterase (AChE). Os resultados mostraram ativação da via de biotransformação devido ao aumento da atividade da CYP1A hepática e da atividade da glutationa S-transferase (GST) hepática e branquial. As defesas antioxidantes foram muito afetadas pela exposição a MA levando a alterações, tanto no fígado como nas brânquias, na atividade da superóxido dismutase (SOD), da catalase (CAT), da glutationa peroxidase (GPx), glutationa redutase (GR) e no conteúdo do tripeptídeo glutationa (GSH). Apesar dessas alterações a peroxidação lipídica não foi detectada em nenhum dos tecidos, mas danos na molécula de DNA foram observados nas células do sangue. Em conclusão, pode-se afirmar que o liofilizado de MA é capaz de promover alterações em parâmetros bioquímicos e genéticos de P. lineatus.

Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase.

To improve phosphorus removal from wastewater, we constructed a high-phosphate-accumulating microorganism, KTPPK, using Pseudomonas putida KT2440 as a host. The expression plasmid was constructed by inserting and expressing polyphosphate kinase gene (ppk) from Microcystis aeruginosa NIES-843 into broad-host-range plasmid, pBBR1MCS-2. KTPPK was then added to a sequencing batch biofilm reactor (SBBFR) using lava as a biological carrier. The results showed that SBBFR with KTPPK not only efficiently removed COD, NH(3)-N, and NO(3)(-)-N but also had a high removal capacity for PO(4)(3-)-P, resulting in a low phosphorus concentration remaining in the outflow of the SBBFR. The biofilm increased by 30-53% on the lava in the SBBFR that contained KTPPK after 11 days when compared with the reactor that contained P. putida KT2440. Real-time quantitative polymerase chain reaction confirmed that the copy of ppk was maintained at about 3.5 × 10(10) copies per µL general DNA in the biofilm after 20 days. Thus, the transgenic bacteria KTPPK could maintain a high density and promote phosphorus removal in the SBBFR. In summary, this study indicates that the use of SBBFR with transgenic bacteria has the potential to remove phosphorus and nitrogen from wastewater.

The cyanobacterial hepatotoxin microcystin binds to proteins and increases the fitness of microcystis under oxidative stress conditions.

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 ΔmcyB 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.

Oxidative damage and antioxidant responses in Microcystis aeruginosa exposed to the allelochemical berberine isolated from golden thread.

Berberine, extracted from golden thread (Coptis chinensis Franch), is an allelochemical exhibiting inhibitory effects on the growth of Microcystis aeruginosa. Berberine-induced oxidative damage and antioxidant responses in M. aeruginosa cells were investigated to elucidate the mechanisms involved in berberine inhibition on algal growth. Malondialdehyde content in M. aeruginosa cells exposed to berberine increased with increased exposure concentration and the prolongation of exposure time. The same changes were observed in O(2)(-) activity of M. aeruginosa cells exposed to berberine. Berberine upregulated superoxide dismutase (SOD) activity at low concentrations while downregulating it at high concentrations. SOD activity transitioned from an increase to a decrease from 0 to 72h exposure to 0.10% berberine. We observed that berberine exposure increased glutathione content in M. aeruginosa cells. The results suggested that berberine-induced oxidative damage might be at least partially responsible for berberine inhibition on M. aeruginosa growth.

Responses of enzymatic antioxidants and non-enzymatic antioxidants in the cyanobacterium Microcystis aeruginosa to the allelochemical ethyl 2-methyl acetoacetate (EMA) isolated from reed (Phragmites communis).

Macrophytic allelochemicals are considered an environment-friendly and promising alternative to control algal bloom. However, studies examining the potential mechanisms of inhibitory allelochemicals on algae are few. The allelochemical ethyl 2-methyl acetoacetate (EMA), isolated from reed (Phragmites communis), was a strong allelopathic inhibitor on the growth of Microcystis aeruginosa. EMA-induced antioxidant responses were investigated in the cyanobacterium M. aeruginosa to understand the mechanism of EMA inhibition on algal growth. The activities of enzymatic antioxidants superoxide dismutase (SOD) and catalase (CAT), and the contents of non-enzymatic antioxidants reduced glutathione (GSH) and ascorbic acid (AsA) of M. aeruginosa cells were analyzed after treatments with different concentrations of EMA. Exposure of M. aeruginosa to EMA caused changes in enzyme activities and contents of non-enzymatic antioxidants in different manners. The decrease in SOD activity occurred first after 4 h of EMA exposure, and more markedly after 40 h. CAT activity did not change after 4 h of EMA exposure, but increased obviously after 40 h. The contents of AsA and GSH were increased greatly by EMA after 4 h. After 60 h, low EMA concentrations still increased the CAT activity and the contents of AsA and GSH, but high EMA concentrations started to impose a marked suppression on them. EMA increased dehydroascorbate (DHAsA) and oxidized glutathione (GSSG) contents during all exposure times. After 60 h, the regeneration rates of AsA and GSH (represented by the AsA/DHAsA ratio and GSH/GSSG ratio, respectively) were reduced by high EMA concentrations. These results suggest that the activation of CAT and the availability of AsA and GSH at early exposure are important to counteract the oxidative stress induced by EMA, and the inactivation of SOD may be crucial to the growth inhibition of M. aeruginosa by EMA.

Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M. aeruginosa.

The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (>60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (>30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes.

Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system.

BACKGROUND: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp.

The unicellular cyanobacterium Microcystis aeruginosa K-81 has two types of restriction barrier, an extracellular nuclease and sequence-specific endonucleases. The nuclease was detected in the culture supernatant and it was easily released from the cells by washing with water or buffer containing Triton X-100. This nuclease was identified as a polypeptide of about 28 kDa that digested covalently closed circular and linear double-stranded DNAs, including chromosomal DNA from M. aeruginosa K-81. Among another 13 Microcystis strains examined, 3 produced an extracellular nuclease. Furthermore, M. aeruginosa K-81 contained two sequence-specific endonucleases, MaeK81I and MaeK81II, which were isoschizomers of SplI and Sau96I, respectively.