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Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis...
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... a PCR template was prepared by rapid cell lysis in the presence of a cation-exchange resin and nonionic detergents (24). PCR annealing step temperatures are shown in Table 1 along with peptide synthetase gene-directed oligonucleotide primer sequences. In capillary or 200-l tubes, the PCR thermal cycling protocol included an initial denaturation at 94°C for 2 min, followed by 35 cycles at 93°C for 10 s, at the annealing temperature (Table 1) for 20 s, and at 72°C for 1 min. ...
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... annealing step temperatures are shown in Table 1 along with peptide synthetase gene-directed oligonucleotide primer sequences. In capillary or 200-l tubes, the PCR thermal cycling protocol included an initial denaturation at 94°C for 2 min, followed by 35 cycles at 93°C for 10 s, at the annealing temperature (Table 1) for 20 s, and at 72°C for 1 min. Amplification reaction components were as previously described (23), and incubations were performed with an FTS-1S capillary thermocycler (Corbett Research, Sydney, Australia) or a PE2400 apparatus (Perkin-Elmer Cetus Corporation, Emeryville, Calif.). ...
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... method of semidegenerate, long PCR was developed to extend sequence information flanking that obtained from the gene libraries. This procedure used the highly redundant primers MTF2 and MTR (Table 1) di- rected to the conserved motifs of known peptide synthetase genes combined with primers specific for the Microcystis genome (19). By modifying and using long- PCR protocols, we found it possible to amplify regions of DNA encoding entire modules of the synthetase. ...
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... resulting PCR product did, however, contain a conserved core sequence (core motif II) which was used to align DNA se- quences for further phylogenetic analyses (Fig. 1B). Specific amplification primers (for MS-PCR) based on the character- ized peptide synthetase gene sequence of M. aeruginosa (4) were designed and directed to the region between conserved peptide synthetase motifs I and III (Fig. 1B and Table 1). The priming sites for MS-PCR did not have a sequence identical in the two Microcystis strains, PCC7806 and HUB524, and were selected to enable amplification of a microcystin synthetase fragment from a broad range of microcystin-producing cya- nobacteria. ...
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... Se utilizaron dos pares de primers específicos para la amplificación de genes de los NRPS y PKS. Estos cebadores apuntan directamente a los dominios de adenilación (A) y cetosintasa (KS), respectivamente: DKF (GTGCCGGTNCCRTGNGYYTC) y DKR GCGATGGAYCCNCARCARMG) para PKS (Moffitt y Neilan, 2001) y MTF2 (GCNGGYGGYGCNTAYGTNCC) y MTR (CCNCGDATYTTNACYTG) para NRPS (Neilan et al., 1999). El ADN de una cepa de Microcystis aeruginosa (LEGE 00063, CIIMAR) se utilizó como control positivo para la presencia de los genes de los dominios de los NRPS y PKS. ...
El presente trabajo colectó, aisló, identificó y caracterizó el potencial antimicrobiano de dos especies filamentosas de cianobacterias de la Amazonia colombiana. Se utilizaron técnicas microbianas de rayado en medio sólido y dilución para obtener las cepas con una sola especie. La identificación se realizó por caracteres morfológicos y secuenciado de la sección conservada Cya de gen 16s. Se identificaron por reacción en cadena de la polimerasa (PCR) la presencia de genes que secuencian policétidos sintasas (PKS) y péptidos no ribosomales sintetasas (NRPS) asociados a rutas productoras de metabolitos secundarios. Se evaluó la actividad antimicrobiana de extractos polares y no polares con una batería Gram positiva, dos Gram negativas y una cepa fúngica. Se identificó la cepa 5 como Limnothrix vacuolifera (Skuja) Komárek y la cepa 9 como Lymnothix planktonica (Wołoszyńska) Meffert. En las dos cepas se encontraron PKS y NRPS. Finalmente, los extractos polares inhibieron el crecimiento de Enterococcus faecalis, Escherichia coli y Klebsiella pneumoniae y una cepa Penicillium sp. Los extractos no polares no mostraron actividad antimicrobiana. Es el primer registro de estas para la Amazonia colombiana y el primer registro de actividad antibiótica de cianobacterias continentales colombianas.
... Present taxa above this 5 % threshold identified as nitrogen fixers and possible producers of microcystins included Aphanizomenon, Anabaena, Leptolyngbya, Nostoc, and Pseudanabaena (Horne, 1979;Lindberg et al., 2004;Halinen et al., 2007;Kurmayer, 2011;Cires and Ballot, 2016;Tsuijimoto et al., 2016;Han et al., 2018;Eigemann et al., 2019). Nitrogen fixers were identified as Cuspidothrix, Cylindrospermopsis, and Spaerospermopsis (Neilan et al., 1999;Willis et al., 2016;Lorenzi et al., 2021). Potential producers of microcystins were identified as Cyanobium, Limnothrix, Microcystis, Phormidium, Planktothrix, Snowella, and Synechococcus (Christiansen et al., 2003;Echenique et al., 2014;Huisman et al., 2018;Oliveira et al., 2019;Pagliara et al., 2021). ...
... (Minamoto et al., 2021). 추출이 완료된 시료는 Table 1과 같다 (Nübel et al., 1997;Neilan et al., 1999;Tsao et al., 2014;Kim et al., 2020). PCR 증폭은 Thermal cycler (A300, (a) (b) 김건희·유경은·호혜인·박채홍·김현진·황순진 100 (Bergaya and Lagaly, 2013;Landman et al., 2014) ...
Cyanobacterial resting cells, such as akinetes, are important seed cells for cyanobacteria's early development and bloom. Due to their importance, various methods have been attempted to isolate resting cells present in the sediment. Ludox is a solution mainly used for cell separation in marine sediments, but finding an accurate method for use in freshwater is difficult. This study compared the two most commonly used Ludox methods (direct sediment treatment and sediment distilled water suspension treatment). Furthermore, we proposed a highly efficient method for isolating cyanobacterial resting cells and eDNA amplification from freshwater sediments. Most of the resting cells found in the sediment were akinete to the Nostocale and were similar to those of Dolichospermum, Cylindrospermum, and Aphanizomenon. Twenty times more akinetes were found in the conical tube column using the sediment that had no treatment than in the sample treated by suspending the sediment in distilled water. Akinete separated through Ludox were mainly spread over the upper and lower layers in the column rather than concentrated at a specific depth in the column layer. The mibC, Geo, and 16S rDNA genes were successfully amplified using the sediment directly in the sample. However, the amplification products of all genes were not found in the sample in which the sediment was suspended in distilled water. Therefore, 5 g to 10 g of sediment is used without pretreatment when isolating cyanobacterial resting cells from freshwater sediment. Cell isolation and gene amplification efficiency are high when four times the volume of Ludox is added. The Ludox treatment method presented in this study isolates cyanobacterial resting cells in freshwater sediment, and the same efficiency may not appear in other biotas. Therefore, to apply Ludox to the separation of other biotas, it is necessary to conduct a pre-experiment to determine the sediment pretreatment method and the water layer where the target organism exists.
... Non-ribosomal peptides (NRPs) are synthetized by a specific nonribosomal peptide synthetase (NRPS) enzyme complex in an RNAindependent synthetic pathway (Neilan et al., 1999). NRPs are small molecular weight cyclic hepta-and penta-peptides (Carmichael et al., 1988), among which microcystins (MCYSTs) and nodularins (NODLNs) are the class leaders. ...
Cyanotoxins are by definition "harmful agents" produced by cyanobacteria. Their toxicity has been extensively studied and reviewed over the years. Cyanotoxins have been commonly classified, based on their poisonous effects on mammals, into three main classes, neurotoxins, hepatotoxins and dermatotoxins, and, considering their chemical features, mainly identified as peptides, alkaloids and lipopolysaccharides. Here we propose a broader subdivision of cyanotoxins into eight distinct classes, taking into account their molecular structures, biosynthesis and modes of action: alkaloids, non-ribosomal peptides, polyketides, non-protein amino acids, indole alkaloids, organophosphates, lipopeptides and lipoglycans. For each class, the structures and primary mechanisms of toxicity of the main representative cyanotoxins are reported. Despite their powerful biological activities, only recently scientists have considered the biotechnological potential of cyanotoxins, and their applications both in medical and in industrial settings, even if only a few of these have reached the biotech market. In this perspective, we discuss the potential uses of cyanotoxins as anticancer, antimicrobial, and biocidal agents, as common applications for cytotoxic compounds. Furthermore, taking into account their mechanisms of action, we describe peculiar potential bioactivities for several cyanotoxin classes, such as local anaesthetics, antithrombotics, neuroplasticity promoters, immunomodulating and antifouling agents. In this review, we aim to stimulate research on the potential beneficial roles of cyanotoxins, which require interdisciplinary cooperation to facilitate the discovery of innovative biotechnologies.
... The mcy cluster in Microcystis aeruginosa PCC 7806 (Microcystis 7806) spans a 55-kb region containing 10 genes, mcyA-J. MCs is a typical nonribosomal peptides (NRPs) and is catalyzed by a hybrid enzyme comprising polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) (Moore et al., 1991;Arment & Carmichael, 1996;Dittmann et al., 1997;Neilan et al., 1999). A bidirectional promoter is present between mcyA and mcyD, and the mcy cluster is divided into two operons (mcyA-C and mcyD-J; Kaebernick et al., 2002). ...
Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. Microcystin‐LR (MC‐LR) produced by Microcystis aeruginosa is the most common cyanotoxins. Due to the limitation of isolation, purification, and genetic manipulation techniques, it is difficult to study and verify in situ the biosynthetic pathways and molecular mechanisms of MC‐LR.
We reassembled the biosynthetic gene cluster (mcy cluster) of MC‐LR in vitro by synthetic biology, designed and constructed the strong bidirectional promoter biPpsbA2, transformed it into Synechococcus 7942, and successfully expressed MC‐LR at a level of 0.006–0.018 fg cell⁻¹ d⁻¹.
We found the expression of MC‐LR led to abnormal cell division and cellular filamentation, further using various methods proved that by irreversibly competing its GTP‐binding site, MC‐LR inhibits assembly of the cell division protein FtsZ.
The study represents the first reconstitution and expression of the mcy cluster and the autotrophic production of MC‐LR in model cyanobacterium, which lays the foundation for resolving the microcystins biosynthesis pathway. The discovered role of MC‐LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their nonblooming counterparts.
... strain AE01, AE02, and Microcystis sp. AE03 was analyzed through PCR amplification of mcyB, anaC, and pks gene cluster, which are indicative of the presence of microcystins, anatoxins, and cylindropsermopsins biosynthesis genes clusters, respectively (Neilan et al., 1999;Fergusson and Saint, 2003;Rantala-Ylinen et al., 2011). The primers listed in Table 1 were used to amplify these cyanotoxin biosynthetic gene clusters. ...
... The primers listed in Table 1 were used to amplify these cyanotoxin biosynthetic gene clusters. The amplification of mcyB gene was carried out by using gene-specific primer sets (FAA/RAA) (Neilan et al., 1999). The thermal cycler was set at an initial denaturation at 94 • C for 3 min, followed by 30 cycles of amplification: 30 s at 95 • C, 30 s at 43 • C, 1 min at 72 • C, and final elongation for 7 min at 72 • C. For pks amplification, a conventional PCR was developed by using the primer sets (pksK18/pksM4) (Fergusson and Saint, 2003). ...
... The "mcyB" gene is part of the MC biosynthetic gene cluster encoding a "peptide synthetase" involved in MC biosynthesis. The peptide synthetase contains two modules with each module performing several processing functions such as, adenylation, thiolation, and condensation (Neilan et al., 1999;Tillett et al., 2000). The encoded products are low molecular weight cyclic heptapeptides known for their toxic effects on liver and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. ...
Cyanobacterial harmful algal blooms (CHABs) are increasing at an alarming rate in different water bodies worldwide. In India, CHAB events in water bodies such as Dal Lake have been sporadically reported with no study done to characterize the cyanobacterial species and their associated toxins. We hypothesized that this Lake is contaminated with toxic cyanobacterial species with the possibility of the presence of cyanotoxin biosynthetic genes. We, therefore, used some of the molecular tools such as 16S ribosomal DNA, PCR, and phylogenetic analysis to explore cyanobacterial species and their associated toxins. A 3-year (2018–2020) survey was conducted at three different sampling sites of Dal Lake namely, Grand Palace Gath (S1), Nigeen basin (S2), and Gagribal basin (S3). Two strains of Dolichospermum sp. AE01 and AE02 (S3 and S1 site) and one strain of Microcystis sp. AE03 (S2 site) was isolated, cultured, and characterized phylogenetically by 16S ribosomal DNA sequencing. The presence of cyanotoxin genes from the isolates was evaluated by PCR of microcystins (mcyB), anatoxins (anaC), and cylindrospermopsins (pks) biosynthesis genes. Results revealed the presence of both mcyB and pks gene in Microcystis sp. AE03, and only anaC gene in Dolichospermum sp. AE02 strain. However, Dolichospermum sp. AE01 strain was not found to harbor any such genes. Our findings, for the first time, reported the coexistence of pks and mcyB in a Microcystis AE03 strain. This study has opened a new door to further characterize the unexplored cyanobacterial species, their associated cyanotoxin biosynthetic genes, and the intervention of high-end proteomic techniques to characterize the cyanotoxins.
... MCs are synthesized nonribosomally by an enzyme complex consisting of 9 or 10 genes, depending on the genus [16][17][18]. These complexes are responsible for the synthesis of the molecular core of all microcystin congeners that a species can produce [16], while the various domains within this complex determine the microcystin congeners being produced [19]. P. agardhii and P. rubescens harmful algal blooms tend to have more microcystin per unit of cyanobacterial biomass than blooms dominated by other microcystin producing species [20]. ...
Planktothrix agardhii is a filamentous cyanobacterial species that dominates harmful algal blooms in Sandusky Bay, Lake Erie and other freshwater basins across the world. P . agardhii isolates were obtained from early (June) blooms via single filament isolation; eight have been characterized from 2016, and 12 additional isolates have been characterized from 2018 for a total of 20 new cultures. These novel isolates were processed for genomic sequencing, where reads were used to generate scaffolds and contigs which were annotated with DIAMOND BLAST hit, Pfam, and GO. Analyses include whole genome alignment to generate phylogenetic trees and comparison of genetic rearrangements between isolates. Nitrogen acquisition and metabolism was compared across isolates. Secondary metabolite production was genetically explored including microcystins, two types of aeruginosin clusters, anabaenopeptins, cyanopeptolins, microviridins, and prenylagaramides. Two common and 4 unique CRISPR-cas islands were analyzed for similar sequences across all isolates and against the known Planktothri x-specific cyanophage, PaV-LD. Overall, the uniqueness of each genome from Planktothrix blooms sampled from the same site and at similar times belies the unexplored diversity of this genus.
... For the detection of biosynthetic gene clusters, PCR amplification was performed with the degenerate primers MDPQQRf (5 -RTRGAYCCNCAGCAICG-3 ) and HGTGTr (5 -VGTNCCNGTGCCRTG-3 ) aimed at the α-keto synthase of PKS-I motif [37] and MTRF2 (5 -GCNGG(C/T)GG(C/T)GCNTA(C/T)GTNCC-3 ) and DKF (5 -GTGCCGGTNCCRTGNGY-YTC-3 ) for core motif-V of NRPS [38]. PCR protocol was performed as described by Graça et al. [39]. ...
Oceans hold a stunning number of unique microorganisms, which remain unstudied by culture-dependent methods due to failures in establishing the right conditions for these organisms to grow. In this work, an isolation effort inspired by the iChip was performed using marine sediments from Memoria beach, Portugal. The isolates obtained were identified by 16S rRNA gene analysis, fingerprinted using BOX-PCR and ERIC-PCR, searched for the putative presence of secondary metabolism genes associated with polyketide synthase I (PKS-I) and non-ribosomal peptide synthetases (NRPS), screened for antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, and had bioactive extracts dereplicated by LC/HRMS. Of the 158 isolated strains, 96 were affiliated with the phylum Actinomycetota, PKS-I and NRPS genes were detected in 53 actinomycetotal strains, and 11 proved to be bioactive (10 against E. coli, 1 against S. aureus and 1 against both pathogens). Further bioactivities were explored using an “one strain many compounds” approach, with six strains showing continued bioactivity and one showing a novel one. Extract dereplication showed the presence of several known bioactive molecules and potential novel ones in the bioactive extracts. These results indicate the use of the bacteria isolated here as sources of new bioactive natural products.
... To amplify β-ketosynthase (KS) domain fragments in Type I Polyketide synthase (PKS-I) genes (Donadio et al. 2007, Jenke-Kodama et al. 2005, the degenerate primers MDPQQRf (5 -RTRGAYCCNCAGCAICG-3 ) and HGTGTr (5 -VGTNCCNGTGCCRTG-3 ) were used (Kim et al. 2005). To amplify non-ribosomal peptide synthetases (NRPS) genes (Wang et al. 2014 (Neilan et al. 1999). The PCR reaction was performed as described previously (Graça et al. 2016, Almeida et al. 2020 in the equipment MyCycler Thermo Cycler (Bio-Rad). ...
The discovery of new bioactive compounds is an invaluable aid to the development of new drugs. Strategies for finding novel molecules can focus on the exploitation of less studied organisms and ecosystems such as planctomycetes and brackish habitats. The unique cell biology of the underexplored Planctomycetota mean it is of particular interest. In this study, we aimed to isolate planctomycetes from the estuary of the Tejo river (Portugal). To reach this goal, macroalgae, water and sediments were sampled and diverse media and isolation techniques applied. Sixty-nine planctomycetal strains were brought into pure culture. An analysis of the 16S rRNA genes found that the majority of the isolates were affiliated to the genus Rhodopirellula. Putative novel taxa belonging to genera Stieleria and Rhodopirellula were also isolated and characterized morphologically. Enterobacterial Repetitive Intergenic Consensus fingerprinting analyses showed higher diversity and different genotypes within close strains. Relevant biosynthetic gene clusters were found in most isolates and acetone extracts from representative strains exhibited mild antimicrobial activities against Escherichia coli and Staphylococcus aureus. Our work has not only enlarged the number and diversity of cultured planctomycetes but also shown the potential for the discovery of bioactive compounds from the novel taxa.
... Among the microbes, the easily evolving bacteria especially possess the diverse ability to produce the diverse secondary metabolites through their biosynthesis pathways. Genome-wide studies revealed that the bacteria species could synthesize groups of secondary metabolites including nonribosomal peptides (NRP), polyketides (PK), terpene (TP), and hybrid PK/NRP [5][6][7]. e bacteria use secondary metabolite as biological weapon against the other species in the same niche; protect themselves from physical environment, communication substances, and symbiosis stimulant, or even are hormones or pheromones to other organisms. Accordingly, novel secondary metabolites identified from the unexplored bacterial strains might be used as beneficial compounds for both industry and medical applications [8,9]. ...
... Diversity of NRPS, PKS, and TPS genes among isolated bacterial genomes was inspected using previously designed degenerate primer pairs. MTF2 (5′-GCNGGYG-GYGCNTAYGTNCC-3′) and MTR (5′-CCNCGDA-TYTTNACYTG-3′) (annealing temperature at 45°C) were used for amplification of A domain of NRPS gene [6]. KS-F (5′-CGCTCCATGGAYCCSCARCA-3′) and KS-R (5′-GTCCCGGTGCCRTGSSHYTCSA-3′) (annealing temperature at 50°C) were used for amplification of KS domain of PKS gene [7]. ...
Thailand was proposed to be rich unexplored source of microorganisms, especially bacterial strains. There should be bacteria with high secondary metabolite production potential in the natural resources that are still unidentified. Moreover, they might not produce secondary metabolites in standard laboratory culture condition after isolation, in which coculture condition would help us pursuing the bacteria to produce bioactive metabolites. Here, we aimed to identify new bacterial strains with high secondary metabolite production potential from Thailand’s natural resources. To achieve the goal, we performed bacteria isolation, phylogenetic analysis, degenerate PCR of secondary metabolism genes, cocultivation, antibacterial analysis, and HPLC chemical profiling. We isolated distinct 40 bacterial strains, which have over 98% 16S rRNA sequence similarity with known species. There were 22, 31, and 29 strains giving positive PCR amplification of NRPS, PKS, and TPS genes, respectively. Among them, Bacillus licheniformis RSUCC0101 had the highest number of PCR products, 26. In standard single culture condition, crude extracts prepared from Bacillus safensis RSUCC0021 and Bacillus amyloliquefaciens RSUCC0282 could inhibit the growth of Staphylococcus aureus ATCC25923. Furthermore, the cocultivation and HPLC analyses showed that the extracts prepared from 3 pairs of culture between Staphylococcus sp. RSUCC0020, Micrococcus luteus RSUCC0053, Staphylococcus sp. RSUCC0087, and Staphylococcus pasteuri RSUCC0090 could inhibit the growth of Staphylococcus aureus ATCC25923 and produced distinct chemical profiles from their single culture condition. Our study led to the isolation and identification of several promising bacterial strains for production of secondary metabolites that might be useful in biomedical applications.
