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1.
BMC Microbiol ; 20(1): 35, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32070286

RESUMO

BACKGROUND: Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. RESULTS: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5' RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions. CONCLUSIONS: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

2.
ACS Synth Biol ; 8(5): 1187-1194, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31042359

RESUMO

The microcystins are a large group of cyclic peptide hepatotoxins produced by several genera of freshwater cyanobacteria. The genes responsible for microcystin biosynthesis are encoded within a large (∼55 kbp) gene cluster, mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [d-Asp3, DMAdda5]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.

3.
FEMS Microbiol Ecol ; 95(4)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30848780

RESUMO

The severity of environmental conditions at Earth's frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils.

4.
Environ Microbiol ; 21(4): 1211-1223, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30689271

RESUMO

The cyanobacterium Raphidiopsis raciborskii is of environmental and social concern in view of its toxicity, bloom-forming characteristics and increasingly widespread occurrence. However, while availability of macronutrients and micronutrients such as N and Fe are critically important for the growth and metabolism of this organism, the physiological response of toxic and non-toxic strains of R. raciborskii to varying Fe and N availabilities remains unclear. By determining physiological parameters as a function of Fe and N availability, we demonstrate that R. raciborskii growth and N2 -fixing activity are facilitated at higher Fe availability under N2 -limited conditions with faster growth of the CS-506 (cylindrospermopsin-producing) strain compared with that of CS-509 (the non-toxic) strain. Radiolabelled Fe uptake assays indicated that R. raciborskii acclimated under Fe-limited conditions acquires Fe at significantly higher rates than under Fe replete conditions, principally via unchelated Fe(II) generated as a result of photoreduction of complexed Fe(III). While N2 -fixation of both strains occurred during both day and night, the CS-506 strain overall exhibited higher N2 -fixing and Fe uptake rates than the CS-509 strain under N-deficient and Fe-limited conditions. The findings of this study highlight that Fe availability is of significance for the ecological advantage of CS-506 over CS-509 in N-deficient freshwaters.

5.
Toxicon ; 158: 1-7, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30471380

RESUMO

To date Paralytic shellfish toxin (PST) variants in cyanobacteria have primarily been characterized using high performance liquid chromatography coupled with fluorescence detection. In this study we re-evaluated the PST profiles of five cyanobacterial cultures (Dolichospermum circinale AWQC131C, Aphanizomenon sp. NH-5, Raphidiopsis raciborskii T3, Scytonema cf. crispum CAWBG524 and CAWBG72) and one environmental sample (Microseria wollei) using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 35 different PST variants were detected. D. circinale contained the highest number of variants (23), followed by S. cf. crispum CAWBG72 (21). Many of the variants detected in the cultures/environmental sample had not been reported from these strains previously: D. circinale (14 variants), S. cf. crispum CAWBG72 (16), S. cf. crispum CAWBG524 (9), Aphanizomenon sp. (9), R. raciborskii (7), and M. wollei (7). Of particular interest was the detection of M-toxins (Aphanizomenon sp., R. raciborskii, D. circinale). These have previously only been identified from shellfish where they were thought to be metabolites. Well-characterized PST variant profiles are essential for research investigating the genetic basis of PST production, and given that the toxicity of each variants differs, it will assist in refining risk assessments.


Assuntos
Cianobactérias/química , Toxinas Marinhas/análise , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas em Tandem/métodos
6.
Environ Microbiol ; 21(2): 702-715, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30589201

RESUMO

Shark Bay, Western Australia is a World Heritage area with extensive microbial mats and stromatolites. Microbial communities that comprise these mats have developed a range of mitigation strategies against changing levels of photosynthetically active and ultraviolet radiation, including the ability to biosynthesise the UV-absorbing natural products scytonemin and mycosporine-like amino acids (MAAs). To this end, the distribution of photoprotective pigments within Shark Bay microbial mats was delineated in the present study. This involved amplicon sequencing of bacterial 16S rDNA from communities at the surface and subsurface in three distinct mat types (smooth, pustular and tufted), and correlating this data with the chemical and molecular distribution of scytonemin and MAAs. Employing UV spectroscopy and MS/MS fragmentation, mycosporine-glycine, asterina and an unknown MAA were identified based on typical fragmentation patterns. Marker genes for scytonemin and MAA production (scyC and mysC) were amplified from microbial mat DNA and placed into phylogenetic context against a broad screen throughout 363 cyanobacterial genomes. Results indicate that occurrence of UV screening compounds is associated with the upper layer of Shark Bay microbial mats, and the occurrence of scytonemin is closely dependent on the abundance of cyanobacteria.

7.
Nat Prod Rep ; 36(8): 1117-1136, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30556569

RESUMO

Covering: up to 2018 Marine and freshwater cyanobacteria produce a variety of toxic compounds that pose a threat to the health of humans, livestock and natural ecosystems world-wide. Significant research efforts have been directed towards understanding the biosynthesis of these cyanotoxins in an attempt to reduce their deleterious effects on water quality and, more recently, to harness their biotechnological potential. While a variety of complementary methods (such as bioinformatic analyses and isotope feeding studies) have been employed over the last three decades to address knowledge gaps in this field, this review focuses on the utility of heterologous expression and biochemical studies, including emerging technologies for engineering and expressing complete cyanotoxin gene clusters.

8.
ACS Chem Biol ; 13(11): 3107-3114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30296060

RESUMO

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.


Assuntos
Família Multigênica , Neurotoxinas/classificação , Neurotoxinas/genética , Saxitoxina/classificação , Saxitoxina/genética , Cianobactérias/genética , Dioxigenases/genética , Genes Bacterianos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Neurotoxinas/química , Fosfoadenosina Fosfossulfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Ligação Proteica , Saxitoxina/análogos & derivados , Saxitoxina/síntese química , Saxitoxina/química , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismo , Transposases/genética
9.
Nat Commun ; 9(1): 3096, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30082706

RESUMO

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed "sequins" (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as reference standards to aid in metagenomic studies.


Assuntos
Metagenoma , Metagenômica , Análise de Sequência de DNA , DNA Bacteriano/análise , Biblioteca Gênica , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Biológicos , Nanoporos , Filogenia , Padrões de Referência , Reprodutibilidade dos Testes , Software
10.
Front Microbiol ; 9: 1223, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29951046

RESUMO

Single stranded DNA viruses have been previously shown to populate the oceans on a global scale, and are endemic in microbialites of both marine and freshwater systems. We undertook for the first time direct viral metagenomic shotgun sequencing to explore the diversity of viruses in the modern stromatolites of Shark Bay Australia. The data indicate that Shark Bay marine stromatolites have similar diversity of ssDNA viruses to that of Highbourne Cay, Bahamas. ssDNA viruses in cluster uniquely in Shark Bay and Highbourne Cay, potentially due to enrichment by phi29-mediated amplification bias. Further, pyrosequencing data was assembled from the Shark Bay systems into two putative viral genomes that are related to Genomoviridae family of ssDNA viruses. In addition, the cellular fraction was shown to be enriched for antiviral defense genes including CRISPR-Cas, BREX (bacteriophage exclusion), and DISARM (defense island system associated with restriction-modification), a potentially novel finding for these systems. This is the first evidence for viruses in the Shark Bay stromatolites, and these viruses may play key roles in modulating microbial diversity as well as potentially impacting ecosystem function through infection and the recycling of key nutrients.

11.
Front Microbiol ; 9: 777, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755429

RESUMO

Within meso/eutrophic freshwater ecosystems the dominance of cyanobacterial blooms during summer months has substantial impacts on ecosystem function with the production of toxins and subsequent induction of hypoxia altering food web structures and biogeochemical cycles. Cyanobacterial aggregates are extensively colonized by heterotrophic bacteria that provide the cyanobacteria with key nutrients and contribute towards remineralisation of organic matter. Here we sampled from five sites within a shallow eutrophic pond over a 6 months period, relating changes in the abundance of particle-associated heterotrophic taxa to phytoplankton abundance, toxin gene copies and physiochemical properties. The abundance of a majority of particle-associated bacteria were stable, in that they persisted despite perturbation. Cyanobacterial species abundance more likely correlated with stable rather than unstable bacteria and unstable bacteria were associated with allochthonous (terrestrial) organic matter. The occurrence of the most stable bacteria was correlated with large numbers of other bacteria suggesting bacteria-bacteria interactions have implications for the stable occurrence of microorganisms on particles. Freshwater ecosystems are frequently inundated with fresh nutrients in the form of surface runoff and experience an increasing number of high temperature days. In addition to increasing the severity and longevity of cyanobacterial blooms, run-off changes the nature of the particle-associated community compromising stability. This disruption has the potential to drive changes in the carbon and nitrogen cycles and requires further attention.

12.
Harmful Algae ; 73: 157-166, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29602504

RESUMO

Cyanobacteria form harmful algal blooms and are highly adapted to a range of habitats, in part due to their phenotype plasticity. This plasticity is partially the result of co-existence of multiple strains within a single population. The toxic cyanobacterium Cylindrospermopsis raciborskii has remarkable phenotypic plasticity, strain variation and environmental adaptation resulting in an expansion of its global range. To understand the genetic basis of the high level of plasticity within a C. raciborskii population, the genomes of nine co-occurring strains were compared. The strains differed in morphology, toxin cell quotas and physiology, despite being obtained from a single water sample. Comparative genomics showed that three coiled strains were 3.9 Mbp in size, with 3544 ±â€¯11 genes, while straight strains were 3.8 Mbp in size, with 3485 ±â€¯20 genes. The core proteome comprised 86% of the genome and consisted of 2891 orthologous groups (OGs), whereas the variable genome comprised ∼14% (847 OGs), and the strain specific genome only ∼1% (433 OGs).There was a high proportion of variable strain-specific genes for the very closely related strains, which may underpin strain differentiation. The variable genes were associated with environmental responses and adaptation, particularly phage defence, DNA repair, membrane transport, and stress, illustrative of the adaptability of the strains in response to environmental and biological stressors. This study shows that high genomic variability exists between co-occurring strains and may be the basis of strain phenotypic differences and plasticity of populations. Therefore management and prediction of blooms of this harmful species requires different approaches to capture this strain variability.


Assuntos
Cianobactérias/genética , Variação Genética , Genoma Bacteriano , Bacteriófagos , Sequência de Bases , Cianobactérias/imunologia , Cianobactérias/virologia , DNA Bacteriano , Filogenia
13.
ACS Synth Biol ; 7(4): 1143-1151, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29562128

RESUMO

Phosphopantetheinyl transferases catalyze the post-translational modification of carrier proteins involved in both primary and secondary metabolism. The functional expression of polyketide synthases and nonribosomal peptide synthetases requires the activation of all carrier protein domains by phosphopantetheinyl transferases. Here we describe the characterization of five bacterial phosphopantetheinyl transferases by their substrate specificity and catalytic efficiency of four cyanobacterial carrier proteins. Comparative in vitro phosphopantetheinylation analysis showed Sfp possesses the highest catalytic efficiency over various carrier proteins. In vivo coexpression of phosphopantetheinyl transferases with carrier proteins revealed a broad range substrate specificity of phosphopantetheinyl transferases; all studied phosphopantetheinyl transferases were capable of converting apo- carrier proteins, sourced from diverse biosynthetic enzymes, to their active holo form. Phosphopantetheinyl transferase coexpression with the hybrid nonribosomal peptide synthetases/polyketide synthases responsible for microcystin biosynthesis confirmed that the higher in vitro activity of Sfp translated in vivo to a higher yield of production.


Assuntos
Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Proteínas de Transporte/metabolismo , Cianobactérias/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Simulação por Computador , Cianobactérias/química , Ligações de Hidrogênio , Microcistinas/genética , Microcistinas/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/genética
14.
Neurotox Res ; 33(1): 153-167, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28836116

RESUMO

Environmental toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their ability to damage several tissues in humans. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA). Furthermore, other toxins such as anatoxin, saxitoxin, microcystin, nodularin and ciguatoxin also have a different range of effects on human tissues, including hepatotoxicity, neurotoxicity and gastrointestinal irritation. However, the vast majority of known environmental toxins have not yet been examined in the context of neurodegenerative disease. This review aims to investigate whether neurotoxic mechanisms can be demonstrated in all aforementioned toxins, and whether there exists a link to neurodegeneration. Management of toxin exposure and potential neuroprotective compounds is also discussed. Collectively, all aforementioned microbial toxins are likely to exert some form of neuronal damage, with many of their modes of action consistent with neurodegeneration. This is important in advancing our current understanding of the cytotoxic potential of environmental toxins upon human brain function, particularly in the context of age-related neurodegenerative disease.


Assuntos
Diamino Aminoácidos , Toxinas Bacterianas , Dinoflagelados/química , Toxinas Marinhas , Microcistinas , Doenças Neurodegenerativas/etiologia , Síndromes Neurotóxicas/etiologia , Diamino Aminoácidos/análise , Diamino Aminoácidos/química , Diamino Aminoácidos/toxicidade , Animais , Toxinas Bacterianas/análise , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Monitoramento Ambiental , Humanos , Toxinas Marinhas/análise , Toxinas Marinhas/química , Toxinas Marinhas/toxicidade , Microcistinas/análise , Microcistinas/química , Microcistinas/toxicidade , Microbiologia da Água
15.
Toxicon ; 138: 68-77, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28797629

RESUMO

The cyanobacterium Dolichospermum circinale (formerly Anabaena circinalis) is responsible for neurotoxic saxitoxin-producing blooms in Australia. Previous studies have reported distinct isolates of toxic D. circinale producing different saxitoxin analogues at varying amounts, but the mechanisms responsible remain poorly understood. To assess the characteristics that may be responsible for this variance, a morphological, molecular and chemical survey of 28 Anabaena isolates was conducted. Morphological characteristics, presence or absence of saxitoxin biosynthetic genes and toxin amount and profile were assessed. The 28 isolates were collected from 16 locations. A correlation between the size of the isolates and its reported toxicity or geographical location could not be found. Molecular screening for the presence of several sxt genes revealed eight out of the 28 strains harboured the sxt gene cluster and all tailoring genes except sxtX. Furthermore, the presence of PSTs was correlated with the presence of the sxt cluster using quantitative pre-column oxidation high performance liquid chromatography with fluorescence detection (HPLC-FLD) and LC-MS/MS. Interestingly, isolates differed in the amount and type of toxins produced, with the eight toxic strains containing the core and tailoring biosynthetic genes while non-toxic strains were devoid of these genes. Moreover, the presence of sxt tailoring genes in toxic strains correlated with the biosynthesis of analogues. A greater understanding of toxin profile/quantity from distinct sites around Australia will aid the management of these at-risk areas and provide information on the molecular control or physiological characteristics responsible for toxin production.


Assuntos
Cianobactérias/genética , Saxitoxina/genética , Austrália , Cianobactérias/classificação , Cianobactérias/citologia , DNA Bacteriano , Toxinas Marinhas/biossíntese , Toxinas Marinhas/genética , Família Multigênica , RNA Ribossômico 16S , Saxitoxina/análogos & derivados , Saxitoxina/biossíntese , Análise de Sequência de DNA , Intoxicação por Frutos do Mar
16.
Bioresour Technol ; 243: 686-692, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28709074

RESUMO

A novel light-to-bioenergy system produced 3.5 times the baseline methane output using a co-culture of cyanobacteria (Oscillatoria sp.) and a methanogenic microbial community. Analysis of micronutrients in the system during the growth phase indicated that cobalt, iron, nickel and zinc were not appreciably consumed. The stable consumption and return of macronutrients calcium and magnesium were also observed. Essential macronutrients nitrogen, in the form of nitrate, and phosphorus showed no cycling during the growth phase and were depleted at rates of 0.35mg/L/day and 0.40µg/L/day, respectively. Biofilm formation increased the resilience of biomass to bacterial degradation in an anaerobic digester, as shown by viability assays of cyanobacterial biofilms in the co-culture.


Assuntos
Cianobactérias , Metano , Biofilmes , Crescimento Quimioautotrófico , Técnicas de Cocultura , Euryarchaeota
17.
ACS Chem Biol ; 12(8): 2021-2029, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28570054

RESUMO

Microcystins are globally the most commonly occurring freshwater cyanotoxins, causing acute poisoning and chronically inducing hepatocellular carcinoma. However, the detection and toxicological study of microcystins is hampered by the limited availability and high cost of pure toxin standards. Biosynthesis of microcystin variants in a fast-growing heterologous host offers a promising method of achieving reliable and economically viable alternative to isolating toxin from slow-growing cyanobacterial cultures. Here, we report the heterologous expression of recombinant microcystin synthetases in Escherichia coli to produce [d-Asp3]microcystin-LR and microcystin-LR. We assembled a 55 kb hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster from Microcystis aeruginosa PCC 7806 using Red/ET recombineering and replaced the native promoters with an inducible PtetO promoter to yield microcystin titers superior to M. aeruginosa. The expression platform described herein can be tailored to heterologously produce a wide variety of microcystin variants, and potentially other cyanobacterial natural products of commercial relevance.


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Cianobactérias/genética , Escherichia coli/genética , Microbiologia Industrial/métodos , Toxinas Marinhas/biossíntese , Toxinas Marinhas/genética , Microcistinas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Cianobactérias/enzimologia , Toxinas Marinhas/metabolismo , Microcistinas/biossíntese , Microcistinas/genética , Microcistinas/metabolismo , Família Multigênica/genética , Peptídeo Sintases/genética , Regiões Promotoras Genéticas/genética
18.
Appl Environ Microbiol ; 83(13)2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28455341

RESUMO

To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions.IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids.


Assuntos
Nostoc/metabolismo , Triterpenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Isomerismo , Metilação , Nostoc/química , Nostoc/genética , Nostoc/crescimento & desenvolvimento , Osmose , Triterpenos/química
19.
Appl Microbiol Biotechnol ; 101(12): 5089-5099, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28341886

RESUMO

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l-1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l-1) together with sorbitol (65 g l-1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l-1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.


Assuntos
Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Gluconatos/metabolismo , Microbiologia Industrial , Lactonas/metabolismo , Zymomonas/enzimologia , Zymomonas/fisiologia , Biomassa , Biotransformação , Etanol/metabolismo , Fermentação , Frutose/metabolismo , Técnicas de Inativação de Genes , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Sorbitol/metabolismo , Estresse Fisiológico , Zymomonas/genética , Zymomonas/crescimento & desenvolvimento
20.
Neurotox Res ; 31(2): 245-258, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27796937

RESUMO

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that α-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated α-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.


Assuntos
Diamino Aminoácidos/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ciguatoxinas/toxicidade , Microcistinas/toxicidade , Saxitoxina/toxicidade , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Microcistinas/antagonistas & inibidores , Degeneração Neural/induzido quimicamente , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Ácido Tióctico/farmacologia
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