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1.
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.

2.
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
3.
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
4.
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
5.
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
6.
Harmful Algae ; 54: 98-111, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-28073484

RESUMO

The production of toxic metabolites by cyanobacterial blooms represents a significant threat to the health of humans and ecosystems worldwide. Here we summarize the current state of the knowledge regarding the genetics, biosynthesis and regulation of well-characterized cyanotoxins, including the microcystins, nodularin, cylindrospermopsin, saxitoxins and anatoxins, as well as the lesser-known marine toxins (e.g. lyngbyatoxin, aplysiatoxin, jamaicamides, barbamide, curacin, hectochlorin and apratoxins).


Assuntos
Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Cianobactérias/fisiologia , Toxinas Bacterianas/metabolismo , Cianobactérias/genética , Ecossistema , Regulação Bacteriana da Expressão Gênica , Humanos
7.
Environ Microbiol Rep ; 8(1): 3-13, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26663762

RESUMO

A common misconception persists that the genomes of toxic and non-toxic cyanobacterial strains are largely conserved with the exception of the presence or absence of the genes responsible for toxin production. Implementation of -omics era technologies has challenged this paradigm, with comparative analyses providing increased insight into the differences between strains of the same species. The implementation of genomic, transcriptomic and proteomic approaches has revealed distinct profiles between toxin-producing and non-toxic strains. Further, metagenomics and metaproteomics highlight the genomic potential and functional state of toxic bloom events over time. In this review, we highlight how these technologies have shaped our understanding of the complex relationship between these molecules, their producers and the environment at large within which they persist.


Assuntos
Toxinas Bacterianas/metabolismo , Cianobactérias/química , Cianobactérias/genética , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Proteômica/métodos , Cianobactérias/metabolismo , Perfilação da Expressão Gênica/tendências , Genômica/tendências , Proteômica/tendências
8.
Environ Microbiol ; 18(2): 427-38, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26347118

RESUMO

Saxitoxins (STX), neurotoxic alkaloids, fall under the umbrella of paralytic shellfish toxins produced by marine dinoflagellates and freshwater cyanobacteria. The genes responsible for the production of STX have been proposed, but factors that influence their expression and induce toxin efflux remain unclear. Here we characterize the putative STX NorM-like MATE transporters SxtF and SxtM. Complementation of the antibiotic-sensitive strain Escherichia coli KAM32 with these transporters decreased fluoroquinolone sensitivity, indicating that while becoming evolutionary specialized for STX transport these transporters retain relaxed specificity typical of this class. The transcriptional response of STX biosynthesis (sxtA) along with that of the STX transporters (sxtM and sxtF from Cylindrospermopsis raciborskii T3, and sxtM from Anabaena circinalis AWQC131C) were assessed in response to ionic stress. These data, coupled with a measure of toxin intracellular to extracellular ratios, provide an insight into the physiology of STX export. Cylindrospermopsis raciborskii and Anabaena circinalis exhibited opposing responses under conditions of ionic stress. High Na(+) (10 mM) induced moderate alterations of transcription and STX localization, whereas high pH (pH 9) stimulated the greatest physiological response. Saxitoxin production and cellular localization are responsive to ionic strength, indicating a role of this molecule in the maintenance of cellular homeostasis.


Assuntos
Anabaena/metabolismo , Cylindrospermopsis/metabolismo , Escherichia coli/genética , Fluoroquinolonas/farmacologia , Saxitoxina/metabolismo , Sódio/metabolismo , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/fisiologia , Dinoflagelados/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Fluoroquinolonas/metabolismo , Água Doce , Concentração de Íons de Hidrogênio , Íons/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Estresse Fisiológico/fisiologia
9.
Nat Prod Rep ; 30(8): 1121-38, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23832108

RESUMO

The heterologous expression of microbial natural product biosynthetic pathways coupled with advanced DNA engineering enables optimisation of product yields, functional elucidation of cryptic gene clusters, and generation of novel derivatives. This review summarises the recent advances in cloning and maintenance of natural product biosynthetic gene clusters for heterologous expression and the efforts fundamental for discovering novel natural products in the post-genomics era, with a focus on polyketide synthases (PKSs) and non-ribosomal polypeptide synthetases (NRPS).


Assuntos
Produtos Biológicos/metabolismo , Vias Biossintéticas , Engenharia Genética/métodos , Família Multigênica , Vias Biossintéticas/genética , Clonagem Molecular , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo
10.
ACS Chem Biol ; 8(9): 1888-93, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23751865

RESUMO

Many chemically complex cyanobacterial polyketides and nonribosomal peptides are of great pharmaceutical interest, but the levels required for exploitation are difficult to achieve from native sources. Here we develop a framework for the expression of these multifunctional cyanobacterial assembly lines in Escherichia coli using the lyngbyatoxin biosynthetic pathway, derived from a marine microbial assemblage dominated by the cyanobacterium Moorea producens. Heterologous expression of this pathway afforded high titers of both lyngbyatoxin A (25.6 mg L(-1)) and its precursor indolactam-V (150 mg L(-1)). Production, isolation, and identification of all expected chemical intermediates of lyngbyatoxin biosynthesis in E. coli also confirmed the previously proposed biosynthetic route, setting a solid chemical foundation for future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in E. coli also opens the possibility for future heterologous expression, characterization, and exploitation of other cyanobacterial natural product pathways.


Assuntos
Vias Biossintéticas , Cianobactérias/metabolismo , Escherichia coli/metabolismo , Toxinas da Lyngbya/metabolismo , Toxinas Marinhas/metabolismo , Cianobactérias/genética , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Microbiologia Industrial/métodos , Toxinas da Lyngbya/genética , Toxinas Marinhas/genética , Proteína Quinase C/metabolismo
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