RESUMO
Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.
Assuntos
Pirimidinas/toxicidade , Compostos de Sulfonilureia/toxicidade , Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Dolichospermum flosaquae/efeitos dos fármacos , Dolichospermum flosaquae/metabolismo , Ésteres/toxicidade , Herbicidas/toxicidade , Fixação de Nitrogênio/efeitos dos fármacos , Anabaena/genética , Dolichospermum flosaquae/genética , Aminoácidos/metabolismo , Nitrogênio/metabolismoRESUMO
Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.
Assuntos
Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Dolichospermum flosaquae/efeitos dos fármacos , Dolichospermum flosaquae/metabolismo , Ésteres/toxicidade , Herbicidas/toxicidade , Fixação de Nitrogênio/efeitos dos fármacos , Pirimidinas/toxicidade , Compostos de Sulfonilureia/toxicidade , Aminoácidos/metabolismo , Anabaena/genética , Dolichospermum flosaquae/genética , Nitrogênio/metabolismoRESUMO
Cyanobacteria are a group of photosynthetic, nitrogen-fixing bacteria present in a wide variety of habitats such as freshwater, marine, and terrestrial ecosystems. In this work, the effects of As(III), a major toxic environmental pollutant, on the lipidomic profiles of two cyanobacteria species (Anabaena and Planktothrix agardhii) were assessed by means of a recently proposed method based on the concept of regions of interest (ROI) in liquid chromatography mass spectroscopy (LC-MS) together with multivariate curve resolution alternating least squares (MCR-ALS). Cyanobacteria were exposed to two concentrations of As(III) for a week, and lipid extracts were analyzed by ultrahigh-performance liquid chromatography/time-of-flight mass spectrometry in full scan mode. The data obtained were compressed by means of the ROI strategy, and the resulting LC-MS data sets were analyzed by the MCR-ALS method. Comparison of profile peak areas resolved by MCR-ALS in control and exposed samples allowed the discrimination of lipids whose concentrations were changed due to As(III) treatment. The tentative identification of these lipids revealed an important reduction of the levels of some galactolipids such as monogalactosyldiacylglycerol, the pigment chlorophyll a and its degradation product, pheophytin a, as well as carotene compounds such as 3-hydroxycarotene and carotene-3,3'-dione, all of these compounds being essential in the photosynthetic process. These results suggested that As(III) induced important changes in the composition of lipids of cyanobacteria, which were able to compromise their energy production processes. Graphical abstract Steps of the proposed LC-MS + MCR-ALS procedure.
Assuntos
Arsênio/metabolismo , Clorofila/metabolismo , Cianobactérias/efeitos dos fármacos , Poluentes Ambientais/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Anabaena/ultraestrutura , Cromatografia Líquida de Alta Pressão/métodos , Cianobactérias/metabolismo , Cianobactérias/ultraestrutura , Análise dos Mínimos Quadrados , Espectrometria de Massas/métodos , Análise MultivariadaRESUMO
Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.
Assuntos
Ferredoxinas/metabolismo , Flavodoxina/genética , Flavodoxina/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Anabaena/genética , Anabaena/metabolismo , Sequência de Bases , DNA de Plantas/genética , Ferredoxinas/deficiência , Ferredoxinas/genética , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Microscopia Eletrônica de Transmissão , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Antissenso/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Nicotiana/ultraestruturaRESUMO
The influence of Anabaena spiroides exopolysaccharides (EPS) on copper speciation (total dissolved, particulate and free Cu(2+) ions) and bioavailability in aquatic organisms was investigated. Bacteria were used as the first trophic level, Paramecium caudatum (protozoan) as the second and the copepod cyclopoid Metacyclops mendocinus as the third level. The organisms were obtained from a freshwater reservoir and held under continuous laboratory controlled conditions. Freshwater media containing EPS excreted by A. spiroides (10mgL(-1)) and copper (1.0x10(-6)molL(-1)) were used for bacteria growth. Contamined bacteria were used as food source to protozoan, which was further furnished to copepods. The results showed a reduction of EPS concentration during bacteria growth and also a smaller copper accumulation by microorganisms in the presence of EPS. We concluded that A. spiroides exopolysaccharides have reduced copper entrance into the experimental aquatic microbial food chain.
Assuntos
Anabaena/metabolismo , Cobre/análise , Cobre/metabolismo , Cadeia Alimentar , Polissacarídeos Bacterianos/química , Anabaena/crescimento & desenvolvimento , Animais , Copépodes/metabolismo , Cobre/toxicidade , Água Doce/química , Água Doce/microbiologia , Paramecium caudatum/metabolismo , Polissacarídeos Bacterianos/análise , Polissacarídeos Bacterianos/isolamento & purificaçãoRESUMO
In the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), it has been shown that spsB and susA, the genes coding for proteins related to sucrose synthesis and cleavage, respectively, exhibit converse expression regarding the nitrogen source. In the nitrogen-fixing filament, spsB expression is mostly localized to the heterocysts and susA is only expressed in vegetative cells. The aim of this work was to investigate the participation of NtcA, a global nitrogen regulator that operates in cyanobacteria, in the regulation of sucrose metabolism genes in Anabaena sp. PCC 7120. The induction of spsB expression observed in the filaments upon combined-nitrogen depletion was abolished in an NtcA-deficient mutant. In vitro experiments showed that NtcA binds specifically but with different affinities to two sites in the spsB promoter region. When susA expression was analyzed after a combined-nitrogen starvation, the levels of mRNA, polypeptide and activity increased in the mutant in comparison with the wild-type strain. Also, NtcA interacted with one site in the promoter region of susA. We conclude that sucrose metabolism is coordinated at the transcriptional level with nitrogen metabolism, suggesting a global metabolism regulating role for NtcA.
Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrogênio/metabolismo , Sacarose/metabolismo , Anabaena/genética , Proteínas de Bactérias/genética , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , RNA Bacteriano/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
Nitrogen (N) available to plants mostly originates from N(2) fixation carried out by prokaryotes. Certain cyanobacterial species contribute to this energetically expensive process related to carbon (C) metabolism. Several filamentous strains differentiate heterocysts, specialized N(2)-fixing cells. To understand how C and N metabolism are regulated in photodiazotrophically grown organisms, we investigated the role of sucrose (Suc) biosynthesis in N(2) fixation in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120). The presence of two Suc-phosphate synthases (SPS), SPS-A and SPS-B, directly involved in Suc synthesis with different glucosyl donor specificity, seems to be important in the N(2)-fixing filament. Measurement of enzyme activity and polypeptide levels plus reverse transcription-polymerase chain reaction experiments showed that total SPS expression is greater in cells grown in N(2) versus combined N conditions. Only SPS-B, however, was seen to be active in the heterocyst, as confirmed by analysis of green fluorescent protein reporters. SPS-B gene expression is likely controlled at the transcriptional initiation level, probably in relation to a global N regulator. Metabolic control analysis indicated that the metabolism of glycogen and Suc is likely interconnected in N(2)-fixing filaments. These findings suggest that N(2) fixation may be spatially compatible with Suc synthesis and support the role of the disaccharide as an intermediate in the reduced C flux in heterocyst-forming cyanobacteria.
Assuntos
Anabaena/metabolismo , Carbono/metabolismo , Fixação de Nitrogênio , Sacarose/metabolismo , Sequência de Aminoácidos , Anabaena/enzimologia , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glucosiltransferases/fisiologia , Proteínas de Fluorescência Verde/análise , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.
Assuntos
Anabaena/genética , Ferredoxinas/fisiologia , Flavodoxina/metabolismo , Nicotiana/metabolismo , Anabaena/metabolismo , Antioxidantes/metabolismo , Cloroplastos/genética , Cloroplastos/fisiologia , Transporte de Elétrons/fisiologia , Meio Ambiente , Flavodoxina/genética , Modelos Biológicos , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Fotossíntese , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Tiorredoxinas/metabolismo , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
In January of 2003, a cyanobacterial bloom in the Patos' Lagoon (Southern Brazil) (32 degrees 05'S-52 degrees 12'W) was observed. Water samples were taken to identify the composition and abundance of the bloom, as well as the occurrence of toxins. The effects of this occurrence on the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) was also evaluated. Predominance of cyanobacteria, particularly Anabaena trichomes ( approximately 2.5.10(6) individuals per liter) was observed, and low concentrations of microcystins and anticholinesterasic toxins were detected. Augmented levels of lipid hydroperoxides (LPO) and glutathione-S-transferase activity, and lowering of total protein content were also observed in organisms collected during the bloom event. Although non-toxic, the cyanobacterial bloom could augment the cycle of hyper-oxygenation and hypoxia in the water. During hyperoxia, L. acuta, an oxyconformer, should consume more oxygen, thus augmenting the rate of reactive oxygen species generation. A repeated cycle of hyper-oxygenation and hypoxia would finally induce oxidative stress, as evidenced by the high levels of LPO and glutathione-S-transferase activity.
Assuntos
Anabaena/crescimento & desenvolvimento , Ecossistema , Poliquetos/metabolismo , Água do Mar/microbiologia , Anabaena/metabolismo , Animais , Brasil , Glutationa Transferase/metabolismo , Peróxidos Lipídicos/metabolismo , Toxinas Marinhas/análise , Microcistinas , Consumo de Oxigênio/fisiologia , Peptídeos Cíclicos/análise , Proteínas/metabolismoRESUMO
The 16S-rDNA from 22 cyanobacteria isolated from biofilms on walls of modern and historic buildings in Brazil was partially sequenced (approximately 350 bp) using specific primers. The cyanobacteria with the closest matching sequences were found using the BLAST tool. The sequences were combined with 52 other cyanobacterial sequences already deposited in public data banks and a dendrogram constructed, after deletion from each sequence of one of the variable 16S rDNA regions (VI). The newly sequenced organisms fitted well within their respective families, but their similarities to other members of the groups were generally low, less than 96%. Close matches were found only with one other terrestrial (hot dry desert) cyanobacterium, Microcoleus sociatus, and with Anabaena variabilis. Phylogenetic analysis suggested that the deletion of the hypervariable regions in the RNA structure is essential for meaningful evolutionary studies. The results support the standard phylogenetic tree based on morphology, but suggest that these terrestrial cyanobacteria are distant relatives of their equivalent aquatic genera and are, indeed, a distinct population.