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1.
Chemosphere ; 201: 772-779, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29550571

RESUMO

This work describes the construction of two novel self-luminescent bioreporter strains of the cyanobacterium Nostoc sp. PCC 7120 by fusing the promoter region of the sodA and sodB genes (encoding the superoxide dismutases MnSod and FeSod, respectively) to luxCDABE from Photorhabdus luminescens aimed at detecting pollutants that generate reactive oxygen species (ROS), particularly O2-. Bioreporters were tested against methyl viologen (MV) as the inducer of superoxide anion (O2-). Both bioreporters were specific for O2- and Limits of detection (LODs) and Maximum Permissive Concentrations (MPCs) were calculated: Nostoc sp. PCC 7120 pBG2154 (sodA) had a range of detection from 400 to 1000 pM of MV and for Nostoc sp. PCC 7120 pBG2165 (sodB) the range of detection was from 500 to 1800 pM of MV after 5 h-exposure. To further validate the bioreporters, they were tested with the emerging pollutant Triclosan which induced bioluminescence in both strains. Furthermore, the bioreporters performance was tested in two real environmental samples with different water matrix complexity, spiked with MV. Both bioreporters were induced by O2- in these environmental samples. In the case of the river water sample, the amount of bioavailable MV as calculated from the bioreporters output was similar to that nominally added. For the waste water sample, the bioavailable MV concentration detected by the bioreporters was one order of magnitude lower than nominal. These differences could be due to MV complexation with organic matter and/or co-occurring organic contaminants. These results confirm their high sensitivity to O2- and their suitability to detect oxidative stress-generating pollutants in fresh-waters.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/enzimologia , Superóxido Dismutase/química , Superóxidos/análise , Poluentes Químicos da Água/análise , Proteínas de Bactérias/genética , Cianobactérias/efeitos dos fármacos , Água Doce/química , Genes Bacterianos , Limite de Detecção , Medições Luminescentes , Oxirredução , Paraquat/química , Regiões Promotoras Genéticas/genética , Superóxido Dismutase/genética
2.
Sci Rep ; 5: 17200, 2015 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-26606975

RESUMO

A novel additivity framework for mixture effect modelling in the context of whole cell inducible biosensors has been mathematically developed and implemented in R. The proposed method is a multivariate extension of the effective dose (EDp) concept. Specifically, the extension accounts for differential maximal effects among analytes and response inhibition beyond the maximum permissive concentrations. This allows a multivariate extension of Loewe additivity, enabling direct application in a biphasic dose-response framework. The proposed additivity definition was validated, and its applicability illustrated by studying the response of the cyanobacterial biosensor Synechococcus elongatus PCC 7942 pBG2120 to binary mixtures of Zn, Cu, Cd, Ag, Co and Hg. The novel method allowed by the first time to model complete dose-response profiles of an inducible whole cell biosensor to mixtures. In addition, the approach also allowed identification and quantification of departures from additivity (interactions) among analytes. The biosensor was found to respond in a near additive way to heavy metal mixtures except when Hg, Co and Ag were present, in which case strong interactions occurred. The method is a useful contribution for the whole cell biosensors discipline and related areas allowing to perform appropriate assessment of mixture effects in non-monotonic dose-response frameworks.


Assuntos
Técnicas Biossensoriais/métodos , Células/metabolismo , Pesquisa , Modelos Teóricos , Análise de Regressão , Reprodutibilidade dos Testes , Synechococcus
3.
Microbiology (Reading) ; 151(Pt 5): 1671-1682, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15870474

RESUMO

Transposon mutagenesis of Anabaena sp. PCC7120 led to the isolation of a mutant strain, PHB11, which grew poorly at pH values above 10. The mutant strain exhibited pronounced Na+ sensitivity; this sensitivity was higher under basic conditions. Mutant PHB11 also showed an inhibition of photosynthesis that was much more pronounced at alkaline pH. Reconstruction of the transposon mutation of PHB11 in the wild-type strain reproduced the phenotype of the original mutant. The wild-type version of the mutated gene was cloned and the mutation complemented. In mutant strain PHB11, the transposon had inserted within an ORF that is part of a seven-ORF operon with significant sequence similarity to a family of bacterial operons that are believed to code for a novel multiprotein cation/proton antiporter primarily involved in resistance to salt stress and adaptation to alkaline pH. The Anabaena operon was denoted mrp (multiple resistance and pH adaptation) following the nomenclature of the Bacillus subtilis operon; the ORF mutated in PHB11 corresponded to mrpA. Computer analysis suggested that all seven predicted Anabaena Mrp proteins were highly hydrophobic with several transmembrane domains; in fact, the predicted protein sequences encoded by mrpA, mrpB and mrpC showed significant similarity to hydrophobic subunits of the proton pumping NADH : ubiquinone oxidoreductase. In vivo expression studies indicated that mrpA is induced with increasing external Na+ concentrations and alkaline pH; mrpA is also upregulated under inorganic carbon (Ci) limitation. The biological significance of a putative cyanobacterial Mrp complex is discussed.


Assuntos
Adaptação Fisiológica , Anabaena/efeitos dos fármacos , Anabaena/fisiologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Sódio/farmacologia , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Análise de Sequência de DNA
4.
Microbiology (Reading) ; 150(Pt 11): 3731-3739, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15528659

RESUMO

The impact of calcium signals in virtually all cells has led to the study of their role in prokaryotic organisms as stress response modulators. Cell differentiation in adverse conditions is a common Ca(2+)-requiring response. Nitrogen starvation induces the differentiation of N(2)-fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. This paper reports the use of a recombinant strain of this organism expressing the photoprotein aequorin to monitor the intracellular free-calcium concentration during the course of heterocyst differentiation. A specific calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and generated by intracellular calcium stores was identified. The intracellular calcium signal was manipulated by treatment with specific calcium drugs, and the effect of such manipulation on the process of heterocyst differentiation was subsequently assessed. Suppression, magnification or poor regulation of this signal prevented the process of heterocyst differentiation, thereby suggesting that a calcium signal with a defined set of kinetic parameters may be required for differentiation. A hetR mutant of Anabaena sp. PCC7120 that cannot differentiate into heterocysts retains, however, the capacity to generate the calcium transient in response to nitrogen deprivation, strongly suggesting that Ca(2+) may be involved in a very early step of the differentiation process.


Assuntos
Anabaena/crescimento & desenvolvimento , Anabaena/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Regulação Bacteriana da Expressão Gênica , Adaptação Fisiológica , Equorina/genética , Equorina/metabolismo , Anabaena/citologia , Anabaena/genética , Proteínas de Bactérias/genética , Citoplasma/química , Deleção de Genes , Genes Reporter , Nitrogênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
J Bacteriol ; 183(2): 628-36, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11133957

RESUMO

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i. e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N(2), are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.


Assuntos
Anabaena/genética , Proteínas de Bactérias , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Genes Bacterianos , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Fixação de Nitrogênio/genética , Peptidoglicano Glicosiltransferase , Peptidil Transferases , Aerobiose , Sequência de Aminoácidos , Anabaena/citologia , Clonagem Molecular , Elementos de DNA Transponíveis , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Fenótipo , Homologia de Sequência de Aminoácidos
7.
Plant Physiol ; 123(1): 161-76, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10806234

RESUMO

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.


Assuntos
Equorina/metabolismo , Cálcio/metabolismo , Cianobactérias/metabolismo , Homeostase , Temperatura Baixa , Temperatura Alta , Proteínas Recombinantes/metabolismo
9.
Microbiology (Reading) ; 144 ( Pt 7): 1799-1805, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9695912

RESUMO

In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.


Assuntos
Aldeído Oxirredutases , Arginina/genética , Proteínas de Bactérias/genética , Cianobactérias/citologia , Cianobactérias/genética , Elementos de DNA Transponíveis/genética , Genes Bacterianos/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/análise , Southern Blotting , Cianobactérias/efeitos dos fármacos , Cianobactérias/crescimento & desenvolvimento , DNA , Dados de Sequência Molecular , Nitrogênio/farmacologia , Nitrogenase/análise , Homologia de Sequência de Aminoácidos
11.
FEMS Microbiol Lett ; 123(1-2): 63-7, 1994 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-7988900

RESUMO

The hepA gene in Anabaena sp. strain PCC 7120 is required for normal formation of the polysaccharide layer of the heterocyst envelope. A plasmid bearing hepA, interrupted by a neomycin-resistance cassette, was transferred by conjugation to wild type Anabaena variabilis ATCC 29413, so that the interrupted hepA gene replaced a homologous sequence. In the recombinant exconjugants, the envelopes of akinetes as well as of heterocysts were altered.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Anabaena/genética , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Anabaena/crescimento & desenvolvimento , Escherichia coli/genética , Plasmídeos , Polissacarídeos Bacterianos/genética
12.
J Bacteriol ; 176(17): 5277-83, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8071202

RESUMO

Mutagenesis of Anabaena sp. strain PCC 7120 with a derivative of transposon Tn5 led to the isolation of a mutant strain, P6, in which heterocysts are not formed (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation of P6 in the wild-type strain reproduced the phenotype of the original mutant. Analysis by pulsed-field gel electrophoresis localized the transposition at ca. 3.44 Mb on the physical map of the chromosome of wild-type Anabaena sp. The transposon was situated within an open reading frame (ORF), which we denote hetP, whose wild-type form was cloned and also sequenced. The predicted HetP protein was not found to show significant sequence similarity to other proteins. The mutation in strain P6 could be complemented by a clone of a fragment of wild-type DNA that includes hetP and at least one additional ORF 3' from hetP, but not by a clone that includes hetP as its only ORF. The latter clone proved highly toxic. The phenotype of the P6 mutant may, therefore, be due to a polar effect of the insertion of the transposon. Filaments of strain P6 and of the wild-type strain, when bearing the complementing fragment on a pDU1-based plasmid, showed an increased frequency of clustered heterocysts compared with that of the wild-type strain.


Assuntos
Anabaena/fisiologia , Proteínas de Bactérias/biossíntese , Elementos de DNA Transponíveis , Genes Bacterianos , Sequência de Aminoácidos , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Bacteriano/isolamento & purificação , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Fenótipo , Plasmídeos , Mapeamento por Restrição
13.
Mol Microbiol ; 12(4): 679-84, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-7934891

RESUMO

Evident differentiation of vegetative cells into heterocysts in Anabaena sp. strain PCC 7120 is prevented by insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored. In contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.


Assuntos
Cianobactérias/genética , Mutação , Cianobactérias/crescimento & desenvolvimento , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genes Bacterianos , Genes Reporter , Luciferases/genética , Plasmídeos/genética
14.
Arch Environ Contam Toxicol ; 22(1): 130-4, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1554245

RESUMO

The effects of the phenoxy acetic herbicides 2,4-dichlorophenoxy acetic acid (2,4D) and methylchlorophenoxy acetic acid (MCPA) on growth, photosynthesis, and nitrogenase activity of cyanobacteria has been investigated. Concentrations ranging from 10(-9) to 10(-3) M did not change significantly the parameters of Anabaena UAM 202. Concentrations higher than 10(-3) M of both herbicides were toxic. The primary toxic action of these herbicides in Anabaena UAM 202 was on photosynthesis.


Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Ácido 2-Metil-4-clorofenoxiacético/farmacologia , Cianobactérias/efeitos dos fármacos , Herbicidas/farmacologia , Nitrogenase/análise , Fotossíntese/efeitos dos fármacos , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Oryza
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