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| Alves,L.P.S.; Almeida,A.T.; Cruz,L.M.; Pedrosa,F.O.; de Souza,E.M.; Chubatsu,L.S.; Müller-Santos,M.; Valdameri,G.. |
| The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method... |
| Tipo: Info:eu-repo/semantics/article |
Palavras-chave: Flow cytometry; Nile red; Poly-3-hydroxybutyrate; Herbaspirillum seropedicae; Azospirillum brasilense. |
| Ano: 2017 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2017000100606 |
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| Araújo,L.M.; Huergo,L.F.; Invitti,A.L.; Gimenes,C.I.; Bonatto,A.C.; Monteiro,R.A.; Souza,E.M.; Pedrosa,F.O.; Chubatsu,L.S.. |
| Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by... |
| Tipo: Info:eu-repo/semantics/other |
Palavras-chave: Azospirillum brasilense; Nitrogen fixation; PII-like protein; GlnD; GlnB; GlnZ. |
| Ano: 2008 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2008000400006 |
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| Sotomaior,P.; Araújo,L.M.; Nishikawa,C.Y.; Huergo,L.F.; Monteiro,R.A.; Pedrosa,F.O.; Chubatsu,L.S.; Souza,E.M.. |
| Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the... |
| Tipo: Info:eu-repo/semantics/article |
Palavras-chave: Azospirillum brasilense; NifA protein; GlnB protein; GAF domain; Nitrogen fixation. |
| Ano: 2012 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012001200005 |
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| Aquino,B.; Stefanello,A.A.; Oliveira,M.A.S.; Pedrosa,F.O.; Souza,E.M.; Monteiro,R.A.; Chubatsu,L.S.. |
| NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein... |
| Tipo: Info:eu-repo/semantics/article |
Palavras-chave: Biological nitrogen fixation; Herbaspirillum seropedicae; NifA. |
| Ano: 2015 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2015000800683 |
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| Nishikawa,C.Y.; Araújo,L.M.; Kadowaki,M.A.S.; Monteiro,R.A.; Steffens,M.B.R.; Pedrosa,F.O.; Souza,E.M.; Chubatsu,L.S.. |
| Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study,... |
| Tipo: Info:eu-repo/semantics/other |
Palavras-chave: Biological nitrogen fixation; Azospirillum brasilense; NifA protein. |
| Ano: 2012 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012000200004 |
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| Galvão,C.W.; Souza,E.M.; Etto,R.M.; Pedrosa,F.O.; Chubatsu,L.S.; Yates,M.G.; Schumacher,J.; Buck,M.; Steffens,M.B.R.. |
| DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than... |
| Tipo: Info:eu-repo/semantics/article |
Palavras-chave: RecA; RecX; Herbaspirillum seropedicae; SOS repair. |
| Ano: 2012 |
URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2012001200004 |
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