RESUMO
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, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.
Assuntos
Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Fixação de Nitrogênio/genética , Fatores de Transcrição/metabolismo , Azospirillum brasilense/química , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
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 σ(54) 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, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.
Assuntos
Azospirillum brasilense/metabolismo , Proteínas de Bactérias/metabolismo , Fixação de Nitrogênio/genética , Fatores de Transcrição/metabolismo , Azospirillum brasilense/química , Azospirillum brasilense/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (± 0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 105 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.PALAVRAS-CHAVE: Boas práticas. Leite cru. Segurança alimentar
RESUMO
Foram estudadas 34 propriedades rurais, no período de outubro a dezembro de 2006, com 68 amostras de leite coletadas em triplicata diretamente dos tanques resfriadores ou vasilhames equivalentes. Foi pesquisada a presença de Listeria spp., comparando-se com o nível de contagem bacteriana total no leite de tanques. Foi utilizado agar-Aloa para isolamento de Listeria spp e de Listeria monocytogenes, com confirmação por PCR. Das amostras de leite cru submetidas a pesquisa de Listeria spp. observou-se que 19 (27,9%) estavam contaminadas, e duas (2,9%) apresentaram resultado positivo para L. monocytogenes. A contagem bacteriana total apresentou média de 5,41 log UFC/mL (±0,57), e 29,3% das amostras estavam acima do limite de 7,5 x 103 UFC/mL. Os resultados permitem afirmar que este patógeno deve ser pesquisado em amostras de leite cru por constituir um importante indicador de segurança alimentar e que os produtores rurais não têm conhecimento adequado sobre os riscos a que estão sujeitos ao manipularem e consumirem este produto.
The presence of Listeria spp., correlated at the level of total bacterial count in bulk tank milk was investigated, about 34 farms in the period of October to December 2006, with 68 triplicate samples of milk collected directly from the bulk tanks or equivalent containers. Aloa-agar was used for isolation of Listeria spp. and Listeria monocytogenes, with confirmation by PCR (polymerase chain reaction). In the samples of raw milk subjected to search of Listeria spp., it was observed that 19 (27.9%) were contaminated, and two (2.9%) were positive for L. monocytogenes. The total bacterial count showed an average of 5.41 log CFU/mL (± 0.57), and 29.3% of samples were above the limit of 7.5 x 10 CFU/mL. The results indicate that this pathogen should be studied in samples of raw milk cause it's an important indicator of food security and farmers don't have adequate knowledge about the risks they are subject to manipulate and consume this product.