Construção e caracterização de estirpes mutantes de Herbaspirillum seropedicae nos genes fnr1, fnr2 e fnr3

Abstract

Resumo: Herbaspirillum seropedicae e uma bacteria fixadora de nitrogenio da classe ƒÀ das Proteobacteria, encontrada em associacao com diferentes Poaceae, como trigo, arroz, sorgo e cana-de-acucar. O genoma de H. seropedicae (GENOPAR) possui tres genes homologos a fnr. As proteinas FNR sao reguladores transcricionais que agem como sensores do estado redox intracelular, regulando um grande numero de genes em resposta a mudancas nos niveis de O2. Neste trabalho, foram construidos sete mutantes por delecao, fnr1, fnr2, fnr3, fnr1fnr2, fnr1fnr3, fnr2fnr3 e fnr1fnr2fnr3. Estes mutantes foram deficientes no crescimento sob condicoes limitantes de oxigenio e o triplo mutante foi menos resistente a NO2 -. Nenhuma evidencia foi encontrada a respeito do envolvimento de FNR na utilizacao de ions amonio ou nitrato como fonte de nitrogenio. Resultados obtidos anteriormente mostraram que a proteina NifA de H. seropedicae, responsavel pela ativacao da expressao dos genes nif, e inativada em uma estirpe mutante de scherichia coli no gene fnr. Devido a esse resultado o possivel envolvimento de FNR na fixacao biologica de nitrogenio de H. seropedicae foi investigado. Os resultados obtidos mostraram que as proteinas FNR nao sao necessarias para a atividade da nitrogenase. Experimentos de determinacao da expressao genica mostraram que FNR reprime a expressao dos genes nifA e nifB de H. seropedicae, e tambem do operon fixNOQP. Experimentos realizados com fusoes entre os genes fnr1, fnr2 e fnr3 com o gene reporter lacZ mostraram que a expressao do gene fnr2 e dependente das proteinas FNR e que a expressao de fnr3 e reprimida pelas outras proteinas FNR. Por fim, os resultados obtidos mostram q e as proteinas FNR de H. seropedicae nao sao essenciais a fixacao biologica de nitrogenio, mas podem estar envolvidas em um fino mecanismo de regulacao da expressao de genes requeridos para fixacao de nitrogenio, permitindo que estes genes sejam expressos quando as concentracoes de oxigenio sao otimas para a fixacao biologica de nitrogenio.

Abstract: Herbaspirillum seropedicae is a nitrogen fixing bacteria, belonging to â-class of Proteobacteria, founded in association with different Poaceae, such as wheat, rice, sorghum and sugar cane. The genome of H. seropedicae (GENOPAR) has three fnr-like genes. FNR proteins are dual transcriptional regulators that act as intracellular redox sensors regulating a wide range of genes in response to changes in the O2 levels. In this work we constructed seven deletion mutants fnr1, fnr2, fnr3, fnr1fnr2, fnr1fnr3, fnr2fnr3 and fnr1fnr2fnr3. These mutants were defective in growth under oxygen limiting conditions, and the triple mutant was also sensitive to NO2 -. No evidence was found about the involvement of FNR in the utilization of ammonium ions or nitrate as nitrogen source. Previous results showed that the H. seropedicae nif gene transcriptional activator NifA is inactive in an fnr mutant strain of Escherichia coli. This result prompted us to investigate the involvement of FNR in H. seropedicae nitrogen fixation. In H. seropedicae none of fnr genes are required for nitrogenase activity. Gene expression experiments in the mutant strains showed that FNR represses the expression of both nifA and nifB in H. seropedicae and the fixNOQP operon is also epressed by FNR. Experiments performed with fusions between fnr1, fnr2 and fnr3 genes with lacZ reporter gene showed that fnr2 gene expression is dependent upon FNR proteins and that the expression of fnr3 is repressed by other FNR proteins. Finally, the results showed that FNR proteins of H. seropedicae are not essential to biological nitrogen fixation, but may be involved in a fine-tunning regulating mechanism of the expression of genes required for nitrogen fixation, allowing these genes to be expressed only when oxygen concentrations are optimal for biological nitrogen fixation.