RESUMEN
Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.
Asunto(s)
Azospirillum brasilense/genética , Regulación Bacteriana de la Expresión Génica , N-Glicosil Hidrolasas , Nitrogenasa/biosíntesis , Procesamiento Proteico-Postraduccional , Compuestos de Amonio Cuaternario/farmacología , ADP Ribosa Transferasas , Azospirillum brasilense/efectos de los fármacos , Azospirillum brasilense/enzimología , Dinitrogenasa Reductasa/genética , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Glicósido Hidrolasas , Mutagénesis Sitio-Dirigida , Fijación del Nitrógeno/genética , Rhodospirillum rubrum/enzimología , Rhodospirillum rubrum/genética , Especificidad de la EspecieRESUMEN
Reversible ADP ribosylation of dinitrogenase reductase, catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase activating glycohydrolase (DRAG) regulatory system, has been characterized in both Rhodospirillum rubrum and Azospirillum brasilense. Although the general functions of DRAT and DRAG are very similar in these two organisms, there are a number of interesting differences, e.g., in the timing and extent of the regulatory response to different stimuli. In this work, the basis of these differences has been studied by the heterologous expression of either draTG or nifH from A. brasilense in R. rubrum mutants that lack these genes, as well as the expression of draTG from R. rubrum in an A. brasilense draTG mutant. In general, these hybrid strains respond to stimuli in a manner similar to that of the wild-type parent of the recipient strain rather than the wild-type source of the introduced genes. These results suggest that the differences seen in the regulatory response in these organisms are not primarily a result of different properties of DRAT, DRAG, or dinitrogenase reductase. Instead, the differences are likely the result of different signal pathways that regulate DRAG and DRAT activities in these two organisms. Our results also suggest that draT and draG are cotranscribed in A. brasilense.
Asunto(s)
ADP Ribosa Transferasas/genética , Azospirillum brasilense/genética , Regulación Bacteriana de la Expresión Génica , Glicósido Hidrolasas/genética , N-Glicosil Hidrolasas , Rhodospirillum rubrum/genética , ADP Ribosa Transferasas/biosíntesis , Adenosina Difosfato Ribosa/metabolismo , Azospirillum brasilense/enzimología , Azospirillum brasilense/efectos de la radiación , Oscuridad , Dinitrogenasa Reductasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/biosíntesis , Luz , Fijación del Nitrógeno/fisiología , Nitrogenasa/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Rhodospirillum rubrum/enzimología , Rhodospirillum rubrum/efectos de la radiación , Especificidad de la Especie , Transcripción GenéticaRESUMEN
The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.