RESUMEN
The bacterial haemoglobin from Vitreoscilla, VHb, displays several unusual properties that are unique among the globin family. When the gene encoding VHb, vgb, is expressed from its natural promoter in either Vitreoscilla or Escherichia coli, the level of VHb increases more than 50-fold under hypoxic conditions and decreases significantly during oxidative stress, suggesting similar functioning of the vgb promoter in both organisms. In the present study we show that expression of VHb in E. coli induced the antioxidant genes katG (catalase-peroxidase G) and sodA (superoxide dismutase A) and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of the oxidative stress regulator OxyR in mediating the protective effect of VHb under oxidative stress. A putative OxyR-binding site was identified within the vgb promoter and a gel-shift assay confirmed its interaction with oxidized OxyR, an interaction which was disrupted by the reduced form of the transcriptional activator Fnr (fumurate and nitrate reductase). This suggested that the redox state of OxyR and Fnr modulates their interaction with the vgb promoter. VHb associated with reduced OxyR in two-hybrid screen experiments and in vitro, converting it into an oxidized state in the presence of NADH, a condition where VHb is known to generate H2O2. These observations unveil a novel mechanism by which VHb may transmit signals to OxyR to autoregulate its own biosynthesis, simultaneously activating oxidative stress functions. The activation of OxyR via VHb, reported in the present paper for the first time, suggests the involvement of VHb in transcriptional control of many other genes as well.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Represoras/metabolismo , Hemoglobinas Truncadas/metabolismo , Anaerobiosis , Proteínas Bacterianas/genética , Secuencia de Bases , Western Blotting , Catalasa/genética , Catalasa/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Hemoglobinas Truncadas/genética , Técnicas del Sistema de Dos Híbridos , Vitreoscilla/efectos de los fármacos , Vitreoscilla/genética , Vitreoscilla/metabolismoRESUMEN
Vitreoscilla becomes resistant to killing by hydrogen peroxide and heat shock when pretreated with nonlethal levels of hydrogen peroxide. The pretreated Vitreoscilla cells (60 microM hydrogen peroxide for 120 min) significantly increased survival of the lethal dose of 20 mM hydrogen peroxide or heat shock (22 degrees C --> 37 degrees C). This indicates the existence of an adaptive response to oxidative stress. However, cells pretreated with 60 microM hydrogen peroxide became nonresistant to a lethal dose of a menadione. This result shows that hydrogen peroxide does not induce cross-resistance to menadione in Vitreoscilla. Furthermore, Vitreoscilla treated with hydrogen peroxide, heat shock, and menadione showed a change in the protein composition, as monitored by a two-dimensional gel analysis. During adaptation to hydrogen peroxide, 12 proteins were induced. Also, 18 new proteins synthesized in response to heat shock were detected by a 2-D gel analysis. The redox-cycling agents also elicited the synthesis of 6 other proteins that were unseen with hydrogen peroxide.
