The expression of nifB gene from Herbaspirillum seropedicae is dependent upon the NifA and RpoN proteins

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Abstract:

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a –24/–12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.Key words: Herbaspirillum seropedicae, nif, nitrogen fixation, NifA, RpoN.

La région promotrice présumée de nifB de Herbaspirillum seropedicae contient deux séquences de liaison de NifA homologues ainsi qu'un promoteur de type –24/–12. Une protéine de fusion nifB::lacZ a été testée chez Escherichia coli et H. seropedicae. Chez E. coli, l'expression de nifB::lacZ est survenue seulement en présence des gènes rpoN et nifA de Klebsiella pneumoniae fonctionnels. De plus, la protéine IHF a stimulé l'expression de nifB::lacZ dans cet environnement. Chez H. seropedicae, l'expression de nifB est survenue uniquement en absence d'ammonium et sous de faibles niveaux d'oxygène, et s'est révélée strictement dépendante de NifA. Des expériences de retard sur gel ont montré que les protéines purifiées RpoN de K. pneumoniae et IHF de E. coli étaient capables de se lier à la région du promoteur de nifB et l'empreinte au DMS in vivo a montré que NifA se lie aux deux sites de liaison de NifA. Ces résultats suggèrent fortement que l'activité du promoteur de nifB chez H. seropedicae est dépendante des protéines NifA et RpoN et que la protéine IHF stimule l'activité de NifA envers le promoteur de nifB.Mots clés : Herbaspirillum seropedicae, nif, fixation d'azote, NifA, RpoN.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: December 1, 2006

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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