Regulation of nif expression by the GlnK and GlnB proteins of Herbaspirillum seropedicae
Noindorf, L; Steffens, M. B. R.; Souza, E. M.; Pedrosa, F.O.; Chubatsu, L. S.
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19046, Curitiba, PR, 81530-350, Brasil
The PII protein family is a group of trimeric proteins that function as transducers of the cellular nitrogen status in prokaryotes. In Azospirillum brasilense, Rhodospirillum rubrum and Rhodobacter capsulatus a PII-like protein, GlnB, is required to activate NifA, the transcription activator of nitrogen fixation.
In H. seropedicae two PII-like proteins, GlnB and GlnK, were identified. The glnB gene is in a monocistronic operon and is expressed constitutively. The glnK gene is located in the orf1glnKamtB operon and is highly expressed under nitrogen-limiting conditions.
To determine the roles of glnB and glnK genes, knock out mutant strains were constructed by inserting TcR and sacB-KmR cassettes, respectively. The glnB mutant had wild-type nitrogenase activity, whereas in the glnK mutant nitrogenase activity was much reduced. The wild-type phenotype of this mutant was restored by the plasmid pLAFR3.18OGA, expressing the orf1glnKamtB operon of H. seropedicae. A plasmid expressing the N-truncated NifA protein of H. seropedicae also restored the wild-type phenotype of the glnK mutant. This result suggests that GlnK is involved in activation of NifA in H. seropedicae, possibly by interaction with the N-terminal domain of this protein.
The glnK mutant was also partially restored by a plasmid expressing the glnB gene of H. seropedicae, suggesting that high levels of GlnB can substitute for GlnK in the activation of NifA.
Supported by CNPq, PRONEX (FINEP/CNPq/MCT), CNPq/PADCTIII, Paraná Tecnologia and Fundação Araucária
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