RESUMO
BACKGROUND: The PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In Herbaspirillum seropedicae, two PII-like proteins (GlnB and GlnK), encoded by the genes glnB and glnK, were identified. The glnB gene is monocistronic and its expression is constitutive, while glnK is located in the nlmAglnKamtB operon and is expressed under nitrogen-limiting conditions. RESULTS: In order to determine the involvement of the H. seropedicae glnB and glnK gene products in nitrogen fixation, a series of mutant strains were constructed and characterized. The glnK- mutants were deficient in nitrogen fixation and they were complemented by plasmids expressing the GlnK protein or an N-truncated form of NifA. The nitrogenase post-translational control by ammonium was studied and the results showed that the glnK mutant is partially defective in nitrogenase inactivation upon addition of ammonium while the glnB mutant has a wild-type phenotype. CONCLUSIONS: Our results indicate that GlnK is mainly responsible for NifA activity regulation and ammonium-dependent post-translational regulation of nitrogenase in H. seropedicae.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum/genética , Herbaspirillum/metabolismo , Fixação de Nitrogênio , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Mutagênese , Nitrogênio/metabolismo , Proteínas PII Reguladoras de Nitrogênio/genética , Regiões Promotoras Genéticas , Compostos de Amônio Quaternário/metabolismoRESUMO
This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.
Assuntos
Membrana Celular/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Herbaspirillum/química , Proteínas de Membrana Transportadoras/análise , Proteoma/análise , Compostos de Amônio Quaternário/metabolismo , Proteínas de Bactérias/análise , Proteínas de Transporte de Cátions/análise , Eletroforese em Gel Bidimensional , Herbaspirillum/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme that has a central role in the general nitrogen regulatory system NTR. In enterobacteria, GlnD uridylylates the PII proteins GlnB and GlnK under low levels of fixed nitrogen or ammonium. Under high ammonium levels, GlnD removes UMP from these proteins (deuridylylation). The PII proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy, and transduce this information to proteins involved in nitrogen metabolism. In Herbaspirillum seropedicae, an endophytic diazotroph isolated from grasses, several genes coding for proteins involved in nitrogen metabolism have been identified and cloned, including glnB, glnK and glnD. In this work, the GlnB, GlnK and GlnD proteins of H. seropedicae were overexpressed in their native forms, purified and used to reconstitute the uridylylation system in vitro. The results show that H. seropedicae GlnD uridylylates GlnB and GlnK trimers producing the forms PII (UMP)(1), PII (UMP)(2) and PII (UMP)(3), in a reaction that requires 2-oxoglutarate and ATP, and is inhibited by glutamine. The quantification of these PII forms indicates that GlnB was more efficiently uridylylated than GlnK in the system used.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Herbaspirillum/metabolismo , Proteínas PII Reguladoras de Nitrogênio/isolamento & purificação , Transdução de Sinais , UDPglucose-Hexose-1-Fosfato Uridiltransferase/isolamento & purificação , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida , Herbaspirillum/enzimologia , Proteínas PII Reguladoras de Nitrogênio/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismoRESUMO
Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium that colonizes economically important grasses. In this organism, the amtB gene is co-transcribed with two other genes: glnK that codes for a PII-like protein and orf1 that codes for a probable periplasmatic protein of unknown function. The expression of the orf1glnKamtB operon is increased under nitrogen-limiting conditions and is dependent on NtrC. An amtB mutant failed to transport methylammonium. Post-translational control of nitrogenase was also partially impaired in this mutant, since a complete switch-off of nitrogenase after ammonium addition was not observed. This result suggests that the AmtB protein is involved in the signaling pathway for the reversible inactivation of nitrogenase in H. seropedicae.