RESUMO
Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.
Assuntos
Proteínas de Bactérias/metabolismo , Herbaspirillum , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Sítio Alostérico , Escherichia coli/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutagênese , Proteínas PII Reguladoras de Nitrogênio/química , Proteínas PII Reguladoras de Nitrogênio/genética , Ligação ProteicaRESUMO
This work aimed to study the influence of pH (3.5 and 7.0) and CaCl2 and MgCl2 addition on heat-set gelation of a quinoa protein isolate at 10% and 15% (w/w). The protein isolate obtained was composed mainly of 11S globulin as was observed by electrophoresis and mass spectrometry analysis. Heat-set gelation occurred at both pH values studied. Nevertheless, the gels formed at pH 3.5 were more viscoelastic and denser than those formed at pH 7.0, that was coarser and presented syneresis. The CaCl2 and MgCl2 addition increased the gel strength during rheological analysis at pH 3.5, possibly due to the formation of fiber-like connections in the gel network. At pH 7.0, the divalent salts resulted in weaker gels formed by agglomerates, suggesting a neutralization of the protein surface charges. The differences in quinoa protein gelation were attributed to solubility, and the flexibility of proteins secondary structure at the pH studied.