The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.
SummaryAmmonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the PII family. In this study we have characterized in vitro complex formation between the AmtB and PII proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-PII complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg 2+ and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P II complex defines a new regulatory role for Amt proteins in Prokaryotes.
The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.
Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.
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