Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Abstract

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.

Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling

“…Effects of adenine nucleotides and 2-OG levels on in vitro uridylylation of A. brasilense P II We showed previously that in vitro uridylylation of the A. brasilense P II proteins by GlnD required ATP and 2-OG (Araú jo et al, 2004(Araú jo et al, , 2008. Given that P II can also bind ADP we investigated whether this molecule could promote or affect P II uridylylation in vitro.…”

“…Uridylylation assays were performed as described previously (Araú jo et al, 2008). The reactions contained 10 mM Tris/HCl pH 7.5, 10 mM KCl, 25 mM MgCl 2 , 1 mM UTP, 3 mM GlnB or GlnZ, 100 nM GlnD and the indicated concentration of ATP, ADP and 2-OG.…”

Mutagenesis and Functional Characterization of the Four Domains of GlnD, a Bifunctional Nitrogen Sensor Protein

“…GlnB represents the unmodified form, while GlnB-UMP, GlnB-(UMP) 2 , and GlnB-(UMP) 3 represent forms with one, two, and three modified subunits, respectively.…”

“…It appears that the uridylylation state of P II is largely dependent on the level of glutamine in the cell (34). Recently, in vitro studies of GlnD activities in other bacteria, such as Azospirillum brasilense, Herbaspirillum seropedicae, and R. rubrum, were also reported (2,3,10,13,38).…”

The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

“…brasilense GlnB (25), GlnZ (26), GlnZQ42HS52VS54D, GlnZS52VS54D, GlnBH42Q, GlnB⌬42-54, His-tagged DraT (27), and His-tagged GlnD (28) and recombinant proteins were overexpressed in E. coli strain BL21(DE3) harboring the plasmids listed in Table 1. The P II proteins (GlnB, GlnZ, and GlnB⌬42-54) were purified using a thermal treatment at 70°C for 15 min prior to loading on the 5-ml HiTrap heparin column (GE Healthcare) (26).…”