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. 2006 Mar;188(5):1866-74.
doi: 10.1128/JB.188.5.1866-1874.2006.

Identification of Rhodospirillum rubrum GlnB variants that are altered in their ability to interact with different targets in response to nitrogen status signals

Yu Zhu  1 , Mary C Conrad, Yaoping Zhang, Gary P Roberts
Affiliations

Identification of Rhodospirillum rubrum GlnB variants that are altered in their ability to interact with different targets in response to nitrogen status signals

Yu Zhu et al. J Bacteriol. 2006 Mar.

Abstract

In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (dinitrogenase reductase ADP-ribosyl transferase)-DRAG (dinitrogenase reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

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Figures

FIG. 1.
FIG. 1.
Model of the structure of R. rubrum GlnB. (A) Ribbon model of the E. coli GlnB trimer (6). The B monomer is shown with a dark backbone. A single ATP molecule, whose position is based on the structure of ATP-bound GlnK of E. coli (60, 61), is shown at the interface of monomers A and B. The T loops are indicated, and at the tip of each is Tyr51, the site of uridylylation. (B) The same view, but with the R. rubrum wild-type residues altered in the GlnB* variants shown as space filled. Only residues on one face of the trimer are shown, and their respective monomer is indicated by the “A” or “B” preceding the residue name.
FIG. 2.
FIG. 2.
Yeast two-hybrid results demonstrate relative interaction between selected GlnB* variants and NifA, DRAT, and NtrB. Strains expressing the proteins indicated were patched onto different plates that are increasingly restrictive for growth and therefore increasingly demanding of GlnB interaction with its receptors, from left to right. In the first four columns, the demand is for growth in SD medium without histidine and in the presence of increasing amounts of 3-amino-1,2,4-triazol (3-AT), a competitive inhibitor of His3 protein (9). The right column demands growth in the absence of adenine (−Ade). WT, wild type. The experiment was performed in quintuplicate, but only a single set of responses is shown.
FIG. 3.
FIG. 3.
Perturbed modification of GS in strains expressing GlnB* variants demonstrates altered regulation of GlnE activity. The figure shows a Western blot of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel developed with antibody to GS, where adenylylated GS appears as the upper of the two bands. All strains are glnB glnD but express wild-type (WT) GlnB (UR1683) or GlnB* variants (UR1685, -1687, -1689, -1691, -1693, and -1695) in single copy from an integrated plasmid. For each pair of lanes, the first shows GS from the cells grown in MG medium (nitrogen deficient, since glutamate serves as sole nitrogen source), and the second shows the GS 60 min after the addition of 10 mM NH4Cl. Lanes 15 and 16 contain similar preparations from UR2 (wild-type R. rubrum).
FIG. 4.
FIG. 4.
GlnJ accumulates in a glnB glnD background when GlnB* variants are expressed in single copy from an integrated plasmid. Cells were grown in MG medium, and proteins from crude extracts were separated by SDS-PAGE and immunoblotted with antibody against R. rubrum GlnJ. UR2 is the R. rubrum wild type (WT). UR1683 contains wild-type GlnB, while UR1685, -1687, -1689, -1691, -1693, and -1695 contain the GlnB* variants.
FIG. 5.
FIG. 5.
In vitro uridylyltransferase activity assays on wild-type (WT) GlnB and GlnB* variants show that many of these variants are dramatically altered in their properties as GlnD substrates. Wild-type GlnB and four GlnB* proteins were overexpressed and purified from E. coli (UQ2549, UQ4308, UQ4310, UQ4311, and UQ4312) and then used as substrates of E. coli GlnD as described in Materials and Methods. The specific GlnB protein used in each portion of the gel is shown at the top, and below that are the specific conditions for the samples in each lane. These are native gels, so that the protein trimer runs at as many as four positions, depending on the number of UMP groups per trimer, with the fully uridylylated form moving the fastest.
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