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Journal of Bacteriology, March 2006, p. 1866-1874, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1866-1874.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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, Mary C. Conrad, Yaoping Zhang, and Gary P. Roberts*

Department of Bacteriology and the Center for the Study of Nitrogen Fixation, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 27 October 2005/ Accepted 19 December 2005

In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three PII 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 NH4+, 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.

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, Madison, WI 53706. Phone: (608) 262-3567. Fax: (608) 262-9865. E-mail: groberts{at}bact.wisc.edu.

Journal of Bacteriology, March 2006, p. 1866-1874, Vol. 188, No. 5
0021-9193/06/$08.00+0     doi:10.1128/JB.188.5.1866-1874.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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