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Journal of Bacteriology, September 1999, p. 5443-5454, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutations Affecting Motifs of Unknown Function in the Central Domain of Nitrogen Regulatory Protein C

Jieli Li, Luciane Passaglia,dagger Irene Rombel,Dagger Dalai Yan, and Sydney Kustu*

Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720-3102

Received 28 April 1999/Accepted 25 June 1999

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of sigma 54 holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated ntrC genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of glnA transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrCWT). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrCWT at low concentrations. Their elevated activation capacities remain to be explained.

* Corresponding author. Mailing address: 111 Koshland Hall, U. C. Berkeley, Berkeley, CA 94720-3102. Phone: (510) 643-9308. Fax: (510) 642-4995. E-mail: kustu{at}nature.berkeley.edu.

dagger Present address: Departamento de Genética/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, CEP 91501-970, Brazil.

Dagger Present address: University of Texas Southwestern Medical Center, Center for Biomedical Inventions and Department of Internal Medicine and Cardiology, Dallas, TX 75235-8573.

Journal of Bacteriology, September 1999, p. 5443-5454, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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