Mutations affecting motifs of unknown function in the central domain of nitrogen regulatory protein C

The positive control function of the bacterial enhancer-binding protein Ntr C resides in its central domain, which is highly conserved among activators of sigma(54) holoenzyme. Previous studies of a small set of mutant forms s pecifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determina nts in the central domain. In this more comprehensive survey, the DNA encod ing a major portion of the central domain was randomly mutagenized and muta ted ntrC genes were introduced into the cell via multicopy expression plasm ids. DNA sequencing of 95 isolates identified by a preliminary phenotypic s creen revealed that the lesions in them caused 55 distinct single amino aci d 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 co ntained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant pr oteins 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 hi gh levels of expression, which apparently decreased activation by these mut ant proteins but not by wild-type NtrC (NtrC(WT)). Of particular interest w ere a subset of these proteins that exhibited greater transcriptional activ ation than NtrC(WT) at low concentrations. Their elevated activation capaci ties remain to be explained.