CHARACTERIZATION OF ESCHERICHIA-COLI GLNL MUTATIONS AFFECTING NITROGEN REGULATION

Nitrogen regulator II (NR(II)), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NR(I). Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NR(I)-phosphate phosphatase activity that is observed when NR(II) and another signal transduction protein known as P(II) (the glnB product) interact. The availability of P(II) for this interaction is controlled by a uridylyltransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies P(II) in response to intracellular signals of nitrogen availability. Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation. The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains. Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NR(II) autophosphorylation at His-139. Complementation experiments with multicopy plasmids encoding NR(II) or P(II) showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NR(II) but was not affected by introduction of the plasmid encoding P(II). Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NR(II) or that encoding P(II). We hypothesize that mutants of the latter type result in a subtle perturbation of the NR(II)-P(II) interaction and suggest two possible mechanisms for their effects.