NITROGEN REGULATION IN AN ESCHERICHIA-COLI STRAIN WITH A TEMPERATURE-SENSITIVE GLUTAMYL-TRANSFER-RNA SYNTHETASE

Escherichia coli cells carrying the gltX351 allele are unable to grow at 42-degrees-C (T(s) phenotype) due to an altered glutamyl-tRNA synth etase. We found that gltX351 cells display a new phenotype termed Gsd- , i.e. an inability to raise glutamine synthetase activity above low c onstitutive levels in minimal medium with 6.8 mM glutamine as sole nit rogen source. When 0.5 mM NH4+ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wi ld-type levels. Northern experiments showed that the Gsd- phenotype is the result of an impairment in transcription initiation from the Ntr- regulated promoter, glnAp2. Intragenic and extragenic secondary mutati ons appeared frequently in gltX351 cells, which suppressed their Gsd- but not their T(s) phenotype. Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd- phenotype and rece ssive for the T(r) phenotype. A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also obse rved but this could not account for the Gsd- phenotype of gltX351 cell s. In cells carrying gltX351 and a suppressor of the Gsd- phenotype, s up-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-o xoglutarate intracellular ratio were even higher than in the gltX351 s ingle mutant. These results indicate that the gltX351 mutant polypepti de may be the direct cause of the Gsd- phenotype. The possibility that it interacts with one or more components that trigger the Ntr respons e is discussed.