Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase

Abstract

Escherichia coli cells carrying the gltX351 allele are unable to grow at 42° C (T^s phenotype) due to an altered glutamyl-tRNA synthetase. We found that gltX351 cells display a new phenotype termed Gsd⁻, i.e. an inability to raise glutamine synthetase activity above low constitutive levels in minimal medium with 6.8 mM glutamine as sole nitrogen source. When 0.5 mM NH ⁺₄ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wildtype 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 mutations 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 recessive for the T^r phenotype. A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also observed but this could not account for the Gsd⁻ phenotype of gltX351 cells. In cells carrying gltX351 and a suppressor of the Gsd⁻ phenotype, sup-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-oxoglutarate intracellular ratio were even higher than in the gltX351 single mutant. These results indicate that the gltX351 mutant polypeptide may be the direct cause of the Gsd⁻ phenotype. The possibility that it interacts with one or more components that trigger the Ntr response is discussed.