Nitrogen regulation in an Escherichia coli strain with a temperature sensitive glutamyl-tRNA synthetase.
Escherichia coli cells carrying the gltX351 allele are unable to grow at 42 degrees C (Ts 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 NH4+ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wild-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 mutations appeared frequently in gltX351 cells, which suppressed their Gsd- but not their Ts phenotype. Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd- phenotype and recessive for the Tr 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.
Osorio
A V
AV
Departamento de Biologia Molecular, Universidad Nacional Autónoma de México, D.F.
Camarena
L
L
Salazar
G
G
Noll-Louzada
M
M
Bastarrachea
F
F
D004794:Enzyme Repression / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005785:Gene Conversion; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005809:Genes, Regulator; D005975:Glutamate-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000032:analysis; D016254:Mutagenesis, Insertional; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D010957:Plasmids; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012352:RNA, Transfer, Glu / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic; D013696:Temperature
D007656:Ketoglutaric Acids; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012352:RNA, Transfer, Glu; D005975:Glutamate-tRNA Ligase; D009584:Nitrogen