Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene.
During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt). Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis. These mutants have less than 10% of the transport activity of the parental strain. Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system. When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase. Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants. Therefore, the mutations are unlikely to lie within genes affecting Ntr elements. Following transformation with plasmid libraries of E. coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated. These plasmids were unable to complement the Amt- phenotype of Ntr- mutants. Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment. Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess. This suggests that the amt product contains domains which are exported to the periplasm.
Jayakumar
A
A
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Hwang
S J
SJ
Fabiny
J M
JM
Chinault
A C
AC
Barnes
E M
EM
D000641:Ammonia / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008744:Methylamines / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D008744:Methylamines; D000641:Ammonia; C027451:methylamine