Complete deficiency of 5'-nucleotidase activity in Escherichia coli leads to loss of growth on purine nucleotides but not of their excretion.
Escherichia coli has many periplasmic phosphatase activities. To test whether it can take up and excrete purine nucleotides, we attempted to completely disrupt periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they were grown with purine nucleotides as the sole carbon source. Using DNA macroarrays to compare global gene expression in wild-type and ushA knockout mutant cells cultured with IMP or GMP as the sole carbon source, we identified two genes that were induced in the ushA knockout mutant cells and encoded signal sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase activity and was induced by IMP or inosine. An ushA aphA double knockout mutant was shown to be unable to grow on purine nucleotides as the sole carbon source. To investigate the excretion of purine nucleotides, we constructed an ushAaphA double knockout mutant of an inosine-producing strain and found that it accumulated IMP in the medium. In addition, when the guaBA operon was introduced into the ushAaphA double knockout IMP producer, GMP was released into the medium. These observations imply the existence of efflux activity for purine nucleotides in E. coli.
Kakehi
Masahiro
M
Fermentation and Biotechnology Laboratories, Ajinomoto Co., Kawasaki, Japan.
Usuda
Yoshihiro
Y
Tabira
Yukiko
Y
Sugimoto
Shinichi
S
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006157:Guanosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007291:Inosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011685:Purine Nucleotides / Q000378:metabolism* / Q000494:pharmacology
D029968:Escherichia coli Proteins; D011685:Purine Nucleotides; C406334:ushA protein, E coli; D007291:Inosine Monophosphate; D006157:Guanosine Monophosphate; D000135:Acid Phosphatase; C508128:AphA protein, E coli; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases