Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity

J Mol Biol. 1990 Mar 20;212(2):319-29. doi: 10.1016/0022-2836(90)90127-8.

Abstract

Escherichia coli strains with abnormally high concentrations of bis(5'-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.

MeSH terms

  • Acid Anhydride Hydrolases*
  • Aminoglycosides*
  • Anti-Bacterial Agents / pharmacology
  • Blotting, Southern
  • Cloning, Molecular
  • Drug Resistance, Microbial / genetics
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Escherichia coli / growth & development
  • Genes, Bacterial
  • Mutation
  • Operon
  • Phenotype
  • Phosphoric Diester Hydrolases / genetics*
  • Phosphoric Diester Hydrolases / metabolism
  • Plasmids
  • Restriction Mapping
  • Temperature

Substances

  • Aminoglycosides
  • Anti-Bacterial Agents
  • Phosphoric Diester Hydrolases
  • Acid Anhydride Hydrolases
  • bis(5'-nucleosyl)tetraphosphatase (symmetrical)
  • kasugamycin