Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity.
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.
Lévĕque
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F
Laboratoire de Biochimie, Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Blanchin-Roland
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S
Fayat
G
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Plateau
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P
Blanquet
S
S
D017766:Acid Anhydride Hydrolases; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D009154:Mutation; D009876:Operon; D010641:Phenotype; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D013696:Temperature
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D010727:Phosphoric Diester Hydrolases; D017766:Acid Anhydride Hydrolases; C036849:bis(5'-nucleosyl)tetraphosphatase (symmetrical); C100305:kasugamycin