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. Various 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−1 ksgA− strains were not sensitive to kasugamycin, the apaH− apaG− ksgA− strain filamented and stopped growing in the presence of this antibiotic at 600 μg/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.