Localization of an amikacin 3'-phosphotransferase in Escherichia coli

Abstract

A plasmid-encoded enzyme reported to phosphorylate amikacin in a laboratory strain of E. coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H332PO4 were shown to utilize internally generated adenosine 5'-triphosphate in the phosphorylation of amikacin. The absence of 32P-phosphorylated amikacin after incubation of [γ-32P]adenosine 5'-triphosphate with spheroplasts confirmed that exogenous adenosine 5'-triphosphate was not used in the reaction. These results suggest an internal location of APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.