CHARACTERIZATION OF GENTAMICIN 2'-N-ACETYLTRANSFERASE FROM PROVIDENCIA-STUARTII - ITS USE OF PEPTIDOGLYCAN METABOLITES FOR ACETYLATION OF BOTH AMINOGLYCOSIDES AND PEPTIDOGLYCAN

The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro. Adaptation of the assay for peptidoglycan N-->O-ace tyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gen tamicin N-acetyltransferase [EC 2.3.1.59; AAC(2')]. The peptidoglycan from cells of P. stuartii PR50 was prelabelled with H-3 by growth in t he presence of N-[acetyl-H-3]glucosamine. Under these conditions, [H-3 ]acetate was confirmed to be transferred to the C-6 position of peptid oglycan-bound N-acetylmuramyl residues. Isolated cells were subsequent ly incubated in the presence of various concentrations of gentamicin a nd tobramycin (0 to 5x MIC). Analysis of various cellular fractions fr om isolated cells and spent culture medium by the aminoglycoside-bindi ng phosphocellulose paper assay revealed increasing levels of radioact ivity associated with the filters used for whole cell sonicates of cel ls treated with gentamicin up to 2x MIC. Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of c ell lysis. Similar results were obtained with tobramycin, but the incr easing trend was less obvious. The transfer of radiolabel to either am inoglycoside was not observed with P. stuartii PR100, a strain that is devoid of AAC(2')-Ia. A high-performance anion exchange chromatograph y-based method was established to further characterize the AAC(2')-Ia- catalyzed acetylation of aminoglycosides. The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation i nto two peeks, both of which were collected and confirmed by H-3 nucle ar magnetic resonance to be the antibiotic. Authentic standards of 2'- N-acetyltobramycin were prepared and were well separated from the pare nt antibiotic when subjected to the HPLC analysis. By applying this te chnique, the transfer of radiolabelled acetate from the cell wall poly mer peptidoglycan to tobramycin was confirmed. In addition, isolated a nd purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, an d N-acetylglucosamine to tobramycin. These data further support the pr oposal that AAC(2')-Ia from P. stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides i s simply fortuitous.