CHARACTERIZATION AND TRANSCRIPTIONAL REGULATION OF THE 2'-N-ACETYLTRANSFERASE GENE FROM PROVIDENCIA-STUARTII

We have cloned the chromosomally encoded 2'-N-acetyltransferase gene [ aac(2')-Ia] from Providencia stuartii. DNA sequence analysis of the cl oned insert identified a single open reading frame, which is capable o f encoding a protein with a predicted molecular mass of 20,073 Da. The deduced AAC(2')-Ia protein showed no significant homology to other pr oteins, including all of the AAC(3) and AAC(6') proteins. Primer exten sion analysis was used to identify the aac(2')-Ia promoter, which cont ained an unusual sequence (CTTTTT) at the - 35 region. Expression of t he aac(2')-Ia gene occurs at low levels in wild-type P. stuartii strai ns; therefore, they are aminoglycoside susceptible. We have isolated m utants with high-level AAC(2')-Ia expression at a frequency of 4.8 x 1 0(-6). Detailed analysis of one mutant demonstrated a 12.2-fold increa se in the accumulation of aac(2')-Ia mRNA. In addition, the levels of beta-galactosidase expression from a plasmid-encoded aac(2')-lacZ tran scriptional fusion were increased 11.5-fold in this mutant relative to those in an isogenic wild-type strain. These results suggested that a trans-acting factor, designated aar (for aminoglycoside acetyltransfe rase regulator), controls AAC(2')-Ia expression in P. stuartii.