9405882 Rather Providencia stuartii is a gram negative member of the Enterobacteriacea and closely related to the Proteus spp. It contains a chromosomal 2'-N-acetyltransferase gene aac(2')-la that was originally identified because overexpression resulted in high-level resistance to the aminoglycoside class of antibiotics. Subsequent studies have demonstrated that aac(2')-la is universally present in Providencia stuartii and may encode an enzyme that acetylates peptidoglycan, a modification proposed to control the activity of autolysins. The ability of the AAC(2')-la enzyme to inactivate aminoglycosides by acetylation may occur due to the structural similarities between peptidoglycan and the aminoglycosides. Wild-type P. stuartii strains express the aac(2')-la gene at low levels and do not exhibit aminoglycoside resistance. However, spontaneous mutants with increased aac(2')-la expression can be selected on media containing aminoglycosides and occur at frequencies between 10-6 and 10-7. Using transposon (mini-Tn5Cm) mutagenesis, insertional mutations have been isolated in three distinct trans acting loci, aarA, aarC, and aarD, which result in high-level aac(2')-la expression. Mutations in these loci also result in dramatic changes in cell morphology, indicating that aac(2')-la expression is also negatively regulated by cell to cell signaling via a diffusible factor termed AR-factor. AR-factor accumulation leads to decreased levels of aac(2')-la expression. Cells defective in a locus designated aarE, fail to produce AR-factor and exhibit high level aac(2')-la expression and a spherical cell morphology instead of the wild-type rod shape. The objective of this research is to characterize the complex negative regulatory mechanisms controlling aac(2')-la expression, and to begin addressing the cellular function of this gene in P. stuartii. This will involve isolation and sequence analysis of the aarA, aarC, aarD, and aarE genes. The role of these genes in aac(2') la regulation will be addressed by determining if they act independently, or in a cascade-like manner. Dependence pathways for expression of the aarA, C, D, and E genes will be examined by probes to these genes in Northern blots using RNA prepared form wild-type and aar A, C, D, and E mutants. The role of AR-factor in expression of these regulatory genes will also be examined by RNA analysis, as will the effect of AR-factor on aac(2')-la transcription. To determine if AR-factor regulates additional genes, random transcriptional fusions to lacZ will be generated using mini-Tn5 Km lacZ and screened for induction or repression by AR-factor. Finally, the role of aac(2')-la in P. stuartii will be addressed by a constructing a P. stuartii strain with the aac(2') deleted and/or constructing a strain with the chromosomal aac(2')-la gene under inducible control. These strains will allow us to directly address the proposed role of aac(2')-la in peptidoglycan acetylation and demonstrate if aac(2')-la overexpression is sufficient to change the morphology of P. stuartii. %%% This research may illuminate unsuspected areas of cell growth and development. ***
