The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2 )-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of ␤-galactosidase from an aac(2 )-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2 )-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD, Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2 )-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2 )-Ia gene (P. N. Rather, E. Orosz, K. J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498, 1993).In gram-negative bacteria, chromosomal acetyltransferases such as the aac(2Ј)-Ia gene product in Providencia stuartii, the aac(6Ј)-Ig gene product in Acinetobacter haemolyticus, and the aac(6Ј)-Ic gene product in Serratia marcescens appear to be intrinsic to these species and confer aminoglycoside resistance when overexpressed (6,30,31,33,40). In P. stuartii, studies on aac(2Ј)-Ia regulation have shown that wild-type strains express the aac(2Ј)-Ia gene at low levels; however, mutants with increased expression can be selected in the presence of aminoglycosides at frequencies of 10 Ϫ6 to 10 Ϫ7 (31). These mutants display increased aac(2Ј)-Ia mRNA accumulation and can result from recessive mutations in the aarA (30), aarB (previously called aar3 [31]), and the aarC (30) genes. Although these mutations lead to increased aac(2Ј)-Ia transcription, it is not known if the aarA, aarB, and aarC gene products directly regulate aac(2Ј)-Ia. Alternatively, they may indirectly regulate aac(2Ј)-Ia by repressing another transcriptional regulator such as an activator.The role of transcriptional activators in the expression of chromosomal antibiotic resistance genes has been clearly established. The chromosomal AmpC ␤-lactamase is under the control of the activator AmpR (23a), and genes involved in multiple antibio...

The 2-N-acetyltransferase [AAC(2)-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2)-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in ␤-galactosidase accumulation from an aac(2)-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2)-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2)-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein.The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

A recessive mutation, aarG1, has been identified that resulted in an 18-fold increase in the expression of beta-galactosidase from an aac(2')-lacZ fusion. Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2')-Ia. The effects of aarG1 on aac(2')-Ia expression were mediated by aarP-dependent and -independent mechanisms. The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance. This Mar phenotype also resulted from aarP-dependent and -independent mechanisms. Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem. The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ. The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase. The AarG product was functionally similar to PhoQ, as it was able to restore wild-type levels of maganin resistance to a Salmonella typhimurium phoQ mutant. However, expression of the aarP and aac(2')-Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.

The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.