Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii

Abstract

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...

aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii

“…The chromosomal 2Ј-N-acetyltransferase [AAC(2Ј)-Ia] has a role in the O acetylation of peptidoglycan in P. stuartii (19) and also acetylates some aminoglycosides (3,20,26). As a result, P. stuartii cells with increased aac(2Ј)-Ia expression become resistant to higher levels of aminoglycosides (14,15,20,21). To identify genes involved in regulating aac(2Ј)-Ia expression, our strategy was to generate mutations that simultaneously increased expression of the chromosomal copy of aac(2Ј)-Ia and of an aac(2Ј)-lacZ fusion present on plasmid pR401 (21).…”

“…Previously, we had identified a transcriptional activator, AarP, which activates the aac(2Ј)-Ia gene (14). The increased aac(2Ј)-Ia expression that was observed in the aarC1 background may have resulted from increased aarP expression.…”

“…A Sau3AI recombinant library of P. stuartii chromosomal DNA in pACYC184 has been described previously (14). Plasmid pACYC184.aarC contains an approximately 1.7-kb Sau3A partial fragment in pACYC184.…”

“…E. coli XL1-Blue (Stratagene) and DH5␣ (Gibco/BRL) were used as hosts for the propagation of plasmids. In addition, to propagate R6K pir mutant derivatives, strains DH5␣ pir (14) and SM10 pir (18) were used. Plasmids pACYC184 (2), pBluescript SK(Ϫ) (Stratagene), pBCKS (Ϫ) (Stratagene), and pKNG101 (13) were used as cloning vectors.…”

Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase

“…To construct pET.TatA-His, the BamHI fragment from pAC.Tat3 was cloned into pET21a such that TatA-His 6 was transcribed from the T7 promoter. The pET.TatA-His 6 plasmid was electroporated into PR50 or PR51, which contained p184.T7 and encoded an IPTG-inducible T7 RNA polymerase gene (33). Transformants were selected on LB agar plates containing 400 g/ml ampicillin and 100 g/ml chloramphenicol.…”