Pseudomonas aeruginosaAmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors

Abstract

In members of the family Enterobacteriaceae, ampC, which encodes a ␤-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR ؉ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal ␤-lactamase levels. However, this increase was not followed by a concomitant increase in the P ampC promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated ␤-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the ␤-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P ampR promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P lasA , P lasI , and P lasR expression. The reduction in the LasB activity was positively correlated with the P rhlR expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…PoxB is a carbapenemase, which affects b-lactam resistance, in the absence of the major b-lactamase AmpC and outer membrane porin OprD (PA0958) [220]. Although no direct relation is seen between cell-wall recycling and poxB, AmpR is found to be a negative regulator of poxB through a yet unknown mechanism [220,221].…”

The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide

“…The genes encoding AmpC and AmpR form a divergent operon (ampR-ampC), which allows AmpR to bind to overlapping promoters within the intergenic region of the operon to control the transcription of both genes (2). Moreover, AmpR has also been found to exert transcriptional control over an additional chromosomal class D ␤-lactamase gene as well as numerous virulence-associated genes in P. aeruginosa (3,4). Accordingly, there has been an increased interest to better understand how AmpR regulates gene expression.…”

“…Induction of chromosomal AmpC ␤-lactamase by the LysRtype transcriptional regulator (LTTR) 4 AmpR is a mechanism of ␤-lactam antibiotic resistance that is common to many enterobacteria as well as Pseudomonas aeruginosa and other nonfermenting Gram-negative bacilli. AmpC enzymes are of considerable medical concern because they confer resistance to a broad range of ␤-lactams, and their activity is not sufficiently suppressed with clinically available ␤-lactamase inhibitors (1).…”

Effects of Null Mutation of the Heat-Shock Gene htpG on the Production of Virulence Factors by Pseudomonas Aeruginosa

“…Then, 100-μl aliquots of the P. aeruginosa supernatants were mixed with 900 μl of the prepared S. aureus cell suspension, and the A 600 values were measured after 0, 10, 20, 30, 40 and 50 min at room temperature. LasA protease activity was determined as A 600 per mg protein total in supernatant [24]. Protein concentration was measured using a Pierce BCA assay (Sigma-Aldrich).…”