Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB beta-lactamases, proteases, quorum sensing, and other virulence factors

Abstract

In members of the family Enterobacteriaceae, ampC, which encodes a beta-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 beta-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 beta-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the beta-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.

FIG. 1.

Physical map of ampR-ampC loci (A) and plasmids (B). (A) The restriction map of the region is based on the PAO1 genome sequence with relevant restriction sites. The 5′ end of ampC (labeled ampC') and the entire ampR open reading frames are indicated. The 1.2-kb fragment has the PAO1 genome coordinates 4594088 to 4592869. SBJ03 and SBJ04 show the position where the primers annealed for the promoter amplification. The striped box at positions 197 to 207 is where ligand-bound AmpR is postulated to bind. (B) The horizontal lines below the map show the amount of PAO1 genome DNA present in each plasmid. The restriction sites, EcoRI and BamHI, flanking the ends were introduced in the primers used for PCR (Table 1). Plasmids pSJ01 and pSJ06 are derivatives of pGEMEX (Promega) and pME6030 (19), respectively. The plasmid pSJ06 is used for complementation analysis and is referred to as pAmpR. The gentamicin cassette (▾) was inserted into the SalI site of pSJ01, creating pSJ05. The ampR::aacCI (ampR::Gm) fragment was subcloned into the suicide vector pEX100T (48) to generate pSJ07, which was introduced into PAO1 to generate PAOampR by homologous recombination. A 342-bp fragment containing the promoters was cloned in both directions, upstream of promoterless lacZ (indicated by the striped arrows) in mini-CTX-lacZ (4) to generate the transcriptional fusions for ampC (pSJ10) and ampR (pSJ11) promoters. These fusions were introduced into PAO1 and PAOampR by site-specific recombination using the attP site (4).