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. 2010 Dec 30:10:328.
doi: 10.1186/1471-2180-10-328.

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

Kok-Fai Kong  1 , Alian Aguila, Lisa Schneper, Kalai Mathee
Affiliations

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

Kok-Fai Kong et al. BMC Microbiol. .

Abstract

Background: In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link.

Results: Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP) and PA4393 (ampG). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP and ampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampP operon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP.

Conclusions: In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampP and ampG dependent pathway, and at high concentrations where although both ampP and ampG play a role, ampG may be of greater importance. Both ampP and ampG are required for maximum induction. Similar to ampC, ampP expression is inducible in an ampR-dependent manner. Importantly, ampP expression is autoregulated and ampP also regulates expression of ampG. Both AmpG and AmpP have topologies consistent with functions in transport. Together, these data suggest that the mechanism of β-lactam resistance of P. aeruginosa is distinct from well characterized systems in Enterobacteriaceae and involves a highly complicated interaction between these putative permeases and known Amp proteins.

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Figures

Figure 1
Figure 1
Alignment of E. coli AmpG, PA4218 and PA4393. The primary sequence of E. coli AmpG, PA4218 (AmpP) and PA4393 (AmpG) were used as an input to M-Coffee, which combines multiple sequence alignments using the T-Coffee platform [45,46]. Identical and similar amino acids were shaded black and gray, respectively, using BOXSHADE.
Figure 2
Figure 2
Physical map of the ampO-ampP (A) and ampF-ampG (B) loci. The restriction map is based on PAO1 genome sequence with relevant restriction sites. (A) The 2779-bp ampO-ampP fragment has the PAO1 coordinates of 4721496 to 4724275. (B) The 2904-bp ampF-ampG fragment corresponds to the PAO1 coordinates of 4921591 to 4924494. The plasmids pKKF03 and pKKF04 are derivatives of pCRII-TOPO (Invitrogen, CA), whereas pKKF157 and pKKF161 are derivatives of pME6030 [41]. The Gm cassette (black inverted triangle) was inserted into the HincII and AscI sites of pKKF03 and pKKF04, respectively.
Figure 3
Figure 3
PCR analysis of ampFG and ampOP operon cDNA. Polyacrylamide gel electrophoresis of PCR products of the junctions of the ampOP and ampFG operons. (A) PCR with primers PA4392_3junctionRTF and PA4392_3junctionRTR to amplify the PA4392 - PA4393 intergenic region. (B) PCR with primers PA4218_9junctionRTF and PA4218_9junctionRTR to amplify the PA4392 - PA4393 intergenic region. (Panels A and B) Lane M: PCR markers (Promega, Madison, WI). Lane 1, cDNA reaction performed with PAO1 RNA, the appropriate buffer and Superscript RT III. Lane 2, cDNA reaction performed with PAO1 RNA, the appropriate buffer without Superscript RT III. Lane 3, P. aeruginosa genomic DNA. The asterisk indicates a nonspecific product. Arrows indicate junction amplicons.
Figure 4
Figure 4
Topology of AmpP and AmpG. The topology of AmpP and AmpG was analyzed by in-frame ampP and ampG fusions to the lacZ and phoA genes, the cytoplasmic and periplasmic markers, respectively. The corresponding points of fusion and qualitative biochemical results of the β-galactosidase (LacZ) and alkaline phosphatase (PhoA) assays [44] are shown for AmpP (A) and AmpG (C). These results, together with transmembrane domain predictions generated using a Kyte-Doolittle algorithm present in Lasergene 7 (DNASTAR, Madison, WI) were used to predict the topology of AmpP (B) and AmpG (D). Solid lines indicate prediction based upon experimental data, dashed lines indicate regions where more than one possibility exists. Cytoplasm and periplasm are denoted by Cyto and Peri, respectively. Fusion sites are indicated by a dot with the corresponding amino acid number. Putative transmembrane domain boundaries were obtained from Lasergene.
Figure 5
Figure 5
Relative β-lactamase activity in PAOampP and PAOampG mutants. Assays were performed on the parental PAO1, and the mutants, PAOampP and PAOampG in the presence of benzyl-penicillin at a concentration gradient of 0 to 125 μg/ml. Cultures at OD600 of 0.6-0.8 were induced for three hours before harvesting. Assays were performed on sonicated lysate using nitrocefin as a chromogenic substrate. The β-lactamase activity of PAO1 at 100 μg/ml of benzyl-penicillin was taken as 100%. Each value is the mean of at least three independent experiments. The asterisk refers to p-values of < 0.05 with respect to PAO1, which were calculated using the two-tailed Student's t-test.
Figure 6
Figure 6
Activity of the ampC promoter. Promoter activity of the ampC gene was analyzed using lacZ transcriptional fusions integrated at the att locus of PAO1, PAOampR, PAOampG and PAOampP (see Materials and Methods and text for details). Cells were grown to an OD600 of 0.6 - 0.8, at which time cultures were divided into two and one set treated with 100 μg/ml benzyl-penicillin. After three hours, cells were harvested and β-galactosidase activity assayed as described [10]. Each value is the mean of at least three independent experiments.
Figure 7
Figure 7
Activity of the ampG and ampP promoters. Promoter activity of the ampG and ampP genes was analyzed using lacZ transcriptional fusions integrated at the att locus of PAO1, PAOampR, PAOampG and PAOampP (see Materials and Methods and text for details). Cells were grown to an OD600 of 0.6 - 0.8, at which time cultures were divided into two and one set treated with 100 μg/ml benzyl-penicillin. After three hours, cells were harvested and β-galactosidase activity assayed as described [10]. All 16 conditions were assayed at the same time but are divided into two panels for visualization purposes. Each value is the mean of at least three independent experiments. The asterisk refers to p-values < 0.05, which were calculated using the two tailed Student's t-test.
Figure 8
Figure 8
Model for regulation of AmpC β-lactamase induction by AmpR, AmpP and AmpG in P. aeruginosa. In Enterobacteriaceae as well as P. aerugniosa, the induction of β-lactamase expression is due to the action of the LysR transcriptional regulator, AmpR. In vitro studies suggest that AmpR can act as either a repressor or an activator, depending upon the presence of different peptidoglycan remodelling intermediates. In this study, it is shown that unlike previously characterized systems, P. aeruginosa has two putative AmpG permease paralogs, AmpG and AmpP. Expression of AmpP is inducible by β-lactam in an ampR-dependent manner. The ampP gene also appears to repress its own expression independent of β-lactam through an unknown mechanism. Although not observed to be induced by β-lactam in a PAO1 background, expression of ampG also appears to be repressed by ampP in the presence of β-lactam (see text for details).
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