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

Background: In Enterobacteriaceae, beta-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 beta-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 beta-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of beta-lactam antibiotics. Low-levels of beta-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 beta-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is beta-lactam and ampR-independent, while ampP operon expression is beta-lactam and ampR-dependent. beta-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions: In P. aeruginosa, beta-lactamase induction occurs in at least three ways, induction at low beta-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 beta-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.