It has been shown in enterobacteria that mutations in ampD provoke hyperproduction of chromosomal ␤-lactamase, which confers to these organisms high levels of resistance to ␤-lactam antibiotics. In this study, we investigated whether this genetic locus was implicated in the altered AmpC ␤-lactamase expression of selected clinical isolates and laboratory mutants of Pseudomonas aeruginosa. The sequences of the ampD genes and promoter regions from these strains were determined and compared to that of wild-type ampD from P. aeruginosa PAO1. Although we identified numerous nucleotide substitutions, they resulted in few amino acid changes. The phenotypes produced by these mutations were ascertained by complementation analysis. The data revealed that the ampD genes of the P. aeruginosa mutants transcomplemented Escherichia coli ampD mutants to the same levels of ␤-lactam resistance and ␤-lactamase expression as wild-type ampD. Furthermore, complementation of the P. aeruginosa mutants with wild-type ampD did not restore the inducibility of ␤-lactamase to wild-type levels. This shows that the amino acid substitutions identified in AmpD do not cause the altered phenotype of AmpC ␤-lactamase expression in the P. aeruginosa mutants. The effects of AmpD inactivation in P. aeruginosa PAO1 were further investigated by gene replacement. This resulted in moderatebasal-level and hyperinducible expression of ␤-lactamase accompanied by high levels of ␤-lactam resistance. This differs from the stably derepressed phenotype reported in AmpD-defective enterobacteria and suggests that further change at another unknown genetic locus may be causing total derepressed AmpC production. This genetic locus could also be altered in the P. aeruginosa mutants studied in this work.

In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAcanhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC ␤-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic ␤-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level ␤-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to ␤-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of ␤-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpChyperproducing clinical isolates.

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Previous research showed that pigeons foraging for preferred grains of maize and less preferred grains of wheat respond to the presence of a competitor by becoming less choosy ( i.e. they more readily choose wheat). We extended this work by disentangling the presence of the competitor from the resource depletion associated with it. In Experiment 1, eight birds foraged for maize and wheat in a flight cage. They were tested both alone and with another bird in different foraging sessions. Two groups were formed and differed in how they were treated when the birds foraged in pairs: both birds had access to food (Both Eat) versus only one bird had access to food (Only One Eats) while the other was placed behind a transparent curtain. For the latter treatment, no effect of the mere presence of the other bird on choice was found. Pigeons were, however, less choosy in the presence of a real competitor that exploited the food. At the end of the experiment, the pigeons that had been tested in the 'Both Eat' treatment, were tested in the 'Only One Eats' treatment. They behaved as if foraging alone. Experiment 2 introduced a new condition in which one bird ate and the other was placed behind the curtain where it pecked at grain but could not eat it. Even though this other bird behaved as if it were a potential competitor, it had no effect on the forager in the pair. The only significant difference in choice proportions was between birds foraging in real competition ( i.e. where the other bird caused resource depletion) or not. In competitive feeding situations, pigeons react to what is most directly associated with the dwindling food conditions but are insensitive to the presence and to the pecking behaviour of the animal that creates them.