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ABSTRACTIn enterobacteria, the ampGgene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampCβ-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosacarries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coliAmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosastrains PAO1 and PAK, inactivation of ampGdrastically repressed the intrinsic β-lactam resistance while ampGh1deletion had little effect on the resistance. Further, deletion of ampGin an ampD-null mutant abolished the high-level β-lactam resistance that is associated with the loss of AmpD activity. The cloned ampGgene is able to complement both the P. aeruginosaand the E. coli ampGmutants, while that of ampGh1failed 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. aeruginosacan effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to β-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 β-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.

Pseudomonas aeruginosa harbors a chromosomal aminoglycoside phosphotransferase gene, aph(3')-IIb, which confers P. aeruginosa resistance to several important aminoglycoside antibiotics, including kanamycin A and B, neomycin B and C, butirosin, and seldomycin F5. The aph(3')-IIb gene has been found to be regulated by an AraC-type transcriptional regulator (HpaA) encoded by a gene located upstream of the aph(3')-IIb gene. In the presence of 4-hydroxyphenylacetic acid (4-HPA), HpaA activates the expression of aph(3')-IIb as well as that of the hpa regulon which encodes metabolic enzymes for the utilization of 4-HPA. hpaA and aph(3')-IIb form an operon, and in response to the presence of 4-HPA, the wild-type P. aeruginosa strain PAK (but not its hpaA mutant strain) displays increased resistance to neomycin. A survey of 39 clinical and 19 environmental isolates of P. aeruginosa demonstrated in all of them the presence of an hpaA-aph gene cluster, while 56 out of the 58 isolates are able to utilize the 4-HPA as a sole carbon source, suggesting a feature common to P. aeruginosa strains. Interestingly, a larger portion of clinical isolates than environmental isolates showed 4-HPA-induced resistance to neomycin. The aph(3')-IIb gene product is likely to function as a metabolic enzyme which has a cross-reactivity with aminoglycosides. These findings provide new insight into the possible mechanism of P. aeruginosa antibiotic resistance.

ABSTRACTPseudomonas aeruginosacauses various acute and chronic infections in humans. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosainfections. By binding to the exit tunnel of the 50S ribosome, AZM causes ribosome stalling and depletion of the intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosacells and repress quorum sensing-regulated virulence factors as well as swarming motility. In P. aeruginosa, the PA5470 gene encodes a putative peptide chain release factor whose expression is highly induced by macrolide antibiotics. However, its function remains unknown. Here, we found that overexpression of PA5470 increased bacterial tolerance against AZM and alleviated the repression of swarming motility. Ribosome pulldown assays revealed that PA5470 contributes to the release of ribosome stalled by AZM. We further demonstrate that overexpression of PA5470 counteracts AZM-mediated repression of the translation of the quorum sensing regulator RhlR. Overall, our results revealed a novel role of PA5470 in the bacterial response to AZM.

ABSTRACTBacterial oligoribonuclease (Orn) is a conserved 3′-to-5′ exonuclease. In Pseudomonas aeruginosa, it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis of P. aeruginosa, indicating a role of Orn in the bacterial response to environmental stimuli. Here we report that Orn is required for the tolerance of P. aeruginosato ciprofloxacin. Transcriptome analysis of an ornmutant revealed the upregulation of pyocin biosynthesis genes. Mutation of genes involved in pyocin biosynthesis in the background of an ornmutant restored bacterial tolerance to ciprofloxacin. We further demonstrate that the upregulation of pyocin biosynthesis genes is due to RecA-mediated autoproteolysis of PrtR, which is the major negative regulator of pyocin biosynthesis genes. In addition, the SOS response genes were upregulated in the ornmutant, indicating a DNA damage stress. Therefore, our results revealed a novel role of Orn in bacterial tolerance to ciprofloxacin.