e Pseudomonas aeruginosa, a human opportunistic pathogen, possesses a number of antioxidant defense enzymes under the control of multiple regulatory systems. We recently reported that inactivation of the P. aeruginosa stringent response (SR), a starvation stress response controlled by the alarmone (p)ppGpp, caused impaired antioxidant defenses and antibiotic tolerance. Since catalases are key antioxidant enzymes in P. aeruginosa, we compared the levels of H 2 O 2 susceptibility and catalase activity in P. aeruginosa wild-type and ⌬relA ⌬spoT (⌬SR) mutant cells. We found that the SR was required for optimal catalase activity and mediated H 2 O 2 tolerance during both planktonic and biofilm growth. Upon amino acid starvation, induction of the SR upregulated catalase activity. Full expression of katA and katB also required the SR, and this regulation occurred through both RpoSindependent and RpoS-dependent mechanisms. Furthermore, overexpression of katA was sufficient to restore H 2 O 2 tolerance and to partially rescue the antibiotic tolerance of ⌬SR cells. All together, these results suggest that the SR regulates catalases and that this is an important mechanism in protecting nutrient-starved and biofilm bacteria from H 2 O 2 -and antibiotic-mediated killing.

SignificanceAntibiotic tolerance causes antibiotic treatment failure and promotes the emergence of genotypic resistance in chronic infections, such as those caused by the pathogen Pseudomonas aeruginosa. Laboratory stationary-phase bacteria exhibit a slow growing and metabolically quiescent state associated with high levels of multidrug tolerance likely analogous to the in vivo environment during chronic infection. We demonstrate that superoxide dismutases confer multidrug tolerance in stationary-phase bacteria, and identify a link between (p)ppGpp-mediated stress responses, superoxide metabolism, and membrane permeability to antibiotics. Inhibition of superoxide dismutase activity may overcome multidrug tolerance and potentiate current bactericidal antibiotics in the treatment of P. aeruginosa chronic infections.

cAs a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (⌬SR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acylhomoserine lactone) quorum-sensing systems, since the ⌬SR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoylhomoserine lactone (C 4 -HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C 12 -HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.

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