“
“Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics. In this study, we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized the phenotype and the gene expression profile using microarray and RT-PCR. PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM)

showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin in the double mutant. A better fitness of the mutator compared Bleomycin datasheet with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has

been shown to provide protection to oxidative stress, was found in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might represent an oxidative stress for the bacteria. The chronic lung infection with Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis (CF) is characterized by the biofilm mode OSI-906 mw of growth and a chronic inflammation dominated by polymorphonuclear leucocytes (PMNs) (Koch & Hoiby, 1993; Bjarnsholt et al., 2009). Pseudomonas aeruginosa are exposed to many reactive oxygen species (ROS), both generated by its

own metabolism and especially from a large number of PMNs which release ROS in response to the chronic CF-lung infection (Brown & Kelly, 1994; Suntres et al., 2002; Kolpen et al., 2010). In addition, exposure of the microorganisms to antipseudomonal antibiotics such as ciprofloxacin, which is an inhibitor of DNA-gyrase, can stimulate the bacterial production of ROS (Dwyer et al., 2007; Kohanski DNA ligase et al., 2007). To combat the mutagenic consequences of the ROS, the MutT, MutY and MutM of the DNA oxidative repair system (GO) play an important role (Mandsberg et al., 2009). The enzymes of the GO system repair and prevent the incorporation of an oxidative damaged form of guanosine, 7,8-dihydro-8-oxo-dGuanine (8-oxodG), in the DNA. The MutT is a nucleoside triphosphate pyrophosphohydrolase catalysing the conversion of 8-oxodGTP to 8-oxodGMP + PPi, preventing oxidized guanine from being incorporated under replication; MutM is a formamidopyrimidine DNA-glycosylase that removes 8-oxodG when base paired with cytosine, and MutY is an adenine glycosylase capable of removing adenine when paired with 8-oxodG minimizing GC : TA transversions(Livingston et al., 2008).