A putative cognate long chain flavodoxin protects Pseudomonas aeruginosa from oxidative stress and plays an antimutator role

MOYANO AJ; RIZZI Y; TOBARES RA; KRAPP A; CARRILLO N; SMANIA AM

Copenague

Congreso; 14th International Symposium of Microbial Ecology (ISME14); 2012

ISME- International society for Microbial Ecology

Oxidative stress is a critical matter which bacteria have to cope with in order to survive. It has been shown in some bacterial species that long chain flavodoxins posses an important protective role against reactive oxygen species (ROS). Pseudomonas aeruginosa is an opportunistic pathogen which has to face ROS in the environment as well as within the host; however the presence of putative flavodoxins and their function have not been investigated so far. Thus, by performing an in silico genome-wide analysis we chose a single ORF among a few candidate genes, here referred to as fldPa, based on its similarities in length, homology and prediction of its secondary structure with well known long chain flavodoxins such as those from Anabaena and Escherichia coli. Subsequently we performed a biochemical characterization of FldPa and tested its expression and function in P. aeruginosa upon oxidative stress. Analysis of purified FldPa which was heterologously expressed in E. coli showed spectral peaks at 385 and 455 nm for the two transitions of the isoalloxazine group, which is typical of flavodoxins. Furthermore, FldPa showed cytochrome c reductase activity, consistent with those activities previously reported for Anabaena and E. coli flavodoxins. In order to functionally characterize FldPa, we exposed P. aeruignosa to H2O2 and observed a strong induction of the fldPa gene. Importantly, the use of 2’ 7’-dichlorofluorescein diacetate dyes as well as the ferrous ion oxidation/xylenol orange method revealed that a strain of P. aeruginosa which was deficient for the fldPa gene showed an increased accumulation of ROS compared to the parental wild type strain upon exposure to H2O2. Accordingly, viability assays showed that the fldPa mutant strain possessed a lower resistance to H2O2, phenomenon which could be reverted by complementation with a vector carrying the cognate fldPa gene, or even by complementation with a vector which heterologously expressed the flavodoxin gene of Anabaena (fldAn). We further tested the protective role of fldPa and fldAn by using a mutT mutant strain of P. aeruginosa, a strain which is particularly vulnerable to oxidative stress because of its deficiency in the 8-Oxoguanine-Repair System. Interestingly, multicopy expression of fldPa and fldAn could partially compensate the increased susceptibility of the mutT deficient strain to H2O2. Since damage in the DNA produced by ROS can lead to an increase mutation frequency and phenotypic diversification, we finally evaluated if overexpression of fldPa and fldAn could also render an antimutator function and neutralize the mutagenesis induced by H2O2 under the mutT deficient background. Notably, overexpression of fldPa and fldAn could avoid the majority although not all the H2O2-induced mutations in this strain. To summarize, the results strongly suggest that fldPa encodes for a cognate long chain flavodoxin-like protein, which is induced when P. aeruginosa is under oxidative stress to exert a protective role against the fisiological and mutational damage produced by ROS. Thus, this study shed light on the adaptive response of P. aeruginosa to one of the primary obstacles in the bacterial world, such as oxidative stress.