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

MOYANO AJ, RIZZI Y, TOBARES RA, KRAPP AR, CARRILLO N AND SMANIA AM

Copenhague

Congreso; ISME14- International society for Microbial Ecology; 2012

ISME- International society for Microbial Ecology

Oxidative stress is a critical challenge which bacteria have to cope with in order to survive in an oxygenic atmosphere. It has been shown in some bacterial species that long chain flavodoxins, ubiquitous electron shuttles containing FMN as prosthetic group, play 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, sequence homology and predicted secondary structure with typical 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 recombinant purified FldPa expressed in E. coli showed spectral peaks at 385 and 455 nm, consistent with the two transitions of the isoalloxazine group of flavin-containing proteins. Furthermore, FldPa was able to mediate cytochrome c reductase activity in vitro, with kinetic parameters comparable to those of bona fide flavodoxins from Anabaena and E. coli. In order to functionally characterize FldPa, we exposed P. aeruginosa to H2O2 and observed a strong induction of the fldPa gene. Moreover, the use of 2? 7?-dichlorofluorescein diacetate (a ROS probe) as well as the ferrous ion oxidation/xylenol orange assay revealed that a mutant strain of P. aeruginosa which was deficient in the fldPa gene showed increased accumulation of ROS compared to the parental wild type strain upon exposure to H2O2. Accordingly, viability assays showed that the fldPa mutant displayed a lower resistance to H2O2, a phenotype 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 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 increased 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. Noteworthy, overexpression of fldPa and fldAn could prevent the majority although not all the H2O2-induced mutations in this strain. The collected 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.