PERSONAL DE APOYO
KRAPP Adriana Del Rosario
congresos y reuniones científicas
Título:
A putative cognate long chain flavodoxin protects Pseudomonas aeruginosa from oxidative stress and plays an antimutator role
Autor/es:
MOYANO AJ, RIZZI Y, TOBARES RA, KRAPP AR, CARRILLO N AND SMANIA AM
Lugar:
Copenhague
Reunión:
Congreso; ISME14- International society for Microbial Ecology; 2012
Institución organizadora:
ISME- International society for Microbial Ecology
Resumen:
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.