The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes.

Pseudomonas aeruginosa ttcA encoding tRNA-thiolating protein requires an iron-sulfur cluster to participate in hydrogen peroxide-mediated stress protection and pathogenicity

“…Adaptive gene expression is a key component of bacterial defence against environmental stresses. The expression of many genes involved in oxidative stress protection and repair processes is frequently induced by exposure to oxidants 7 , 34 – 36 . The expression patterns of ttcA in PAO1 cultivated under inducing concentrations of various oxidants were determined using real time RT-PCR.…”

“…6His-tagged TtcA from P . aeruginosa was expressed using the pQE-30Xa expression system (Qiagen, Germany) under oxygen-limited conditions as previously described 34 . The full-length ttcA gene was amplified from PAO1 genomic DNA with the primers EBI1035 and EBI1036.…”

“…coli M15 strain harbouring pQE-30Xa- ttcA was grown to an OD 600 of 1.0 before being induced with 100 μM IPTG for 60 min with 40 rpm shaking. Purification of 6His-tagged TtcA was carried out using a nickel-nitrilotriacetic acid (Ni-NTA) agarose column as previously described 34 . The purity of the TtcA protein was more than 95%, as judged by a major band corresponding to the 32.3-kDa protein observed on SDS-PAGE.…”

Response of the Anaerobic Methanotroph “ Candidatus Methanoperedens nitroreducens” to Oxygen Stress

“…The self-repair response and the deactivation of oxygenic mechanisms is enhanced by the upregulation of genes encoding type A flavoproteins, known to be involved in chain reactions for oxygen reduction. This mechanism has been studied for obligate anaerobic bacteria from the genus Clostridium, and it is generally involved in the maintenance of the redox balance by influencing the ratio of NADP ϩ /NADPH in cellular processes (73,74).…”

Pseudomonas aeruginosa glutathione biosynthesis genes play multiple roles in stress protection, bacterial virulence and biofilm formation

“…To determine the oxidant resistance level, a plate sensitivity assay was performed as previously described [ 56 ]. Briefly, exponential-phase cells (OD 600nm of approximately 0.5 after 3 h of growth) were adjusted to an OD 600nm of 0.1 prior to a 10-fold serial dilution in LB medium.…”