Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+) Reductase Gene (fprB) Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response

Abstract

P. aeruginosa (PAO1) has two putative genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein’s physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

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

“…Total RNA was treated with DNase I (Thermo Scientific, USA) prior to performing cDNA synthesis using 5 μg DNase I-treated RNA, RevertAid Reverse Transcriptase, and random hexamer primers (Thermo Scientific, USA). Real-time PCR was performed as previously described [ 54 ] using 10 ng cDNA to determine gene expression levels using primers EBI11 ( ) and EBI12 ( ) for gshA , BT5458 ( ) and BT5459 ( ) for gshB , EBI315 ( ) and EBI316 ( ) for phzA1 , and EBI316 and EBI317 ( ) for phzA2 . The 16S rRNA gene was amplified using primers BT2781 ( ), and BT2782 ( ) was used as an internal control to normalize cDNA samples.…”

Iron availability and oxygen tension regulate the Yersinia Ysc type III secretion system to enable disseminated infection

“…Since IscR has been shown to differentially regulate gene expression in response to ironlimiting conditions in E. coli and Pseudomonas aeruginosa [19,27,28], we sought to determine the effect of iron on the expression of both iscR and T3SS genes in Yersinia. We previously developed a protocol for limiting Y. pseudotuberculosis for iron and demonstrated induction of a Fur-repressed gene [29], indicating efficacy of iron limitation.…”

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

“…FprA achieves higher catalytic efficiency when flavodoxin is its redox partner. FprB is important in defenses against multiple stresses including metal, oxidative, and osmotic stresses and is required for the full function of iron-sulfur cluster ([Fe-S])-containing enzymes via its redox partner, Fdx2, involving in the ISC [Fe-S] biogenesis system [ 29 ]. For example, in an iron storage complex, the [Fe-S] cluster of bacterioferritin-associated ferredoxin (Bdf) transfers electrons to the heme in bacterioferritin (BfrB) and promotes the release of Fe 2+ from BfrB by mediating electrons from FprA to BfrB [ 30 ].…”