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.
Highlights
Ferredoxin NADP(+) reductase (Fpr) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase enzyme
The signature amino acid residues for Fpr sub-class II, which include the tyrosine-247 [24] and tryptophan-248 residues of E. coli Fpr [6], are conserved in P. aeruginosa FprB (Fig 1A)
The regulation and patterns of stress-induced fprB expression suggests a correlation between levels of FprB, [4Fe-4S] clusters, and the ability of bacteria to respond to stress
Summary
Ferredoxin NADP(+) reductase (Fpr) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase enzyme. It catalyzes reversible electron transfer between NADPH and electron carrier [Fe-S] proteins such as ferredoxin and flavodoxin [1, 2]. The expression of fprA (encoding subclass I Fpr) is induced in response to superoxide stress and is regulated by FinR, a LysR-type transcriptional regulator [10]. Both FprA and FinR have important roles in cellular defense against stress due to superoxide and ferrous iron depletion [10,11,12]. The disruption of fprB in P. putida exhibits growth defects under high osmotic stress conditions and slower rate of recovery of oxidatively damaged aconitase (an [4Fe-4S]-containing enzyme) activity than that of the wild type [13]
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