Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa

We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa ha rboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to e ncode, respectively, (i) the or subunit of RNA polymerase; (ii) the L17 rib osomal protein; (iii) the major catalase, KatA; and (iv) one of two iron st orage proteins called bacterioferritin A (BfrA; cytochrome b(1) or b(557)). Our goal was to determine the contributions of KatA and BfrA to the resist ance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multi copy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two transla tional start codons encoding a heteromultimer of similar to 160 to 170 kDa and having an apparent K-m for H2O2 of 44.7 mM. Isogenic katA and bfrA muta nts were hypersusceptible to H2O2, while a katA bfrA double mutant demonstr ated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only s imilar to 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase ac tivity could not restore wild type catalase activity in the bfrA mutant. RN ase protection assays revealed that katA and bfrA are on different transcri pts, the levels of which are increased by both iron and H2O2. Mass spectrom etry analysis of whole cells revealed no significant difference in total ce llular iron levels in the bfrA, katA, and katA bfrA mutants relative to wil d-type bacteria. Our results suggest that P. aeruginosa BfrA may be require d as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.