The iron-storage protein bacterioferritin (Bfr) from strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Bfr. The DNA sequence of was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial sequence) was located within the 5’ flanking region of in front of a putative −35 hexamer. The homology between the DNA sequences of and was 557%; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 397% identity, and showed 413% and 561% identity, respectively, to Bfr. Expression of recombinant BfrA and BfrB in strain DH5α was detected on Western blots probed with polyclonal anti- Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the Ω fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by HO and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress.


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