Ferric reductase A is essential for effective iron acquisition in Paracoccus denitrificans

1 Department of Biochemistry, Faculty of Science, Masaryk University, Czech Republic, CZ-611 37 Brno, Czech Republic 2 Department of Molecular Cell Physiology, Faculty of Earth and Life Science, VU University Amsterdam, NL-1081 HV Amsterdam, The Netherlands Correspondence Igor Ku era ikucera{at}chemi.muni.cz Based on N-terminal sequences obtained from the purified cytoplasmic ferric reductases FerA and FerB, their corresponding genes were identified in the published genome sequence of Paracoccus denitrificans Pd1222. The ferA and ferB genes were cloned and individually inactivated by insertion of a kanamycin resistance marker, and then returned to P. denitrificans for exchange with their wild-type copies. The resulting ferA and ferB mutant strains showed normal growth in brain heart infusion broth. Unlike the ferB mutant, the strain lacking FerA did not grow on succinate minimal medium with ferric 2,3-dihydroxybenzoate as the iron source, and grew only poorly in the presence of ferric sulfate, chloride, citrate, NTA, EDTA and EGTA. Moreover, the ferA mutant strain was unable to produce catechols, which are normally detectable in supernatants from iron-limited wild-type cultures. Complementation of the ferA mutation using a derivative of the conjugative broad-host-range plasmid pEG400 that contained the whole ferA gene and its putative promoter region largely restored the wild-type phenotype. Partial, though significant, restoration could also be achieved with 1 mM chorismate added to the growth medium. The purified FerA protein acted as an NADH : FMN oxidoreductase and catalysed the FMN-mediated reductive release of iron from the ferric complex of parabactin, the major catecholate siderophore of P. denitrificans . The deduced amino acid sequence of the FerA protein has closest similarity to flavin reductases that form part of the flavin-dependent two-component monooxygenases. Taken together, our results demonstrate an essential role of reduced flavins in the utilization of exogenous ferric iron. These flavins not only provide the electrons for Fe(III) reduction but most probably also affect the rate of siderophore production. A sequence comparison of FerA with some homologous proteins identified by BLAST searching is available with the online version of this paper.