The root-colonizing pseudomonad Pseudomonas putida (Pp) appears to produce two subunits, alpha and beta, of the iron-binding protein, bacterioferritin. A gene encoding the alpha-bacterioferritin subunit was located adjacent to the major catalase in Pp. The deduced protein sequence of the Pp bfralpha gene had a very high identity with other alpha-subunits, possessing conserved amino acids responsible for ferroxidase activity. The gene also lacked a deduced methionine at residue 52, associated with heme binding in beta-subunits. An antibody generated toward the Escherichia coli (E. coli) multifunctional single subunit bacterioferritin recognized two proteins in the Pp extract, a 22 kDa protein likely to be a beta-subunit and, to a lesser extent, a 23 kDa band. The 23 kDa band was absent in a Pp mutant in which the bfralpha gene was disrupted. Loss of alpha-bacterioferritin stimulated production of fluorescent siderophore. Growth on media and on root surfaces was not impaired by deletion of the alpha-bacterioferritin. Transcription of bfralpha was independent of the catalase gene and was dependent on iron. The transcript levels from bfralpha decreased in iron deficiency experienced during stationary-phase or upon treatment during growth with an iron chelator.