The biological catalysis of Mn(II) oxidation is thought to be responsible for the formation of most naturally occurring insoluble Mn(III, IV) oxides (40, 41) and consequently plays a key role in the biogeochemical cycling of Mn. The resulting biogenic Mn oxides have high adsorptive capacities for toxic metals (16, 17) and can oxidize both natural organic compounds (38) and organic contaminants (36). The binding of transition metals to biogenic Mn oxides can, in turn, greatly affect the phase distributions and residence times of these transition metals in many natural systems (16,17). An understanding of environmental conditions that favor or inhibit the production of the extracellular enzyme responsible for Mn(II) oxidation will contribute to insight into this important ecological process and is also a prerequisite for the design of any successful technology that uses microorganisms for the production of Mn oxides.A variety of phylogenetically distinct microorganisms are capable of the extracellular oxidation of Mn(II) (5). Based on the presence of conserved predicted amino acid motifs, the genes that encode putative Mn(II)-oxidizing enzymes (mofA in Leptothrix discophora [9], cumA in Pseudomonas putida GB-1 [7], and moxA in Pedomicrobium sp. strain ACM 3067 [33]) are all thought to produce multicopper oxidases. Recently, further support for the role of putative multicopper oxidases in Mn(II) oxidation has come from the recovery and sequencing of peptides excised from Mn(II)-oxidizing bands in polyacrylamide gel analyses of proteins from three Mn(II)-oxidizing Bacillus species (15). Specifically, Mn(II)-oxidizing bands from the exosporia of two of the three Bacillus species tested were shown by tandem mass spectrometric analyses to contain peptides with homology to the predicted C terminus of the putative multicopper Mn(II) oxidase MnxG.Despite this growing body of evidence regarding the role of multicopper oxidases in Mn(II) oxidation, little is known about how the concentrations of different nutrients (e.g., iron, carbon, and nitrogen) or growth conditions such as pH and the oxygen concentration regulate the production of the enzyme(s) which oxidizes Mn(II). Nelson et al. (26) evaluated the minimal growth conditions needed for Mn(II) oxidation and found that the addition of 0.1 M Fe(II) to the defined minimal mineral salt (MMS) medium used for growth was necessary for the complete oxidation of Mn(II) by L. discophora SS-1; however, adding 0.1 M Fe(II) to stationary-phase cells did not allow complete Mn(II) oxidation.In the present study, we grew L. discophora SS-1 in a controlled-reactor system and evaluated the time courses of Mn(II) oxidation and mofA transcript levels in batch cultures of cells with limited and sufficient iron. Parker et al. (30) observed that retarded Mn(IV) formation by iron-starved P. putida is a consequence of the binding of the Mn(III) intermediate (42) to the siderophore pyoverdine. Therefore, siderophore production was also evaluated as part of this research.
MATERIALS AND METHODS...