California Sea Grant College Program

The marine Bacillus sp. strain SG-1 produces dormant spores that catalyze the oxidation of soluble Mn(II) to insoluble Mn(IV) oxides. The Mn(ll)-oxidizing activity was localized to the ridged outermost spore layer, which could be physically removed and retain activity. MnxG, the product of the most downstream gene in the previously identified mnx gene cluster, is a multicopper oxidase believed to be the Mn(II) oxidase. MnxG was immunologically localized to the outermost layer of wild-type spores but was absent in non-oxidizing mutants within the mnx cluster, suggesting that MnxG may be the only protein directly involved in Mn(II) oxidation.

To explore the phylogenetic diversity of marine Bacillus strains capable of producing Mn(II)-oxidizing spores, 16S rRNA and mnxG sequences were PCR-amplified from 14 Mn(II)-oxidizing spore-formers isolated from coastal marine sediments. Phylogenetic trees based on both genes revealed extensive diversity of Mn(II)-oxidizers within the genus Bacillus, including several distinct clusters and lineages. SDS-PAGE analysis of spore surface proteins revealed the presence of active Mn(II)-oxidizing bands which were inhibited by o-phenanthroline and azide, consistent with the involvement of a multicopper oxidase.

In Pseudomonas putida GB-l, a multicopper oxidase gene, cumA, is believed to encode an essential component of the outer membrane Mn(II)-oxidizing complex. Using degenerate PCR primers, homologous cumA sequences were amplified from a variety of Mn(II)-oxidizing and non-oxidizing Pseudomonas strains. Phylogenetic trees based on CumA and l6S rRNA sequences had similar topologies and revealed that the cumA gene and the capacity to oxidize Mn(II) occur in diverse Pseudomonas strains. However, cumA may have an alternative function or be functionally inactive in non-oxidizing strains.

A yellow-pigmented Mn(II)-oxidizing a-proteobacterium, strain SD-21, was isolated from surface sediments of San Diego Bay, CA. During logarithmic growth phase, this organism produces Mn(II)-oxidizing proteins which are heat labile and inhibited by azide and o-phenanthroline, suggesting the possible involvement of a multicopper oxidase. This is the first report of Mn(II) oxidation within the alpha-4-Proteobacteria as well as the first MN(II)-oxidizing protein(s) identified in a marine Gram-negative bacterium.

Overall, these studies indicate that 1) Mn(II)-oxidizing bacteria are phylogenetically diverse and that 2) copper may play a universal role in Mn(II) oxidation.