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Summary Soluble ligand‐bound Mn(III) can support anaerobic microbial respiration in diverse aquatic environments. Thus far, Mn(III) reduction has only been associated with certain Gammaproteobacteria. Here, we characterized microbial communities enriched from Mn‐replete sediments of Lake Matano, Indonesia. Our results provide the first evidence for the biological reduction of soluble Mn(III) outside the Gammaproteobacteria. Metagenome assembly and binning revealed a novel betaproteobacterium, which we designate ‘Candidatus Dechloromonas occultata.’ This organism dominated the enrichment and expressed a porin‐cytochrome c complex typically associated with iron‐oxidizing Betaproteobacteria and a novel cytochrome c‐rich protein cluster (Occ), including an undecaheme putatively involved in extracellular electron transfer. This occ gene cluster was also detected in diverse aquatic bacteria, including uncultivated Betaproteobacteria from the deep subsurface. These observations provide new insight into the taxonomic and functional diversity of microbially driven Mn(III) reduction in natural environments.

Manganese oxide minerals (Mn(III/IV)Ox) are ubiquitous in natural environments and interactions between Mn(III/IV)Ox and microbes play important roles in biogeochemical cycles. Current techniques for determining the spatial distribution of microbes with Mn(III/IV)Ox include electron microscopy and synchrotron radiation analyses. However, these techniques may not be readily available in most laboratories or may be cost prohibitive. Here we present a rapid, cost‐effective “simul‐staining” method for imaging particulate Mn(III/IV)Ox and cells on the same filter using epifluorescence microscopy with differential interference contrast (DIC) capability as a prescreening tool before higher resolution and/or more time‐intensive analyses. This method uses leucoberbelin blue (LBB) dye, which turns blue when oxidized by particulate Mn(III/IV)Ox on filters, and the fluorescent nucleic acid stain, SYBR Green, which fluoresces when bound to nucleic acids. First, the DIC configuration is used to locate blue “haloes” of oxidized LBB around Mn(III/IV)Ox particles. Second, a filter set that excites dyes with blue light and emits green fluorescence is used to image SYBR Green bound to nucleic acids in cells. Third, ImageJ is used for image analysis to associate Mn(III/IV)Ox particles and microbes. We demonstrate that this simul‐staining is suitable for laboratory cultures of manganese‐oxidizing bacteria as well as environmental samples from a marine oxycline.

SummarySoluble ligand-bound Mn(III) can support anaerobic microbial respiration in diverse aquatic environments. Thus far, Mn(III) reduction has only been associated with certain Gammaproteobacteria. Here, we characterized microbial communities enriched from Mn-replete sediments of Lake Matano, Indonesia. Our results provide the first evidence for biological reduction of soluble Mn(III) outside the Gammaproteobacteria. Metagenome assembly and binning revealed a novel betaproteobacterium, which we designate “Candidatus Dechloromonas occultata.” This organism dominated the enrichment and expressed a porin-cytochrome c complex typically associated with iron-oxidizing Betaproteobacteria and a novel cytochrome c-rich protein cluster (Occ), including an undecaheme putatively involved in extracellular electron transfer. This occ gene cluster was also detected in diverse aquatic bacteria, including uncultivated Betaproteobacteria from the deep subsurface. These observations provide new insight into the taxonomic and functional diversity of microbially-driven Mn(III) reduction in natural environments.Originality-Significance StatementRecent observations suggest that Mn(III)-ligand complexes are geochemically important in diverse aquatic environments. Thus far, microbially-driven Mn(III) reduction has only been associated with Gammaproteobacteria encoding three-component outer-membrane porin-cytochrome c conduits. Here, we demonstrate that Betaproteobacteria dominate in abundance and with respect to protein expression during biologically-mediated Mn(III) reduction in an enrichment culture from an anoxic lacustrine system. Using metaproteomics, we detect for the first time that Betaproteobacteria express a two-component porin-cytochrome c conduit, and an uncharacterized extracellular undecaheme (11-heme) c-type cytochrome. Although this is the first definitive report of an undecaheme within the Betaproteobacteria, we find evidence that they are widespread in uncultivated strains. These results widen the phylogenetic diversity of Mn(III)-reducing bacteria, and provide new insights into potential molecular mechanisms for soluble Mn(III) reduction