Effect of Cu(II) and Conserved Copper Binding Sites on Multicopper Oxidase CopA and Characterization of the BioMnOx

The microbial manganese removal process is believed to be the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and found to have high manganese oxidation activity in vitro and Cu(II) can significantly enhance its manganese oxidation activity. The gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA and then obtain four mutant strains. The manganese removal efficiency of the four strains was determined to find that H120 is the catalytic active site of the CopA. Protein modification analysis of CopA obtained under different conditions by mass spectrometry revealed that the loss of Cu(Ⅱ) and the mutation of the conserved copper binding site H120 resulted in the loss of modification of ethoxyformyl and quinone, the number of modifications was reduced and the position of modification was changed, eventually causing a decrease in protein activity. It reveals that Cu(II) and H120 play an indispensable role in the manganese oxidation of the multicopper oxidase CopA. The Mn valence state of BioMnOx was analyzed by XPS, finding that both the strain-mediated product and the CopA-mediated product were composed of MnO2 and Mn3O4 and the average valence of Mn is 3.2.

Comparative genomics on cultivated and uncultivated, freshwater and marine Candidatus Manganitrophaceae species implies their worldwide reach in manganese chemolithoautotrophy

Chemolithoautotrophic manganese oxidation has long been theorized, but only recently demonstrated in a bacterial co-culture. The majority member of the co-culture, Candidatus Manganitrophus noduliformans, is a distinct but not yet isolated lineage in the phylum Nitrospirota (Nitrospirae). Here, we established two additional MnCO3-oxidizing cultures using inocula from Santa Barbara (USA) and Boetsap (South Africa). Both cultures were dominated by strains of a new species, designated Candidatus Manganitrophus morganii. The next abundant members differed in the available cultures, suggesting that while Ca. Manganitrophus species have not been isolated in pure culture, they may not require a specific syntrophic relationship with another species. Phylogeny of cultivated Ca. Manganitrophus and related metagenome-assembled genomes revealed a coherent taxonomic family, Candidatus Manganitrophaceae, from both freshwater and marine environments and distributed globally. Comparative genomic analyses support this family being Mn(II)-oxidizing chemolithoautotrophs. Among the 895 shared genes were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse Complex 1 containing 2 additional pumping subunits was also shared by the family, as were genes for the reverse TCA carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the family lacked genes for nitrification found in Nitrospira species. The results suggest that Ca. Manganitrophaceae share a core set of candidate genes for the newly discovered manganese dependent chemolithoautotrophic lifestyle, and likely have a broad, global distribution.

Start-up strategies for nitrification and manganese oxidation on a single stage RSF for drinking water production

In drinking water production from groundwaters, biological rapid sand filters can be used for ammonium and manganese removal in aerobic conditions. However, in some boreholes, a start-up duration of several months is required to reach the required removal capacity, leading to significant water losses. Moreover, in specific industrial cases no exogenous biomass under the form of backwash water or activated sludge can be added to accelerate the process, and different approaches are seldom considered in literature. With the aim of saving water, start-up strategies coupling water temperature increase and substrate dosing were studied to accelerate the installation of biological activities, in a pilot plant fed with borehole water. These set-ups enabled a substantial acceleration of nitrification but no improvement of manganese oxidation in the experimental conditions, although the experiments showed no clear negative influence of nitrification, through nitrite accumulation, on biological manganese oxidation. To further save energy and reduce water loss, outlet water recirculation at a rate of 75% during the start-up phase was validated. The proposed start-up strategy enabled the complete installation of active biofilms with a mean start-up time reduction of 36% and water use reduction of 84% compared to the reference natural conditions.

Genome analysis of Pseudomonas sp. OF001 and Rubrivivax sp. A210 suggests multicopper oxidases catalyze manganese oxidation required for cylindrospermopsin transformation

Background Cylindrospermopsin is a highly persistent cyanobacterial secondary metabolite toxic to humans and other living organisms. Strain OF001 and A210 are manganese-oxidizing bacteria (MOB) able to transform cylindrospermopsin during the oxidation of Mn2+. So far, the enzymes involved in manganese oxidation in strain OF001 and A210 are unknown. Therefore, we analyze the genomes of two cylindrospermopsin-transforming MOB, Pseudomonas sp. OF001 and Rubrivivax sp. A210, to identify enzymes that could catalyze the oxidation of Mn2+. We also investigated specific metabolic features related to pollutant degradation and explored the metabolic potential of these two MOB with respect to the role they may play in biotechnological applications and/or in the environment. Results Strain OF001 encodes two multicopper oxidases and one haem peroxidase potentially involved in Mn2+ oxidation, with a high similarity to manganese-oxidizing enzymes described for Pseudomonas putida GB-1 (80, 83 and 42% respectively). Strain A210 encodes one multicopper oxidase potentially involved in Mn2+ oxidation, with a high similarity (59%) to the manganese-oxidizing multicopper oxidase in Leptothrix discophora SS-1. Strain OF001 and A210 have genes that might confer them the ability to remove aromatic compounds via the catechol meta- and ortho-cleavage pathway, respectively. Based on the genomic content, both strains may grow over a wide range of O2 concentrations, including microaerophilic conditions, fix nitrogen, and reduce nitrate and sulfate in an assimilatory fashion. Moreover, the strain A210 encodes genes which may convey the ability to reduce nitrate in a dissimilatory manner, and fix carbon via the Calvin cycle. Both MOB encode CRISPR-Cas systems, several predicted genomic islands, and phage proteins, which likely contribute to their genome plasticity. Conclusions The genomes of Pseudomonas sp. OF001 and Rubrivivax sp. A210 encode sequences with high similarity to already described MCOs which may catalyze manganese oxidation required for cylindrospermopsin transformation. Furthermore, the analysis of the general metabolism of two MOB strains may contribute to a better understanding of the niches of cylindrospermopsin-removing MOB in natural habitats and their implementation in biotechnological applications to treat water.