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Bacterial chemolithoautotrophy via manganese oxidation

Abstract

Manganese is one of the most abundant elements on Earth. The oxidation of manganese has long been theorized1—yet has not been demonstrated2,3,4—to fuel the growth of chemolithoautotrophic microorganisms. Here we refine an enrichment culture that exhibits exponential growth dependent on Mn(II) oxidation to a co-culture of two microbial species. Oxidation required viable bacteria at permissive temperatures, which resulted in the generation of small nodules of manganese oxide with which the cells associated. The majority member of the culture—which we designate ‘Candidatus Manganitrophus noduliformans’—is affiliated to the phylum Nitrospirae (also known as Nitrospirota), but is distantly related to known species of Nitrospira and Leptospirillum. We isolated the minority member, a betaproteobacterium that does not oxidize Mn(II) alone, and designate it Ramlibacter lithotrophicus. Stable-isotope probing revealed 13CO2 fixation into cellular biomass that was dependent upon Mn(II) oxidation. Transcriptomic analysis revealed candidate pathways for coupling extracellular manganese oxidation to aerobic energy conservation and autotrophic CO2 fixation. These findings expand the known diversity of inorganic metabolisms that support life, and complete a biogeochemical energy cycle for manganese5,6 that may interface with other major global elemental cycles.

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Fig. 1: Bio-oxidation of MnCO3 produces manganese oxide nodules to which two species associate.
Fig. 2: Mn(II) oxidation coupled to co-culture growth of species A and species B.
Fig. 3: Phylogenetic analysis and metabolic reconstruction of species A (‘Candidatus Manganitrophus noduliformans’).
Fig. 4: Stable isotope probing of autotrophic CO2 fixation.

Data availability

All sequencing data has been deposited at the NCBI under BioProject PRJNA562312. The cloned 16S rRNA gene sequences of ‘Candidatus Manganitrophus noduliformans’ (species A) and R. lithotrophicus (species B) from the co-culture have been deposited at GenBank under accession numbers MN381734 and MN381735, respectively. The iTAG sequences from the different enrichments have been deposited at the Sequence Read Archive (SRA) under accession numbers SRR10031198, SRR10031199 and SRR10031200. Genome sequences of the co-culture, from which the genome of ‘Candidatus Manganitrophus noduliformans’ was reconstructed, have been deposited under BioSample SAMN12638105 with raw sequences deposited at SRA under accession number SRR10032644; the reconstructed genome of ‘Candidatus Manganitrophus noduliformans’ has been deposited at DDBJ/ENA/GenBank under accession number VTOW00000000. Genome sequences of R. lithotrophicus strain RBP-1 have been deposited under BioSample SAMN12638106, with raw sequences deposited at SRA under accession number SRR10031379; the reconstructed genome of R. lithotrophicus strain RBP-1 has been deposited at DDBJ/ENA/GenBank under accession number VTOX00000000. Additionally, reconstructed genomes have been deposited in Joint Genome Institute (JGI) Genomes Online Database Study ID Gs0134339, with Integrated Microbial Genome ID 2784132095 for ‘Candidatus Manganitrophus noduliformans’ and ID 2778260901 for R. lithotrophicus strain RBP-1. Transcriptome sequence data for the seven biological replicates have been deposited at SRA under accession numbers SRR10060009, SRR10060010, SRR10060011, SRR10060012, SRR10060013, SRR10060017 and SRR10060018. Unique biological materials are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by NASA Astrobiology Institute Exobiology grant #80NSSC19K0480; and by Caltech’s Center for Environmental Microbial Interactions and Division of Geological and Planetary Sciences. We thank S. Connon for assistance with iTag sequencing preparations; G. Rossman and U. Lingappa for spectroscopic analyses and minerology insights; G. Chadwick for discussions on physiology and bioenergetics; I. Antoshechkin and V. Kumar for assistance with nucleic acid library preparation and sequencing at the Millard and Muriel Jacobs Genetics and Genomics Laboratory; N. Dalleska for assistance with ICP–MS analyses at the Environmental Analysis Center; F. Gao for inputs on RNA data analysis using kallisto software at the Bioinformatics Resource Center in the Beckman Institute; C. Ma for assistance with SEM analyses at the GPS Analytical Facility; Y. Guan for assistance with nanoSIMS analyses at the GPS Microanalysis Center; and multiple colleagues for feedback before publication.

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H.Y. and J.R.L. together applied for funding, designed and conducted the experiments, performed data analyses, prepared the figures and wrote the manuscript.

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Correspondence to Jared R. Leadbetter.

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Peer review information Nature thanks Edward F. DeLong, Philip Hugenholtz, Bradley M. Tebo, Michael Wagner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Effect of temperature, anti-bacterials and Mn(II)Cl2 on biological Mn(II)CO3 oxidation.

a, Incubation temperature influences oxidation. An optimum between 34 °C and 40 °C was observed, but above these temperatures oxidation was inhibited. By contrast, non-biological reactions would generally be predicted to continue to increase in rate with increasing temperature. b, Sensitivity of Mn(II) oxidation to the presence of either of two antibiotics, or to prior pasteurization before extended incubation at 32 °C. c, When amended to active co-cultures at concentrations >2.0 mM, MnCl2 appeared to inhibit MnCO3 oxidation when an active culture containing about 2.2 mM unreacted MnCO3 was used as the inoculum. The number of points for each experimental condition represents independent cultivation experiments.

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Extended Data Fig. 2 Mn(II) oxidation and growth by the co-culture.

a, DNA yield of the two-species co-culture incubated in MOPS-buffered basal medium in the absence of Mn(II) substrate. No statistically significant changes in the mean DNA yields (P = 0.06, day 0 vs 10; P = 0.70, day 10 vs 21; P = 0.20, day 0 vs 21; two-tailed t-test with equal variance) are observed over the incubation period. b, c, Exponential increase in bacteria and biomass yields in a Mn(II)-oxidizing culture, which is coupled to exponential increases Mn(II) oxidation (same culture analysed in Fig. 2). Bacteria were measured via 16S rRNA gene copies using a general bacteria probe in quantitative PCR; points represent 3 technical replicates. Biomass was measured via DNA yield from same culture volumes. d, Exponential increases in Mn(II) oxidation (Fig. 2a) and DNA yields by this same culture (1 mM nitrate replicate 1, c) correlate. Similar relationships were observed in samples from independent cultivation experiments (n = 2). el, Kinetics of Mn(II) oxidation by the co-culture in basal medium; two phases of exponential Mn(II) oxidation were observed. eg, Basal medium with 1 mM nitrate (n = 4; for replicate 1, see b–d and Fig. 2). hl, Basal medium with 1 mM ammonia (n = 5). m, Exponential growth of species A and species B paralleled Mn(II) oxidation in basal medium with 1 mM ammonia as the nitrogen source (1 mM ammonia replicate 5, l), rather than 1 mM nitrate. n, Linear relationship between cell growth and the amount of Mn(II) oxidized (1 mM ammonia replicate 5, l and m). Values in n were normalized by subtracting the initial cell number and Mn oxide concentrations at the onset of the experiment, and negative values after normalization were excluded from the analysis. All data points included in the line fits are used to calculate the doubling times (Td), unless otherwise noted by ‘x’ symbols.

Source data

Extended Data Fig. 3 Properties of the refined co-culture.

a, Estimations of the relative ratio between species A and species B. Slow-growing microorganisms, in particular species A (which also has a smaller cell volume than species B or Escherichia coli) could have a lower number of ribosomes, resulting in lower signal intensity from rRNA-targeted fluorescent probes, relative to the fluorescent signal from DNA stain DAPI. The two species together account for 99.7% of assigned sequence reads (Supplementary Table 1). The two species together account for 97.54% of the sequence reads in the metagenome (f). §The two species together account for 99.576% (s.d. = 0.005%, n = 7) of the rRNA sequence reads and 100.1700% (s.d. = 0.0005%, n = 7) of the non-rRNA sequence reads in the co-culture metatranscriptomes (h). be, Possible metabolic interactions that may be occurring between species A (orange) and species B (blue). f, Genome statistics for species A and species B. g, Observed rates and yields of Mn(II) oxidation by the co-culture, in comparison to the literature values5,23,26,39,95,96,97,98,99,100 reported for other physiologically or phylogenetically related lithotrophs or metal-active heterotrophs. ||Conversion estimate based on Escherichia coli biomass of 2.8 × 10−13 g dry cell weight per cell, of which 55% is protein27. Co-culture values correspond to results from the single independent culture with nitrate as the nitrogen source for which extensive data on both oxidation kinetics and growth (genome copies) were collected. h, Transcriptome statistics for 7 co-cultures sampled at different degrees of Mn(II) oxidation.

Extended Data Fig. 4 Microscopy of Mn oxide nodules formed by the co-culture.

ae, Epifluorescence microscopy reveals distribution of cells of species A and species B associated with dissolved Mn oxide nodules. DAPI (blue) was used to stain DNA, in addition to applying species-specific FISH probes targeting the 16S rRNA of species A (magenta) and species B (green). Probe fluorescence for species A was dim and faded rapidly, but was associated with the cells that otherwise appear in photomicrographs to only be DAPI-stained. No third species is present, as observed in independent cultivation experiments (n = 2), and confirmed via independent methods (Extended Data Fig. 3a). fp, Scanning electron micrographs of Mn(II)CO3 substrate (f, g) and Mn oxide nodules collected from liquid cultures (hp). Representative nodules are from independent cultivation experiments (n = 4).

Extended Data Fig. 5 Phylogenetic analyses on species A.

a, 16S rRNA gene phylogram, based on a Bayesian analysis of 1,532 aligned nucleotide positions. NCBI82 taxonomic classifications are used, and sequences shown are all from the phylum Nitrospirae. The names and known physiologies for the previously described genera in this phylum are shown on the right. NCBI accession numbers for 16S rRNA sequences are included in the node names. Source environment for the sequences are shown in brackets. b, Multilocus phylogram, based on a Bayesian analysis of 5,036 aligned amino acid positions concatenated from 120 bacterial protein markers62. GTDB62 taxonomic classifications are used, and sequences shown are from the phylum under the headings ‘Nitrospirota’ and Nitrospirota_A’. The names and known physiologies for the previously described classes in this phylum are shown on the right. NCBI accession numbers for genome assemblies are included in the node names. For a, b, the dots on the branches indicate posterior probabilities greater than 0.80. c, Phylogenetic analyses of the phylum Nitrospirae (Nitrospirota) limited to only those species with reconstructed genomes yield a topology different from that observed in a and Fig. 3a. Bayesian phylogram based on 1,532 aligned 16S rRNA nucleotide positions (left); multilocus Bayesian phylogram, based on 5,036 aligned amino acid positions of 120 concatenated bacterial protein markers (right). Sequences clustering within the three previously described classes within this phylum are collapsed into separate nodes. d, Protein sequence phylogeny of dihydroxy-acid and 6-phosphogluconate dehydratases. Sequences were selected based on a previous study101, with the addition of homologues found in Nitrospira inopinata, Leptospirillum ferriphilum and species A (red). All 770 aligned amino acid positions were used in the maximum likelihood analysis. Protein accession numbers from the NCBI database or gene identifiers from the IMG database of the 3 new sequences are shown in parentheses. Black dots on the branches represent bootstrap values equal to 100%. Although dihydroxy-acid dehydratase and 6-phosphogluconate dehydratase are homologous, they form separate clusters phylogenetically as reported101. The homologues in Nitrospirae all belong to the dihydroxy-acid dehydratase clade, therefore are unlikely candidates for 6-phosphogluconate dehydratase activity and function in the ED pathway. All scale bars show evolutionary distance (0.1 substitutions-per-site average).

Extended Data Fig. 6 Phylogenetic analyses and aerobic heterotrophic growth of isolated species B.

a, 16S rRNA gene phylogram, based on a Bayesian analysis of 1,532 aligned nucleotide positions. NCBI82 taxonomic classifications are used, with sequences selected from the class Betaproteobacteria. The genus Ramlibacter, consistently identified in two phylogenetic approaches, is shaded in grey, with species B in bold. Source environments for the species in Ramlibacter are shown in brackets. The order and family classifications are included to the right separated by a semicolon. The black dots on the branches indicate posterior probabilities greater than 0.90. b, Multilocus phylogram, based on a maximum-likelihood analysis of 5,035 aligned amino acid positions concatenated from 120 bacterial protein markers62. GTDB62 taxonomic classifications are used, and sequences shown are from the order Betaproteobacteriales. The GTDB family classifications are included to the right of species names. NCBI accession numbers for 16S rRNA sequences or the genome assemblies are included after the species names. The black dots on the branches indicate bootstrap values greater than 90%. Scale bars shown evolutionary distance (0.1 substitutions-per-site average). c, d, Kinetics of species B growth basal media with either 5 g/l of tryptone (n = 3 biological replicates) (c) or 10 mM acetate (n = 2 biological replicates) (d).

Source data

Extended Data Fig. 7 Phylogenetic analyses of cytochrome bd oxidase subunit I and cytochrome bd-like oxidases.

Only cytochrome bd-like oxidases were identified in species A, in contrast to other classes in the phylum Nitrospirae (Nitrospirota). a, Unrooted maximum-likelihood tree, constructed using 242 amino acid positions shared between cytochrome bd and bd-like oxidases, using RAxML89 (model LGF). Deduced proteins from the genome of species A are in red, with their IMG gene identifiers and clade numbering (as shown in Fig. 3b) included in brackets. Other proteins from the phylum Nitrospirae (Nitrospirota) are coloured blue, orange or brown for classes Nitrospiria, Leptospirillia or Thermodesulfovibrionia, respectively. Cytochrome bd oxidase of species B, with its IMG identifier, is in green; it belongs to the cyanide insensitive oxidase clade in purple. b, Phylogenetic analysis of cytochrome bd-like oxidases from species A. Unrooted maximum-likelihood tree was constructed using 242 amino acid positions shared between different clades of cytochrome bd-like oxidases. Cytochrome bd-like oxidases are assigned to different clades, based on the phylogeny and their gene cluster structures. Species A encodes 8 cytochrome bd-like oxidases (bold), representing clades I, II, IIIb, Va and Vb; clade numbering as shown in Fig. 3b are included in brackets after the IMG identifiers. Black dots on branches represent bootstrap values greater than 90%. Scale bars show evolutionary distance (substitutions-per-site average).

Extended Data Fig. 8 Sequence alignment of cytochrome bd and bd-like oxidases.

Cytochrome bd-like oxidase in species A (sequence names starting with A, followed by their IMG gene identifier and clade numbering as shown in brackets in Fig. 3b) and cytochrome bd oxidase subunit I in species B (sequence name starting with B, followed by its IMG gene identifier) are aligned to characterized cytochrome bd oxidases in Escherichia coli (sequence name starting with Eco, followed by its NCBI identifier) and Geobacillus thermodenitrificans (sequence name starting with Geo, followed by its NCBI identifier). Key features as revealed by structure102 are indicated at the top of the alignment, using E. coli protein residue numbering. The cytochrome bd oxidase subunit I sequence from species B shows conservation of all key residues. By contrast, cytochrome bd-like oxidases in species A do not show conservation of many key residues; instead, they are predicted to have up to 14 transmembrane helixes (compared to 9 in E. coli). One cytochrome bd-like oxidase in species A has a C-terminus extension with a haem c binding motif (CXXCH).

Extended Data Fig. 9 Stable isotope probing of Mn(II)-oxidizing co-culture measured using nanoSIMS.

a, Summary of stable isotope probing analysis of cells dissolved from Mn oxide nodules, either with paraformaldehyde fixation and FISH, or without (to avoid dilution with natural abundance isotopes). Cells of species A and species B were either identified by FISH or by elemental composition (species B cells were observed to have higher 14N/15N ratios), and their isotopic compositions were obtained via nanoSIMS (n = the total number of cell regions of interest analysed in the nanoSIMS images). For FISH–nanoSIMS analyses, a total of 2 and 5 nanoSIMS images from single cultures incubated with either MnCO3 or Mn13CO3, respectively, was examined. For nanoSIMS analyses without paraformaldehyde fixation and FISH, a total of 3 and 17 nanoSIMS images from single cultures incubated with either MnCO3 or Mn13CO3, respectively, was examined. bu, Individual secondary ion images from nanoSIMS showing incorporation of inorganic 13C and 15N into the cells of both species (dissolved from Mn oxide nodules grown in the presence of MnCO3 and 15NO3 (bk) or Mn13CO3 and 15NO3 (lu)), and species B cells could have higher 14N content than species A. Secondary ions 12C2 (mass 24 for 12C), 13C12C (mass 25 for 13C), 14N12C (mass 26 for 14N), 15N12C (mass 27 for 15N), 32S (mass 32 for 32S) were simultaneously measured. The counts of the secondary ions are shown in brackets (minimum–maximum) and displayed using the colour scale shown to the right of the images. bf and lp correspond to the top and bottom panels in Fig. 4, respectively. White arrows indicate species B cells identified in FISH showing high 14N in nanoSIMS. v, NanoSIMS measurement of residual Mn associated with cells grown with Mn13CO3 and 15NO3, after dissolving from Mn oxide nodules. The same nanoSIMS image area was analysed as in lp, except 55Mn16O (mass 71 for 55Mn) was measured (n = 1 nanoSIMS image) in addition to other secondary ions. Negligible amount of Mn was found in the biomass, indicating that any remaining Mn13CO3 substrate had been completely dissolved away during sample preparation, and thus did not interfere with the 13C analyses.

Extended Data Fig. 10 Evaluations of experimental methods.

ad, Evaluation of FISH oligonucleotide probes. Three probes (NLT499, black circles; BET359, white circles; BET867, white squares) were tested in different probe combinations and formamide concentrations, using 16S rRNA gene clones of species A (a, b) or species B (c, d). Each point in the dissociation profile represents the mean of fluorescence intensities of at least 100 different single cells in 5 distinct microscopic fields of 1 biological replicate. Lines connect the 95% confidence intervals of the points. No interference was found when targeting either species A or species B with different probe combinations and formamide concentrations. RU, relative units of fluorescence intensity. e, Evaluation of ICP–MS method to measure Mn compounds with different oxidation states. Mn(II) in its various forms can be almost entirely measured in the acid-soluble fraction with little in the acid-insoluble fraction, and any increase in the acid-insoluble fraction is an indication of oxidized Mn(II). We refer to the ‘acid-soluble fraction’ as Mn(II), and the ‘acid-insoluble fraction’ as Mn(II) oxidized representing Mn(III/IV). Supplementary Note 4 provides more details. MnO2 was synthesized according to two previously published methods103,104. f, Evaluation of transcriptome analysis software kallisto93. Average fragment length for RNA libraries was measured to be 230 bp. However, using 230 bp as the input parameter for fragment length caused a kallisto93 expression evaluation issue for genes <230 bp in length; thus, the fragment length was adjusted downward to 100 bp to evaluate the expression of genes <230 bp. This parameter change does not affect the overall transcript expression for genes >230 bp as seen in the correlation analysis, performed using transcriptome sample Mn03. g, h, Evaluation of quantification range and efficiency of quantitative PCR oligonucleotide probes. Three quantitative PCR oligonucleotide probes (bacteria (g) or species A- or species B-specific (h)) were tested using cloned 16S rRNA gene of either species A (open squares, solid lines) or species B (open triangles, dashed lines) as DNA templates. Threshold cycle (CT) versus gene copies show that all three probes had amplification efficiencies between 90–105% in the quantification ranges plotted. Points represent 3 technical replicates. i, j, Evaluation of specificity of quantitative PCR oligonucleotide probes. The percentage of species A (i) and species B (j) was estimated in reactions containing a mixture of cloned 16S rRNA genes from both species A and species B as DNA templates. Dashed lines represent theoretical 100% match in the expected versus measured values. The results indicate that the species-specific probes quantified their targeted species with minimal interference. Points represent 4 technical replicates.

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Supplementary information

Supplementary Information

This file contains the Supplementary Taxonomic Proposal; Supplementary Notes; and Supplementary Information References. Together, these provide additional support and information underlying the main text and methods.

Reporting Summary

Supplementary Table 1

This table contains community profiles of Mn-oxidising enrichments by iTag sequencing of 16S rRNA genes.

Supplementary Table 2

This table contains a percent identity matrix of representative 16S rRNA gene sequences in the phylum Nitrospirae.

Supplementary Table 3

This table contains transcriptomics analyses of multiple biological replicates of the co-culture after oxidising different amounts of Mn(II).

Supplementary Table 4

This table contains key genomic features and their expression in Species A.

Supplementary Table 5

This table contains key genomic features and their expression in Species B.

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Yu, H., Leadbetter, J.R. Bacterial chemolithoautotrophy via manganese oxidation. Nature 583, 453–458 (2020). https://doi.org/10.1038/s41586-020-2468-5

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