Bacterial chemolithoautotrophy via manganese oxidation

Media and culture enrichment, refinement, and maintenance.

A fortuitous enrichment culture for Mn(II) lithotrophs was established over the Summer and Fall of 2015. A dense slurry of freshly precipitated MnCO₃ (see below) was distributed onto the internal surface of wide mouth glass jar and the coat was allowed to dry. The jar was filled completely with unsterilised Pasadena municipal drinking water (typically a blend from surface and aquifer sources) collected and allowed to stand for approximately 10 weeks, open and without agitation in an unoccupied room maintained at ca. 21°C (GPS 34°08’12.1”N 118°07’38.3”W). Additional freshly precipitated MnCO₃ was added as a dense suspension after the cream-coloured coating had blackened, and the jar was covered with a loose fitting lid and allowed to incubate for several more months, after which a small amount (ca. 5% v/v) of the dark product was used to inoculate flasks of MnCO₃ suspended in municipal tap water. After this, a separate line of cultures was initiated in a defined, deionized water medium (below) incubated in the laboratory at 37 °C.

The basal medium used for routine growth and maintenance of this line and in related experiments was adapted and modified from prior formulations^51,52. The medium contained, per liter deionized H₂O: NaCl, 1 g; MgCl₂·6H₂O, 400 mg; CaCl₂·2H₂O, 1 g; KCl, 5 g; Na₂SO₄, 142 mg; FeCl₃·6H₂O, 2 mg; H₃BO_3, 30 µg; MnCl₂·4H₂O, 100 µg; CoCl₂·6H₂O, 190 µg; NiCl₂·6H₂O, 24 µg; CuCl₂·2H₂O, 2 µg; ZnCl₂, 68 µg; Na₂SeO₃, 4 µg; Na₂MoO₄, 31 µg; riboflavin, 100 µg; biotin, 30 µg; thiamine HCl, 100 µg; L-ascorbic acid, 100 µg; d-Ca-pantothenate, 100 µg; folic acid, 100 µg; nicotinic acid, 100 µg; niacinamide, 100 µg; 4-aminobenzoic acid, 100 µg; pyridoxine HCl, 100 µg; lipoic acid, 100 µg; NAD, 100 µg; thiamine pyrophosphate, 100 µg; cyanocobalamin, 10 µg. As P source, a solution of potassium phosphate (pH 7.2) was added to a final concentration of 0.15 mM. As N source, NaNO₃ was added to a final concentration of 1 mM; alternatively, 1 mM NH₄Cl or other N sources were employed as noted. MOPS at its pKa was added as a buffer to a final concentration of 5 mM, when noted. MnCO₃, or alternative, heat-stable substrate for growth was added to the basal medium prior to steam sterilisation; alternatively, heat-unstable substrate for growth was added using filter sterilised stock solutions after media had adequately cooled.

Freshly precipitated MnCO₃ was employed, for the initial enrichment culture, for routine Mn(II)-dependent cultures in small volumes; or for the serial dilution-to-extinction resolution of complex cultures; or for stock and starter cultures. To prepare this, 25 g MnCl₂ (Sigma-Aldrich #221279) was dissolved into 100 ml deionized water, yielding 125 ml of a 1.59 M MnCl₂ solution. Over the course of several minutes, this solution was slowly poured into 3 liters of 0.33 M NaHCO₃ while vigorously stirring. After the cessation of stirring after ca. 1 hour, the resultant precipitate was allowed to settle by gravity. Thereafter, the overlying reaction fluid was decanted and discarded. The precipitate was resuspended in 3 liters of deionized water, and the stirring, settling, decanting, and resuspension steps were repeated at least 10 times. After the final wash, the precipitate was resuspended in deionized water to a final volume of 100 ml, stored in a clean glass bottle, and refrigerated at 4°C in the dark. Initially, the precipitate appeared white to light pink, but aged to a light tan within ca. 24 h. Thereafter, the material remained stable for months to years. Alternatively, a hydrated MnCO₃ substrate (Sigma-Aldrich #63539) was employed for larger culture volumes, and/or for reproducible mass balances, as noted.

Cells were typically cultured in 10 ml of medium in 18-mm diameter culture tubes, or in 100 ml of medium in 250-ml Erlenmeyer flasks at 37 °C, with 25 to 200 mM MnCO₃, as noted. To prevent dehydration of cultures over the long periods of incubation, cut strips of Parafilm (Heathrow Scientific, Vernon Hills, IL, USA) were used to seal the bottom edge of the 18-mm plastic test tube enclosures. Cultures were incubated stationary without agitation, or with shaking at 200 rpm, as noted.

To refine the number of species in the complex manganese-oxidising enrichment, 5 successive rounds of serial tenfold dilution-to-extinction series were performed using 9 ml of MnCO₃ nitrate basal media in 18-mm culture tubes, incubated at 32 °C. Culture tubes in each dilution series were scored as positive for the presence of lithotrophic manganese oxidisers when, after 2–12 weeks of incubation, the small and easily dispersed particles of the MnCO₃ substrate (light cream in colour) were converted to larger Mn oxide nodules or a single continuous Mn oxide coating (dark brownish-black in colour) that typically tightly adhered to the bottom of the glass culture tubes. Mn oxides from the final dilution tube showing such oxidation were used as the inocula for the next serial-dilution-to-extinction series.

To examine whether Mn(II) oxidation in the cultures was biological in nature, an active co-culture with Mn oxides was used to inoculate 18-mm culture tubes containing the basal media with 50 mM MnCO₃. Cultures were amended with either of two antibiotics, kanamycin (30 µg/ml) or vancomycin (20 µg/ml), or pasteurised overnight at 50 °C before incubation at 32 °C. To examine the impact of incubation temperature on oxidation, cultures were incubated without agitation in different incubators set at a diversity of temperatures; incubation temperatures were regularly and independently confirmed with >2 thermometers. After 2 weeks, 2 ml mixture from the cultures were sampled and stored at −80 °C for later ICP-MS analysis (below). Reported values were corrected for Mn oxides carried over in the inoculum, as ascertained by the lowest amount determined in the 50 °C pasteurisation experiments.

To examine the growth of the culture in the absence of Mn(II) substrate, a stationary co-culture (confirmed separately to be viable) was used to inoculate 4 replicates of 250-ml Erlenmeyer flasks containing 120 ml of the basal media without MnCO₃. The flasks were incubated at 37 °C with shaking at 200 rpm. After inoculation and after 10 and 21 days, 20 ml mixture from the cultures were sampled and centrifuged at 5250 × g for 10 min; the pellet was stored at −80 °C. DNA was extracted from the thawed pellets using the DNeasy PowerSoil kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions, with the bead beating option using FastPrep FP120 (Thermo Electron Corporation, Milford, MA, USA) at setting 5.5 for 45 s instead of the 10 min vortex step. DNA concentration was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

To examine whether MnCl₂ can be oxidised by, or otherwise impacts growth: an active co-culture, which had oxidized 38% of the 35 mM MnCO₃ initially provided (measured using the ICP-MS method below), was used to inoculate (10% v/v) 18-mm culture tubes containing 10 ml of the basal media with 0.5–20 mM MnCl₂ instead of MnCO₃. After inoculation, and after days 5 and 10, 0.5 ml of the oxides and culture fluid mixture was sampled and stored at −80 °C for later ICP-MS analysis (below). Reported values were corrected for Mn oxides carried over in the inoculum, as ascertained by the lowest amount determined in the 0.5 mM MnCl₂ culture.

For attempts to observe single colony formation by Mn(II) lithotrophs on agar media, the basal medium was adjusted to contain 200 mM MnCO₃ and 1.5% washed agar (BD Difco), and distributed into petri dishes after steam sterilisation.

Genomics predicts that each species in the co-culture may be able to produce compatible solutes (e.g. trehalose and hydroxyectoine, by Species A), and thus may be able to grow under a range of salinities. To examine the impact of increased salinity on the Mn(II)-oxidising lithotrophs, the basal medium was amended with NaCl to achieve final salt concentrations of 2 ppt, 2.8 ppt, 3.8 ppt, 4.6 ppt, 5.5 ppt, 9 ppt, 16 ppt, 23 ppt, 30 ppt, and 37 ppt (equivalent to 6%, 8%, 11%, 13%, 16%, 26%, 45%, 65%, 85%, and 105% of the salinity of seawater, respectively). After inoculation, oxidation in the tubes was monitored visually over time.

For isolation and maintenance of strains of “Species B” (Ramlibacter lithotrophicus) from the co-culture (Supplementary Note 2), plates of agar-solidified (1.5% agar w/v, BD Life Sciences) basal media were employed except that sodium succinate (10 mM, final conc.) or tryptone (0.5% w/v, BD Life Sciences) were used in place of MnCO_3. Viable cells of this bacterium could rarely be retrieved from the co-culture as planktonic cells overlying Mn oxide nodules. For clonal isolation, 200 μl of a dense suspension of the dark Mn oxides produced by the co-culture were spotted onto the surface of succinate agar medium and allowed to dry, after which the Mn oxides were vigorously and heavily streaked over the agar surface and monitored for the development of colonies thereafter. After 3–5 days of incubation, colonies of Species B appeared small, leathery, and adherent to the agar surface; transfer of cells to new plates or liquid media was facilitated by the use of a sterile syringe needle for the removal of an entire single colony from the agar surface. In liquid, newly isolated strains of Species B can be grown with tryptone (0.5% w/v) or acetate (10 mM, final conc.) (Extended Data Fig. 6c,d), and can form fabric-like biofilms blanketing at the bottom of culture tubes. Transfer of such material proved challenging, as the fabric like biomass typically adhered to the insides of both plastic and glass pipette surfaces, leading to the rapid selection for strain variants that do not form flocs.

For the examination of anaerobic growth of Species B, 10 ml of basal medium is dispensed into 18-mm glass “Balch” tubes (Bellco, Vineland, NJ, USA) and stoppered with 1 cm butyl rubber stoppers under an N₂ headspace. Autotrophic growth of the isolate using H₂ + O₂ + CO₂ was examined similarly, except that an air headspace, periodically spiked with 1 ml of H₂ + CO₂ (80%/20%, v/v), was used. Colonies of Species B did not develop on standard lysogen broth (LB) agar, or on plates of the basal medium amended with 5 g/L yeast extract (BD Biosciences), traits that were used to monitor for culture purity or contamination.

For growth of manganese-oxidising lithotrophs in agarose tubes, the basal medium was amended to contain 150 mM freshly prepared MnCO₃ and 0.38% w/v agarose (Aquapor LE Ultrapure; National Diagnostics, Atlanta, GA, USA). After steam sterilisation, 15 ml aliquots of the molten agar medium was dispensed into sterile 18-mm glass culture tubes fitted with plastic caps. After cooling to 45°C in a water bath, the molten agar was inoculated with 0.5 ml of a dense suspension of Mn oxides from an actively growing lithotrophic culture, gently vortexed, and allowed to harden on ice before incubation at 37°C. Tubes were sealed with parafilm to avoid desiccation over the long periods of incubation, and monitored for both clearing of MnCO₃ as well as formation (or changes in the size, shape, and distribution) of Mn oxide nodules.

Examination of Mn oxide nodules

In all cases examined, the generation of the dark, granular product was coincident with the generation of Mn oxides from Mn(II)CO₃, as determined via colourimetry⁵³, FTIR-ATR spectroscopy (below), reactivity with H₂O₂, and/or ICP-MS (below).

To visualize, with minimal disturbance, cells on Mn oxide nodules generated in agarose tubes, cores of regions where the carbonate cleared and nodules developed were sampled with sterile glass Pasteur pipettes. Agar cores containing undisrupted nodules were extruded carefully into a plastic weight boat and soaked in the basal medium (without MnCO₃) amended to contain 10 μg/μl acridine orange⁵⁴ for 30 minutes. The dye solution was decanted, and the core was soaked for 30 minutes in buffered medium without dye. The core was carefully transferred to glass microscope slides and gently covered with coverslips before examination via light microscopy. Light microscopy of Mn oxide nodules in agarose cultures was performed using both a Zeiss Stemmi 2000-C stereomicroscope and a Zeiss Axioplan 2 Imaging Microscope fitted with a HBO 100 mercury arc lamp housing and an ebq 100 lamp power supply (Lighting & Electronics Jena, Jena, Germany). For epifluorescence microscopy of acridine orange stained nodules, a blue (FITC) long pass filter set was employed [excitation, filter D470/40 Lot 33820 (Chroma Technology, Bellows Falls, VT, USA); emission, long pass filter OG515 (515 nm cut-on; Schott, Elmsford, NY, USA)]. Photodocumentation was with Panasonic Lumix GH3 and G85 microfourthirds-lens-mount cameras, mounted onto the research microscope via a combination of Zeiss to c-mount (1.0 X D10ZNC; Diagnostic Instruments, Sterling Heights, MI, USA) and “c-mount to microfourthirds-mount” (Fotodiox, Gurnee, IL, USA) adaptors. RAW images were imported into Lightroom 9 (Adobe, San Jose, CA, USA). Global adjustments to RAW images were made in Lightroom, e.g. to improve clarity through the opaque agarose. The global changes to RAW images in this line of experiments involved correction for white balance and adjustments via the texture-, clarity-, dehazing-, sharpening-, and “colour-noise reduction”- sliders.

For mineralogical analyses, Mn oxide nodules were harvested from cultures and allowed to settle by gravity, washed three times with distilled water, incubated in consumer-grade concentrated bleach for 2 hours at room temperature (to remove organic biomass carbon), washed three times with distilled water, and then dried at 80 °C. Spectroscopic analyses were performed on Nicolet Magna 860 Fourier transform infrared (FTIR) spectrophotometer (Thermo Nicolet, Madison, WI, USA) with Attenuated Total Reflectance (ATR) accessory (Durascope, SensIR Technologies, Danbury, CT, USA), and Nicolet iS50 FTIR spectrophotometer (Thermo Nicolet) with GladiATR ATR accessory (Pike Technologies, Madison, WI, USA).

For scanning electron microscopy (SEM) of the Mn oxide nodules, samples were chemically fixed on ice for 3–4 hrs in the basal medium amended with 2.5% glutaraldehyde and HEPES (25 mM, pH 7.5). Fixed samples were washed twice in the same buffered solution but without glutaraldehyde, and the final pellet was resuspended in the buffered solution with 50% (v/v) ethanol. Dehydration series were performed by increasing the ethanol concentration every 15 min to 70, 90, and finally 100% (v/v). Critical point drying (Quorum Technologies, Newhaven, UK) was performed as follows: ethanol was replaced by liquid CO₂ at a pressure of 55 bar at <10 °C. Fifteen minutes after reaching the critical point, the pressure was released slowly at >35 °C until ambient pressure was reached. The samples were then deposited onto double coated carbon conductive tape (Ted Pella, Redding, CA, USA), and in some cases, were sputter coated with 10 nm of Pt/Pd (80/20 by weight, Cressington, UK). SEM was performed using a field emission SEM (1550VP, Zeiss, Germany) with SE2 detector at an operating voltage of 10 kV.

Community analysis

An iTag 16S rRNA gene sequencing approach was employed to obtain microbial community profiles. For such, a 2 ml culture sample containing Mn oxide nodules was centrifuged at 6000 × g for 5 min. DNA was extracted from the pellets and quantified as above. The V4-V5 region of the 16S rRNA genes was amplified using primers 515F-Y (5’-GTG YCA GCM GCC GCG GTA A-3’) and 926R (5’-GGA CTA CNV GGG TWT CTA AT-3’)⁵⁵ with Illumina adaptor overhangs⁵⁶ added to the oligonucleotides. PCR amplification was performed using 7.5 µL of Q5 High Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, USA), 0.75 µL of each forward and reverse primers (10 µM), 5 µL of PCR-grade water and 1 µL of DNA with a concentration about 1 ng/µL. PCR cycling condition was as following: initial denaturation at 98 °C for 2 min, 25 cycles of 98 °C for 10 sec, 54 °C for 20 sec, 72 °C for 20 sec, and a final extension of 72 °C for 2 min before cooling down to 6 °C. Duplicate PCR reactions for each samples were run; after confirming successful and comparable amplification using gel electrophoresis, the duplicates were combined for Illumina Nextera XT barcoding⁵⁶ using PCR condition as above except in 25 µL reaction volume and 10 cycles with annealing at 66 °C. Barcoded samples were quantified using QuantIT PicoGreen dsDNA Assay (Thermo Fisher Scientific) on the C1000 Thermal Cycler with CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA), combined in equal molar amounts, and purified with QIAquick PCR Purification Kit (Qiagen). Sequencing was performed on the MiSeq platform (Illumina, San Diego, CA, USA) with paired 250 bp reads after PhiX addition of 15–20% (Laragen, Culver City, CA, USA).

Sequence data was processed using QIIME⁵⁷ v1.8.0. Raw sequences were first joined and quality-trimmed using the default read pair joining and quality-trimming parameters. Processed sequences were clustered into de novo operational taxonomic units (OTUs) at 99% sequence identity using UCLUST⁵⁸ v7.11.0.667, with the most abundant sequence picked as the representative sequence for each OTU. OTUs with only a single sequence (“singletons”) was removed. Taxonomic identification for the representative sequences were done using SILVA⁵⁹ Ref NR 99 Database Release 119.

Cloning of near full-length 16S rRNA genes of Species A and Species B

Near full-length 16S rRNA genes were amplified and cloned from the co-culture. First, DNA was extracted as above. Second, 16S rRNA gene was amplified using primers BACT27F (5’-AGA GTT TGA TYM TGG CTC-3’) and U1492R (5’-GGY TAC CTT GTT ACG ACT T-3’) modified from published versions⁶⁰. PCR was performed using the Expand High Fidelity System (Roche Molecular Systems, Pleasanton, CA, USA) with the following conditions: 2.5 µl of 10 × buffer, 0.35 µl of Taq polymerase, 0.55 µl of 10 mM dNTP, 0.50 µl of each forward and reverse primer, 18.6 µl of PCR-grade water and 2 µl of a DNA extract. The cycling condition was as following: 95 °C for 2 min, followed by 30 cycles of 94 °C for 15 sec, annealing at 54 °C for 30 sec and extension at 72 °C for 45 sec, and a final extension step at 72 °C for 4 min before cooling down to 4 °C. Third, the PCR products were immediately purified using QIAquick PCR purification kit (Qiagen), ligated into pGem-T Easy Vector (Promega, Madison, WI, USA), and the resulting plasmid was transformed into JM209 competent Escherichia coli cells (Promega) following the manufacturer’s instructions. Over 50 white colonies were observed on X-gal containing plates from 50 µl of transformed cells. Clones were grown overnight at 37 °C in LB medium with 10% glycerol and 0.1 mg/ml ampicillin following the manufacturer’s instructions (Promega). Lastly, PCR was performed using the NEB Taq Polymerase kit (New England Biolabs) with the following conditions: 3.0 μl of 10 × buffer, 0.66 μl of dNTP, 0.30 μl of Taq polymerase, 0.60 μl of each M13 forward and reverse primers (10 µM), 0.30 µl of 10 µg/µl bovine serum albumin, 23.95 μl of PCR water, and 0.60 μl of cells. The cycling conditions were the same as the cloning reaction above. The PCR products were purified using Multiscreen HTS PCR 96-well plates (Millipore, Billerica, MA, USA). Sanger sequencing was performed on the purified PCR products to confirm their sequence identities using both M13 forward and reverse primers (Laragen).

Fluorescence in situ hybridisation (FISH)

Three oligonucleotide probes were developed or tested in this study in order to visualize and differentiate cells representing Species A and Species B in Mn oxide nodules (Supplementary Note 3; Extended Data Fig. 10a–d). SILVA⁵⁹ Ref NR 99 Database Release 128 and the PT Server function of ARB software⁶¹ v6.0.2 was used for the development. The Species A oligonucleotide probe (NLT499: 5’-ACA GAG TTA GCC GTG GCT-3’) was developed and designed to target the 16S rRNA genes from members of the classes Leptospirillia, Thermodesulfovibrionia, as well as other uncultivated classes within the phylum Nitrospirota⁶². Two oligonucleotide probes were used to target members of the order Betaproteobacteriales, including Species B. Previously, a probe (BETA359 or Beta1: 5’-CCC ATT GTC CAA AAT TCC CC-3’)^63,64 had been reported, but the optimal hybridisation conditions were not. A second probe was designed for this study (BETA867: 5’-AGG CGG TCA ACT TCA CGC-3’). A previously developed probe that targets most all Bacteria (EUB338: 5’-GCT GCC TCC CGT AGG AGT-3’)⁶⁵ was also employed for FISH. All probes were double labelled⁶⁶: with Cy3 dye for NLT499, FITC dye for BETA359 and BETA867, and Cy5 dye for EUB338 (Integrated DNA Technologies, San Diego, CA, USA).

The Clone-FISH method⁶⁷ was used to evaluate the specificity and hybridisation condition of these three oligonucleotide probes. Transformed JM209 E. coli (above) containing either 16S rRNA gene of Species A or Species B were grown overnight at 37 °C with shaking at 200 rpm in ampicillin-containing LB media. Plasmids were isolated using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer’s instructions. Isolated plasmids were then transformed into NovaBlue(DE3) E. coli competent cells (EMD Millipore, Billerica, MA, USA) following manufacturer’s instructions. 16S rRNA gene sequence of transformed NovaBlue(DE3) cells were confirmed as above by Sanger sequencing (Laragen). Cells for Clone-FISH were prepared as previously described⁶⁷. Briefly, overnight cultures of OD₆₀₀≈0.4 were induced with IPTG (1 mM) for 2 h, and chloramphenicol (170 mg l⁻¹) was added to the cultures and incubated for 5 h. The cells were then harvested by centrifuging at 8000 × g for 10 min, fixed with 4% paraformaldehyde in 1 × PBS, washed twice with PBS and stored in PBS:ethanol (1:1) at −20 °C. FISH reactions were performed as previously described⁶⁸ using a BX51 epifluorescence microscope (Olympus, Shinjuku, Japan) with 100× (UPlanFL N) oil immersion objective. Each point in the dissociation profile represents the mean of fluorescence intensities of at least 100 single cells in 5 microscopic fields evaluated using the software daime⁶⁹ v2.1 with default automatic segmentation settings of threshold algorithm Isodata and manually size filtering resulting regions of interest for single cell analysis.

To prepare Mn oxide nodules for FISH visualisation, Superfrost Micro Slide (VWR Scientific, Radnor, PA, USA) were dipped in 0.2% UltraPure agarose (Thermo Fisher Scientific) at 45 °C. Mn oxide nodules were fixed with paraformaldehyde (above), except stored in PBS at 4 °C instead for no longer than 2 weeks. Fixed Mn oxide nodules in PBS were immediately pipetted onto the slide and cooled to room temperature. After this, the slide was dipped in freshly prepared DCBE buffer (0.05 M sodium dithionite, 0.1 M sodium citrate, 0.1 M sodium bicarbonate and 0.1 M EDTA at pH 7; a modification of DCB buffer⁷⁰) for 5 min at room temperature to dissolve away the Mn, leaving nodule-associated cells constrained within the agarose. The cells were then permeabilized with protease K (15 µg/ml) for 10 min at room temperature, and lysozyme (10 mg/ml) for 30 min at 37 °C. The slide was washed once in 0.01 M HCl for 15 min and three times in water for 1 min each before drying at 37 °C. The permeabilisation and HCl washing is not necessary for FISH reaction to work, and can be omitted. FISH reactions, using NLT499-Cy3, BETA867-FITC, and EUB338-Cy5 probes, were performed in 35% formamide hybridisation buffer⁶⁸ with corresponding salt concentration in the washing buffer⁶⁸. DAPI-citifluor (5 μg/ml) was added to the FISH samples prior to epifluorescence microscopy using a BX51 epifluorescence microscope (Olympus) with 100× (UPlanFL N) oil immersion objective.

Stable isotope probing and nanoSIMS analyses

Mn-oxidising co-cultures were grown in basal medium containing Mn¹³CO₃ and ¹⁵NO₃⁻ to visualize the assimilation of ¹³C and ¹⁵N into the cells of Species A and Species B. For these, 0.5 ml samples from of a stationary culture grown in basal medium with 26 mM unlabelled MnCO₃ was transferred into 18-mm glass “Balch” tubes containing 9.6 ml of autoclaved basal media with 13 mM Mn¹³CO₃ (prepared using ¹³C-NaHCO₃, 99 atom % ¹³C, Cambridge Isotope Laboratories, Tewksbury, MA, USA). For N source, the media contained 1 mM Na¹⁵NO₃ (98 atom % ¹⁵N, Sigma-Aldrich Isotec, #364606). The culture tubes were stoppered under ca. 20 ml of air headspace with 1 cm butyl rubber stoppers, and incubated at 37 °C without shaking until, by visual examination, no MnCO₃ appeared to remain. Resulting Mn oxide nodules were fixed with paraformaldehyde, dissolved with DCBE buffer (above) by shaking at 37 °C for 1 hr, and filtered onto 0.2 µm polycarbonate filters (Isopore GTBP02500, Millipore) that have been sputter coated with 10 nm of Au. FISH reactions were performed on the filters (as describe above but without permeabilisation and HCl washing) in 25% formamide hybridisation buffer. As evaluated in Clone-FISH, no cross-reaction was observed in 25% formamide hybridisation buffer and may increase signal intensity. A culture grown in the presence of 13 mM unlabelled MnCO₃ (prepared using NaHCO₃, Thermo Fisher Scientific, #S233–500) and 1 mM Na¹⁵NO₃ was used for comparison and as a control. DAPI-citifluor (5 μg/ml) was added to the FISH samples prior to epifluorescence microscopy using a Elyra S1 (Zeiss, Jena, Germany) fitted with a Plan-Apochromat 63×/1.4 Oil immersion DIC objective. In parallel, in order to both assess and to minimize the dilution of signal ¹³C and ¹⁵N in labelled biomass (e.g. by certain unlabelled reagents such as paraformaldehyde and reagents for FISH) during post-incubation sample preparations: Mn oxide nodules were dissolved as above with DCBE buffer but without paraformaldehyde fixation, with the resultant cells subsequently stained for epifluorescence microscopy with DAPI-citifluor only (i.e. no FISH reagents). However, the dissolution of nodules for analyses required the use of DCBE buffer which contains unlabelled citric acid, bicarbonate, and EDTA, residuum from which, if associated with biomass from the nodules, would artifactually lower estimates of the extent of the labelling especially for ¹³C. Finally, filters were washed in ethanol with increasing concentrations (50%, 80%, 100%) each for 3 minutes before mounting on nanoSIMS holder using double coated carbon conductive tape (Ted Pella).

NanoSIMS analysis was performed using a CAMECA NanoSIMS 50L at the Caltech Microanalysis Center. A focused primary Cs⁺ beam of 100–1000 pA was used for pre-sputtering the sample until ¹²C¹⁴N^– counts stabilized, and 2–4 pA was used for image collection with rasters of 512 × 512 pixels. Secondary ions ¹²C¹²C⁻, ¹³C¹²C^–, ¹²C¹⁴N^–, ¹²C¹⁵N^– and ³²S⁻ were measured simultaneously for at least 3 image frames. In one instance, a region was measured a second time with ⁵⁵Mn¹⁶O⁻ added to the analysis to confirm the absence of Mn in the sample. Individual ion image frames in the resulting data were aligned using the ¹²C¹⁴N⁻ ion and epifluorescence microscopy images were transformed to match that of nanoSIMS ion images using Look@NanoSIMS⁷¹ v2019-05-14; regions of interest (ROIs) corresponding to cells of Species A or Species B were defined manually. Final ion counts per ROI were calculated by summation of ion counts over all image frames. Atom percent of ¹³C or ¹⁵N was calculated from the individual ion counts as described in Supplementary Note 9.

Kinetics of Mn(II) oxidation and cell growth

Glassware used in the kinetic experiments were acid washed with 3.7% HCl and combusted at 550 °C to eliminate residue metals and organic carbon. All cultures were incubated at 37 °C with shaking at 200 rpm.

The kinetic experiments were performed in 1 L flasks with 0.4 L of the basal media using commercial MnCO₃ (Sigma-Aldrich) with a final content of 100 mM Mn(II) as determined using the ICP-MS method (below). The basal media contain either 1 mM NaNO₃ (n=4) or 1 mM NH₄Cl (n=5) as the N source. Each culture flask was inoculated with 5 ml of a Mn oxide slurry sampled from a flask of an active co-culture grown with 1 mM nitrate and 1 mM urea as the N source. Culture material was sampled from each flask daily for the first 36 days, followed by 3 final, well separated time points over the final 30 days. At each time point, culture flasks were removed from the shaking incubator and swirled. Immediately thereafter, a total of 3 ml of oxides and culture fluid mixture was aseptically sampled from the flask via 5 ml disposable pipette. Of this, 1 ml of the sample was saved at −80 °C for later ICP-MS analysis (below); 2 ml of the sample was centrifuged at 8000 × g for 5 min and the pellet was stored at −80 °C for later quantitative PCR analyses. DNA was extracted from the pellets and quantified as above.

Chemical analysis of Mn

Reduced Mn(II) and oxidised Mn(III)/Mn(IV) pools were measured using a previously described method⁶ and evaluated (Supplementary Note 4; Extended Data Fig. 10e). Briefly: 0.1 ml of oxide and culture fluid mixture was mixed with 0.9 ml of 0.5 N HCl. After reacting for at least 10 min, the mixture was centrifuged at 16100 × g for 3 min. The supernatant (“acid-soluble fraction”, representing Mn(II)) was pipetted out into a separate tube. The pellet (“acid-insoluble fraction”, representing Mn(III/IV)) was then reacted with 1 ml of 0.25 N NH₂OH⋅HCl in 0.25 N HCl. The acid-soluble and acid-insoluble fractions were then centrifuged again at 16100 × g for 3 min to avoid any carryover. From each fraction, 0.1 ml was sampled and then diluted into 10 ml of 2% HNO₃. The Mn contents were measured using an Agilent 8800 inductively coupled plasma mass spectrometer (ICP-MS; Agilent Technologies, Santa Clara, CA, USA) with the helium gas collision mode and quantified using a Mn standard solution (Sigma-Aldrich, Supelco 1.19789). Attempts were also made to measure total Mn content using the formaldoxime method⁷², and oxidised Mn(III)/Mn(IV) content using the leucoberbelin blue dye^53,73. However, both methods resulted in underestimates of the manganese content when comparing to the ICP-MS method and standards. In large part, this was due to relatively large Mn oxide nodules be challenging to both dissolve and to react with those reagents to completion.

For determining dissolved Mn concentrations in particle free fluids associated with MnCO₃ and spent cultures: fluids were sampled from uninoculated and inoculated cultures containing 20–100 mM MnCO₃. From each sample, 0.1 ml was subsampled after centrifuging at 16000 × g for 3 min, subsequently filtered through a 0.22 µm filter, and each fraction dissolved into 10 ml of 2% HNO₃. The ICP-MS measurements for both Mn(II) and Mn(III/IV) fractions were performed as above.

Quantitative PCR

To obtain standards and test quantitative PCR specificity, plasmids with 16S rRNA gene of either Species A or Species B were purified from transformed JM209 E. coli as above. Purified plasmids were then linearized using 150 units of restriction enzyme SacI in 50 µl reactions containing 1 × NEB Buffer 1 (New England Biolabs) overnight at 37 °C. The restriction digest reactions were heat inactivated at 65 °C for 20 min and purified using Multiscreen HTS PCR 96-well plates (Millipore). Concentration of the linearized and purified plasmids were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) to make DNA template standards containing known copies of 16S rRNA genes of Species A or Species B.

To track the growth of all Bacteria in Mn-oxidising cultures, quantitative PCR assays were performed based on an assay developed and optimized previously⁷⁴ (Supplementary Note 5; Extended Data Fig. 10g–j). All primers and probes were obtained from Integrated DNA Technologies (San Diego, CA, USA) and diluted in 10 mM Tris·HCl (pH 8.0). The forward and reverse primers used, BACT1369F1 (5’-CGG TGA ATA CGT TCC CGG-3’) and PROK1492R1 (5’-GGC TAC CTT GTT ACG ACT T-3’), were one version of the previous degenerate primers BACT1369F and PROK1492R. The TaqMan probe for prokaryotes, TM1389F (TM1389F-FAM-ZEN, 5’-CTT GTA CAC ACC GCC CGT C-3’), was modified on the 5’ with 6-FAM fluorophore, 3’ with Iowa Black FQ dark quencher and internally with ZEN quencher. Also, TaqMan probes that specifically target Species A and Species B were developed. The specific Species A TaqMan probe TM1484R-A (5’-ATC ACC AAT CAT ACC TTG GGT GCC TG-3’) was modified on the 5’ with HEX fluorophore, 3’ with Iowa Black FQ dark quencher and internally with ZEN quencher. The specific Species B TaqMan probe TM1484R-B (5’-GTC ACG AAC CCT GCC GTG GTA ATC-3’) was modified at the 5’ with Texas Red-X fluorophore and 3’ with Iowa Black RQ dark quencher. Optimized quantitative PCR reaction mixtures contain 10 µl of PrimeTime Gene Expression Master Mix (Integrated DNA Technologies), 1 µl of each forward and reverse primer (10 µM), 0.5 µl of each of the three TaqMan probes, 5.5 µl of PCR-grade water, and 1 µl of template DNA. The reactions were run in triplicates at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 sec and 62 °C for 30 sec, on the C1000 Thermal Cycler with CFX96 Real-Time System (Bio-Rad). The amplification efficiency was calculated [Amplification Efficiency = 10^(−1/slope) – 1]. The upper and lower limits of quantification ranges were determined based on dilution of standards with amplification efficiencies in the log-linear phase between 90%−105%. To convert to cell number, the 16S rRNA gene copies quantified in quantitative PCR assays were divided by 1 or 2 for Species A or Species B, respectively, based on the number of 16S rRNA gene copy per genome.

Genomics

To obtain genomic DNA from Species B, a flask with 200 ml of the basal medium containing 5 g/L of both tryptone and yeast extract was inoculated with a single colony of isolated Species B from a succinate nitrate medium plate. The culture was grown at 37 °C with shaking at 200 rpm until early stationary phase, then harvested by centrifuging at 5250 × g for 30 min at room temperature. DNA from the cell pellets was extracted following the Bacterial genomic DNA isolation using CTAB protocol version 3 as described⁷⁵. Ethanol-precipitated DNA was additionally purified using the PureLink PCR Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. To obtain genomic DNA from the co-culture, Mn oxide nodules were harvested from an early stationary phase culture: 2 ml of culture containing ca. 0.15 g of Mn oxide nodules was centrifuged at 5000 × g for 10 min at room temperature. DNA was extracted from the pellet using the DNeasy PowerSoil kit (Qiagen) as above. DNA extract was purified and concentrated using Protocol A of the CleanAll DNA/RNA Clean-up and Concentration Micro Kit (Norgen Biotek, Thorold, Ontario, Canada) following the manufacturer’s instructions.

Purified genomic DNA samples (2–50 ng) were fragmented to the average size of 600 bp via use of a Qsonica Q800R sonicator (power: 20%; pulse: 15 sec on/15 sec off; sonication time: 3 min). Libraries were constructed using the NEBNext Ultra™ II DNA Library Prep Kit (New England Biolabs) following the manufacturer’s instructions. Briefly, fragmented DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3’ ‘A’ base for ligation of NEBNext Multiplex Oligos for Illumina (New England Biolabs) which have a single 3’ overhanging ‘T’ base and a hairpin structure. After ligation, adapters were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN, USA) to generate fragment sizes between 500 and 700 bp. Adaptor-ligated DNA was PCR amplified with 8 to 12 cycles depending on the input amount followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (Thermo Fisher Scientific) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on HiSeq2500 platform (Illumina) with paired 250 bp reads following manufacturer’s instructions. Base calls were performed with RTA v1.18.64 followed by conversion to FASTQ with bcl2fastq v1.8.4 (Illumina). In addition, reads that did not pass the Illumina chastity filter as identified by the Y flag in their fastq headers were discarded. The resulting reads were uploaded to the KBase platform⁷⁶, trimmed using Trimmomatic⁷⁷ v0.36 with default settings and adaptor clipping profile Truseq3-PE, and assembled using Spades⁷⁸ v3.11.1 with default settings for standard dataset. Manual binning and scaffolding were performed using mmgenome v0.7.1⁷⁹, using differential coverage from isolated Species B vs the Species A + Species B co-culture, to generate genome bins for Species A and Species B. Trimmed reads were aligned to either the Species A or Species B genome bin using bowtie2⁸⁰ v2.3.4.1 with default settings. Finally, the resulting reads were reassembled using Spades⁷⁸ v3.11.1 with --careful setting and manually binned with mmgenome⁷⁹ v0.7.1 once again, excluding and contigs <500 bp. Reconstructed genomes were annotated using the IMG⁸¹ Microbial Genome Annotation and NCBI⁸² Prokaryotic Genome Annotation Pipelines; beta-barrel protein prediction was performed using PRED-TMBB⁸³.

Phylogenetic analyses

For Species A phylogenies, 275 publicly available genome assemblies in NCBI Assembly Database⁸² (as of March 26, 2019) were analysed. These fell within the phylum Nitrospirae (Taxonomy ID 40117)⁸⁴, which corresponded to the phylum under the headings Nitrospirota and Nitrospirota_A in the Genome Taxonomy Database (GTDB)⁶² v0.2.2. Genome assemblies with estimated completeness of <60% and contamination of >5% (based on CheckM⁸⁵ v1.0.6) were excluded. For 16S rRNA gene phylogeny, 16s rRNA genes from the genome of Species A or Species B, as well as the genome assemblies were retrieved using CheckM⁸⁵ v1.0.6 ssu_finder utility. Sequences less than 900 bp and contain more than 2 N were excluded. The 16S rRNA gene sequences were aligned using SINA⁸⁶ v1.2.11 and imported into SILVA⁵⁹ Ref NR 99 Database Release 128. 60 16S rRNA gene sequences, including 5 different outgroup sequences (Desulfovibrio vulgaris, Ramlibacter tataouinensis TTB310, Nitrospina gracilis 3/211, Acidobacterium capsulatum, Candidatus Methylomirabilis oxyfera), with 1532 nucleotide positions were exported with bacteria filter in the SILVA Database. Bayesian phylogenetic trees were constructed using MrBayes⁸⁷ v3.2.6 with evolutionary model set to GTR + I + gamma, burn-in set to 25% and stop value set to 0.01, and edited in iTOL⁸⁸. For concatenated multilocus protein phylogeny, marker proteins from 40 genomes including the same 5 outgroup species were identified and aligned using a set of 120 ubiquitous single-copy bacterial proteins in GTDB⁶² v0.2.2. The protein alignment was filtered using default parameters in GTDB⁶² v0.2.2 (the full alignment of 34744 columns from 120 protein markers were evenly subsampled with a maximum of 42 columns retained per protein; a column was retained only when the column was in at least 50% of the sequences, and contained at least 25% and at most 95% of one amino acid). The resulting alignment with 5036 amino acid positions was used to construct the multilocus protein phylogeny using MrBayes⁸⁷ v3.2.6 as above except the evolutionary model was set to invgamma and a mixed amino acid model.

For Species B phylogenies, 60 publicly available genome assemblies were selected from the NCBI Assembly Database⁸² under the class Betaproteobacteria (Taxonomy ID 28216)⁸⁴, which corresponded to the order Betaproteobacteriales in GTDB⁶² v0.2.2. These 60 genomes assemblies had >93% completeness and <3% contamination based on CheckM⁸⁵ v1.0.6, and represented different genera. The same 5 outgroups were used as above, except Ramlibacter tataouinensis TTB310 was replaced with Nitrospira inopinata. Bayesian 16S rRNA gene phylogeny was performed as above with 1532 aligned nucleoic acid positions. Maximum likelihood multilocus protein phylogeny was constructed using 5035 amino acid positions using RAxML⁸⁹ v8.1.7 with protein model GAMMALGF and rapid bootstrapping of 100 replicates.

For functional gene phylogeny, protein sequences were aligned using ClustalO⁹⁰ v1.2.4 and maximum likelihood trees were constructed using RAxML⁸⁹ v8.1.7 as above.

Transcriptomics

Glassware treatment and culturing condition for the transcriptomics experiments were as described in the kinetics experiments (above). Three replicates from different, actively Mn-oxidising cultures were used as inocula for RNA experiments. A 5% v/v inoculum from two active Mn-oxidising cultures was transferred into triplicate flasks containing the basal medium amended with 100 mM freshly prepared MnCO₃, for a total of 6 flasks (sample IDs: Mn03/Mn06/Mn08 and Mn09/Mn10/Mn11). A 1% v/v inoculum from another active Mn-oxidising culture was transferred into a 7^th flask of the same medium (sample ID Mn12). For harvesting biomass before complete oxidation of Mn(II) (Extended Data Fig. 3h), shaking of the incubator was paused for 3 minutes to allow Mn oxide nodules to settle by gravity. The overlaying medium was then decanted off until about 10 ml of culture and oxides remained. First 35 ml and then 15 ml of LifeGuard Soil Preservation Solution (Qiagen) were added, sequentially, to maximize transfer efficiency of Mn oxide nodules from the culture into a new 100 ml centrifuge tube. The culture-LifeGuard mixture was stored at 4 °C for less than a week, a duration for which RNA was expected to remain well preserved, according to the manufacturer. Initially, a series of different trials were performed [using multiple methods and variations, including the RNeasy Mini Kit (Qiagen), the RNeasy Powersoil Kit (Qiagen), and a previous customized procedure⁹¹] to directly extract RNA from Mn oxide nodules. These all failed to extract measurable RNA from samples. When an early chemical dissolution step was added to the protocol extraction of RNA from Mn oxide nodules was successful in combination with the procedures below. Before RNA extraction: the culture-Lifeguard mixture was first centrifuging at 5250 × g for 10 min at 4 °C. 400 ml of freshly prepared 0.22 µm filtered DCBE solution (recipe above) was added to the pellet and incubated at 37 °C with shaking at 200 rpm for 10 min to dissolve the Mn oxide nodules. The dissolved samples were then centrifuged at 5250 × g for 10 min at 4 °C. The supernatant was decanted, and the remaining 5 ml material was mixed with 10 ml of 0.22 µm filtered and autoclaved CBE solution (similar to the DCBE solution, but without sodium dithionite). The mixture was then centrifuged at 5250 × g for 10 min at 4 °C. The supernatant was discarded, and 0.9 ml of RLT solution with 2-mercaptoethanol from the RNeasy Mini Kit (Qiagen) was added to the pellet. The mixture was transferred into Glass 0.1 mm PowerBead Tubes (Qiagen) and bead beat using FastPrep FP120 (Thermo Electron Corporation) at setting 5.5 for 45 s. RNA extraction then proceeded using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. DNA from the extracted RNA was removed using the DNase Max Kit (Qiagen). The RNA extracts were purified and concentrated using Protocol C of the CleanAll DNA/RNA Clean-up and Concentration Micro Kit (Norgen Biotek) and stored at −80 °C until sequencing library preparation.

For RNA sequencing library preparation, RNA integrity was first assessed using RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies). RNA-seq libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturer’s instructions. Briefly, 1–10 ng of total RNA was fragmented to the average size of 200 bp by incubating at 94 °C for 15 min in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors (New England Biolabs). After ligation, adaptors were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (Thermo Fisher Scientific), and the fragments were determined to a mean of 350 bp with standard deviation of 70 bp using High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies). Sequencing was performed on the HiSeq2500 platform (Illumina) with single-end 50 and 100 bp reads following manufacturer’s instructions. Base calls were performed with RTA 1.18.64 followed by conversion to FASTQ with bcl2fastq 1.8.4 (Illumina). Low quality reads and TruSeq3-SE adaptors were removed using Trimmomatic⁷⁷ v0.36 with default settings. rRNA was removed using sortmerna⁹² v2.0 with supplied SILVA databases and default settings. Read mapping of the non-rRNA was performed using kallisto⁹³ v0.44.0 with 100 bootstraps, fragment mean of 130 bp and standard deviation of 70 bp. Despite that the fragment mean was determined to be 230 bp using Agilent Bioanalyzer above after adaptor removal (120 bp), this caused issue in evaluating gene expression of genes smaller than 230 bp in kallisto⁹³ v0.44.0. The fragment mean was therefore decreased while not affecting the overall transcript expression (Extended Data Fig. 10f). Final analysis and normalisation of different RNA samples was performed using sleuth⁹⁴ v0.30.0, with either the reconstructed genomes of both species or only of Species B. Supplementary Table 5 includes RNA processing statistics of the processed files where reads mapping was done using BBMap (https://sourceforge.net/projects/bbmap/) v37.93 with minid=0.97 ambiguous=toss settings.