Purple sulfur bacteria fix N2 via molybdenum-nitrogenase in a low molybdenum Proterozoic ocean analogue
SamplingSamples were collected on 28 August 2018 during a field campaign to Lake Cadagno29, Switzerland. In situ measurements and water collection was performed at the deepest part of the lake (21 m). Water was collected using a pump CTD system as described in Di Nezio et al.36. Online in situ data were obtained during a continuous downcast of the CTD-system from the water surface down to ~17.5 m depth. During the upcast, discrete water samples were collected from a total of 20 depths (between 12 m and 17 m) above, in, and below the chemocline for chemical analyses and from 3 depths for incubation experiments (13.7 m, 14 m, and 15.5 m).In Lake Cadagno, wind-driven internal waves lead to vertical shifts of the water masses and their corresponding physicochemical parameters56. While sampling, it was apparent that the depths of the individual water masses had slightly shifted between the down- and the upcast. Therefore, we corrected the water depths of the samples collected during the upcast so that the physicochemical parameters during sampling best matched those of the continuous downcast, to ensure that samples were assigned to the respective water mass that they originated from. A custom R script was employed for the depth correction. In brief, all parameters measured by the CTD-system during the upcast and the downcast were normalized to percent (with 100% as the maximum observed value, and 0% the minimum observed value). Per individual sampling depth (during the upcast, where the pump cast CTD remained stationary for some time), average values of conductivity, temperature, and pressure were calculated and converted to percent values. Then, the depth from the downcast profile was identified that best matched all calculated percent values. This was achieved by subtracting the percent values per parameter from all respective data points of the downcast profile. Absolute values of the calculated differences per data row were summed. The depth with the lowest resulting sum, i.e., with the most similar physicochemical parameters, was then chosen as the corrected depth.Chemical analyses, flux calculations, and rate determinationsFor chemical analyses, lake water from the individual sampling depths was sterile-filtered (0.2 µm, cellulose acetate filter) and frozen at −20 °C until analysis. Samples were analyzed with a QuAAtro39 autoanalyzer (Seal Analytical) using the methods described in Strickland and Parsons57 to determine concentrations of dissolved inorganic phosphorus (PO43−), nitrite (NO2−), nitrate (NO3−), and reactive silica (Si). Ammonium concentrations were determined from the same filtered samples using the colorimetric analysis described in Kempers et al.58. Molybdenum concentrations were determined from filtered samples after acidification with 1% HNO3 (69%, ROTIPURAN®, Roth) using an ICP-MS 7900 (Agilent, Santa Clara, USA). Molybdenum was analyzed on mass 95 in He-mode using a multi-element calibration SRM (21 elements, Bernd Kraft). The SRM NIST 1643f was analyzed in parallel to guarantee the quality of analyses. Concentrations of sulfide were determined colorimetrically from unfiltered Lake water samples, following Cline59.To calculate the turbulent flux (J) of ammonium into the chemocline, we assumed a steady-state using Fick’s first law: J = −D∂C/∂x. A turbulent diffusion coefficient (D) of 1.6 × 10−6 m2 s−1 was used, corresponding to turbulence at the Lake Cadagno chemocline boundaries60. The change in concentration (∂C) was calculated over 14.25 m to 14.77 m depth, where the steepest ammonium gradient was observed. Ammonium uptake rates were calculated for the chemocline by integrating this flux over the chemocline from 13.45 m to 14.45 m depth.To quantify N2 fixation and primary production (i.e., CO2 fixation) rates, stable isotope incubations with 15N2 and 13CO2 were performed using established protocols61. Briefly, lake water from three different depths of the chemocline was sampled directly from the CTD pump system into five 250 ml serum bottles per depth. Water was filled into the bottles from bottom to top, allowing 1–2 bottle volumes to overflow to minimize oxygen contamination before crimp-sealing the bottles headspace-free with butyl rubber stoppers. Back in the field laboratory, no more than 8 h after sampling, one bottle per depth was filtered onto pre-combusted (460 °C, 6 h) glass microfiber filters (GF/F, Whatman®, UK) for in situ natural abundance of C and N. 13C-labeled sodium bicarbonate (NaH13CO3, 98 atom% 13C, dissolved in autoclaved MilliQ water; Sigma-Aldrich) was injected (320 µL) into three bottles per depth, to achieve a final concentration of 160 µmol L−1. Then, a volume of 5 ml 15N2 gas (Cambridge Isotope Laboratories, >98 atom% 15N, Lot #: I-19197/AR0586172) was injected as a bubble into the same bottles and shaken for 20 min to equilibrate the 15N2 gas. Sulfide solution was injected aiming for a final concentration of approximately 2 µM to remove trace oxygen contamination in the incubation bottles. Finally, the 15N2 gas bubble was replaced by anoxic in situ lake water from the respective depth. The bottles, together with one untreated control bottle per depth (containing unamended lake water), were incubated for a full light-dark cycle (13 h light, 11 h dark) under natural light conditions (0–8267 Lux, average: 247 Lux, median: 10.8 Lux, as determined by a HOBO pendant data logger, Onset Computer Corporation, Bourne, USA) in a water bath kept at ~12 °C.After incubation, samples were filtered onto pre-combusted GF/F filters. The filters were dried at room temperature and frozen at −20 °C for transport and storage. In addition, subsamples for nanoscale secondary ion mass spectrometry (nanoSIMS) analysis and for the determination of 13C and 15N enrichments in the substrate pools were taken from all bottles amended with 13C and 15N. NanoSIMS samples were fixed with 2% (final w/v) formaldehyde solution for 1 h at room temperature, prior to filtration onto gold-sputtered 0.22 µm polycarbonate membrane filters (GTTP IsoporeTM, Merck Millipore, USA). Subsamples for label% determinations were taken in gas-tight glass vials (Exetainer Labco, UK) and biological activity was terminated with HgCl2.Samples on GF/F filters were analyzed for C and N content and the respective isotopic composition by an elemental analyzer (Thermo Flash EA, 1112 Series) coupled to a continuous-flow isotope ratio mass spectrometer (Delta Plus XP IRMS; Thermo Finnigan, Dreieich, Germany). Enrichment of 15N in the N2 pool was determined using a membrane inlet mass spectrometer (MIMS; GAM200, IPI). Enrichment of 13C in the dissolved inorganic carbon pool was determined from 13C/12C-CO2 ratios after sample acidification with phosphoric acid using cavity ring-down spectroscopy (G2201-I coupled to a Liaison A0301, Picarro Inc., connected to an AutoMate Prep Device, Bushnell, USA). In addition, we tested the used 15N2 gas bottle for contamination with 15N-ammonia62. Briefly, a 2 ml subsample of the used 15N2 gas was injected into a 12 ml gas-tight glass vial (Exetainer) filled with MilliQ (pH 95% sequence identity to any of the MAG NifD/NifK sequences were identified with a blastp search74 to the NCBI nr database. Multiple sequence alignments were obtained with MAFFT87. All full-length sequences were used to construct base trees with RAxML88 and 100 bootstraps in ARB90. The ARB Parsimony function was employed to add partial sequences to the base trees.The resulting trees were visualized in iTOL91.FISH, cell counts, and cell sizesFrom each incubation depth, 10–30 ml lake water was filtered onto 0.22 µm polycarbonate membrane filters (GTTP IsoporeTM, Merck Millipore, USA). The filters were fixed in 2% formaldehyde solution in sterile-filtered lake water for 10–12 h at 4 °C and then washed with MilliQ water. The filters were frozen and stored at −20 °C until further processing.The 16S rRNA FISH probe “Thiosyn459” (Table S7), exclusively targeting T. syntrophicum Cad16, was designed in ARB90. In addition, two competitor probes and four helper probes92 were designed (Table S7) to ensure efficient and specific binding of the probe to the target. All FISH probes and respective formamide concentrations are listed in Table S7. Probes, but not helpers and competitors, were double-labeled with either Atto488 or Atto594 fluorophores. Samples were embedded in 0.05% low melting point agarose. Cells were permeabilized with lysozyme (1.5 mg ml−1) for 30 min at 37 °C. Hybridization was performed for 2–4.5 h at 46 °C. Washing included 15 min in washing buffer at 48 °C and 20 min in 1× PBS buffer at room temperature. We used the hybridization and washing buffers described in Barrero-Canosa et al.93 to reduce background fluorescence. Cells were counterstained with DAPI.Samples were analyzed using a Zeiss Axio Imager.M2 microscope equipped with a Zeiss Axiocam 506 mono camera. Z-stack images were taken and the number of fluorescently labeled cells per image was counted for the individual probes. For each PSB population, we analyzed ≥38 randomly selected fields of view and ≥54 target cells, on one filter replicate each (see Supplementary File S1). Total cell counts were obtained in triplicates through flow cytometry as described in Danza et al.94.For cluster-forming organisms (Thiodictyon syntrophicum, Lamprocystis purpurea, Lamprocystis roseopersicina, and Lamprocystis spp.), the cell size (length and width, for biovolume and C-content calculations, see section below) of 100 cells per population was determined from the maximum-intensity projection of the z-stack images using the Zeiss Zen blue software 3.2.Single-cell analysis with nanoSIMSFor nanoSIMS analyses, we chose the replicate sample from 13.7 m depth that exhibited the highest bulk N2 fixation rate. Random spots were marked with a laser microdissection microscope (6000 B, Leica) on the gold-sputtered GTTP filter covered with cells incubated with 15N2 and 13CO2. After laser marking, FISH was performed as described above. For analysis of Thiodictyon cells, no permeabilization was performed, while for analysis of the other population’s permeabilization was reduced to 15 min at 37 °C using 2 mg ml−1 Lysozyme. Within one hybridization reaction, we simultaneously applied Apur453 with S453D and Laro453 with Cmok453, each probe double labeled with different fluorescent dyes (Atto488 and Atto594).Single-cell 15N- and 13C-assimilation from incubation experiments with 15N2 and 13CO2 was measured using a nanoSIMS 50 L instrument (CAMECA), as described in Martínez-Pérez et al.53. Briefly, instrument precision was monitored regularly on graphite planchet. Samples were pre-sputtered with a Cs+ beam (~300 pA) before the measurements with a beam current of around 1.5 pA. The diameter of the primary beam was tuned More
