Purple sulfur bacteria fix N2 via molybdenum-nitrogenase in a low molybdenum Proterozoic ocean analogue

Abstract

Biological N₂ fixation was key to the expansion of life on early Earth. The N₂-fixing microorganisms and the nitrogenase type used in the Proterozoic are unknown, although it has been proposed that the canonical molybdenum-nitrogenase was not used due to low molybdenum availability. We investigate N₂ fixation in Lake Cadagno, an analogue system to the sulfidic Proterozoic continental margins, using a combination of biogeochemical, molecular and single cell techniques. In Lake Cadagno, purple sulfur bacteria (PSB) are responsible for high N₂ fixation rates, to our knowledge providing the first direct evidence for PSB in situ N₂ fixation. Surprisingly, no alternative nitrogenases are detectable, and N₂ fixation is exclusively catalyzed by molybdenum-nitrogenase. Our results show that molybdenum-nitrogenase is functional at low molybdenum conditions in situ and that in contrast to previous beliefs, PSB may have driven N₂ fixation in the Proterozoic ocean.

Fig. 1. Lake Cadagno chemocline nutrient, δ¹⁵N and turbidity depth profile, bulk CO₂ and N₂ fixation rates, and PSB abundance.

a Molybdenum (light blue) and sulfide (yellow) depth profiles. b Ammonium (dark blue) and δ¹⁵N values of bulk biomass (red circles, duplicate measurements at depths 13.7 m and 14 m, single measurement at 15.5 m). c Bulk N₂ fixation rates (green) and bulk CO₂ fixation rates (blue, determined in the same samples as N₂ fixation rates). All rate measurements were performed in biological triplicates (except 15.5 m where one replicate was lost), individual replicates are shown as small circles, and the average rate is shown in large circles with the respective standard deviation. N₂ fixation rates at 15.5 m depth were below the detection limit. d Abundances of key PSB populations (stacked bars, cell numbers based on FISH counts) and turbidity (gray line, a measure of total cell abundance). The location of the chemocline, as determined from the conductivity profile, is indicated by gray shading (a–d).

Fig. 2. NifH diversity in Lake Cadagno metagenomes and metatranscriptomes.

Tree shows all verified NifH amino acid sequences obtained from the assembled metagenome from 2014 (MAG sequences and unbinned mg_nifH sequences) and the three metatranscriptomes from 2018 (nifH_read sequences). Reference sequences obtained from NCBI are indicated in gray. Transcription (presence) is indicated by filled boxes in 13.7 m, 14 m, and 15.5 m water depths from inner to outer circle, respectively, while empty boxes indicate that no transcription of the gene was detected. Detailed read counts are listed in Table S3. Sequences originating from GSB and PSB are highlighted in green and purple, respectively. MAG taxonomy was inferred from GTDB-Tk classification. The lowest taxonomic rank assigned is shown, with no taxonomic information indicating that classification based on GTDB-Tk was not possible due to a lack of sufficient marker genes (see also Fig. S1).

Fig. 3. In situ abundance and activity profile of the microbial community in Lake Cadagno, based on relative small subunit rRNA read abundance in 2018.

a Relative small subunit rRNA read abundance of the entire microbial community in Lake Cadagno chemocline classified to order level. Chromatiales (PSB) are indicated in purple, Chlorobiales (GSB) in green. b Relative small subunit rRNA read abundance of Chromatiales genera. c Relative small subunit rRNA read abundance of Chlorobiales genera.

Fig. 4. Single cell N₂ fixation activity determined by nanoSIMS.

a Single-cell N₂ fixation rates of individual PSB populations in 13.7 m water depth. The number of cells analyzed per population (n) is shown above each box plot. Boxplots depict the 25–75% quantile range, with the centerline depicting the median (50% quantile); whiskers encompass data points within 1.5× of the interquartile range. Y-axis is in log scale. b Representative epifluorescence and c nanoSIMS images of the individual PSB populations. Scale bars (b and c) represent 4 µm. Individual target PSB populations (in purple) were identified using specific FISH probes. All cells were counterstained with DAPI (white). PSB in nanoSIMS images are outlined in white. Note the presence of one C. okenii cell in the L. roseopersicina images. For each population, four independent FISH experiments were conducted, with similar results as shown in panel (b).

Fig. 5. Total N₂ fixation rates of investigated PSB in comparison to bulk N₂ fixation rate in 13.7 m water depth.

The bulk rate is shown as the average of the triplicate incubations, with the error bar representing the standard deviation. Gray circles represent the individual replicates. Population-specific rates were calculated based on the average cell abundance and average single-cell N₂ fixation rate. Here, error bars represent the propagated standard error of the cell abundance and the single-cell N₂ fixation rate.