Non-cyanobacterial diazotrophs: global diversity, distribution, ecophysiology, and activity in marine waters

Abstract

Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research.

Keywords: diazotrophs; marine nitrogen cycle; nitrogen fixation; non-cyanobacterial diazotrophs.

Figure 1.

NCD diversity includes taxa found using PCR-based and PCR-free approaches. Phylogenetic tree represents the NCD nifH gene catalog based on amino acid sequences from OTU representatives. Sequences were aligned to the NifH/frxC family (Fer4_NifH; PF00142) using HMMalign in HMMER software v2.4 (Finn et al. 2011). Tree topology was calculated using FastTree 2.1.11 (Price et al. 2010) using maximum-likelihood rearrangements and the JTT model for nucleotide evolution. Branch support was determined using the Shimodaira–Hasegawa test (>50% support indicated with small gray squares on branches). iTOL 6.5.2 (Letunic and Bork 2021) was used to visualize the tree and display the source(s) of sequences in each cluster. nifH clusters are defined according to the convention established in Zehr et al. (2003) and are indicated in gray text in the center of the tree. Representative sequences affiliated with NCD qPCR/ddPCR assays are in bold. OTUs that contain sequences derived from both PCR-based and PCR-free approaches are in shaded boxes. Branches with multiple names indicate OTUs that contain sequences targeted by more than one qPCR assay. Thick outer color bars show if the sequences were acquired through PCR-based (teal) or PCR-free (yellow) methods, while the thinner color bars correspond to the specific method (PCR-based) or sampling campaign (PCR-free). Interactive tree publicly available at https://itol.embl.de/shared/1HrZrblPr7p4s. See Table S2 (Supporting Information) for supporting data.

Figure 2.

NCDs occupy diverse marine habitats. NCDs in marine waters may be free-living and motile or nonmotile (a), associated with various particles including self-aggregates (b), suspended or sinking particles (c), plankton holobionts (d), or live in symbiosis with copepods (e) or other protists (f). Presumed habitats of some NCD taxa which are discussed throughout this review are indicated using symbols described in the legend. Created with BioRender.com.

Figure 3.

NCD taxa have distinct global and depth distribution patterns. Maps (A), (C), (E), and (G) show the global nifH gene abundances of four NCD taxa at all sampling depths (0–4000 m) while depth plots (B), (D), (F), and (H) show the upper 300 m only. Observations of no detect are represented by gray squares; observations of detect but not quantifiable (DNQ) were given a nominal value of 1 nifH gene l^–1 in the database and are represented by gray triangles. Dataset doi: 10.5281/zenodo.6537451.

Figure 4.

Environmental predictors vary among NCD taxa. NCD nifH gene abundances are plotted against environmental metadata from World Ocean Atlas monthly climatology (temperature, nitrate, phosphate, N:P, and O₂ concentration) and Pisces model output of phytoplankton biomass in units of carbon (modeled phytoplankton C). Tolerances for colocalization are presented in Table S4 (Supporting Information). Each point represents an individual nifH gene abundance sample, with sample depth shown in color. Note that a small fraction of data with outlying x-axis values were excluded from plots. Black lines and gray shading represent the smoothed conditional mean and 95% confidence intervals. Symbols (+/-) reflect positive and negative correlations (Spearman rank with Bonferroni correction for 28 comparisons).

Figure 5.

Ecophysiological features of NCDs include pathways involved in energy generation and transport of fixed N not present in cyanobacterial diazotrophs. Color represents presence/absence of metabolic pathways (black: all genes; gray: one missing gene; and white: no genes) related to energy acquisition (A), N uptake (B), and regulation of nitrogenase synthesis and activity (C) of NCDs and cyanobacterial diazotrophs as inferred from reconstructed Tara Oceans MAGs (Delmont et al. 2022). Alpha = ɑ-proteobacteria; Beta = β-proteobacteria; Delta = δ-proteobacteria; Epsilon = ε-proteobacteria; Gamma = γ-proteobacteria; Plancto = Planctomycetes; and Verruco = Verrucomicrobiota. See Fig. 6 for more details about these regulatory pathways and the proteins involved.

Figure 6.

NCDs use diverse regulatory pathways for N₂ fixation. Circular diagrams next to each regulatory pathway represent the presence or absence of the corresponding genes in at least one MAG from each NCD group (Alpha = ɑ-proteobacteria; Beta = β-proteobacteria; Delta = δ-proteobacteria; Epsilon = ε-proteobacteria; Gamma = γ-proteobacteria; Plancto = Planctomycetes; and Verruco = Verrucomicrobiota) as indicated by color (presence) or white (absence).

Figure 7.

Predicated features of euphotic marine NCD communities are likely influenced by habitats. Potential strategies used by NCDs to acquire energy and C needed to support N₂ fixation with energetic constraints and O₂ inactivation of nitrogenase. Question marks emphasize where many open questions remain. Notably, there is recent evidence that NCDs may be fixing N₂ on particles, as discussed in this review (Geisler et al. 2019). EPS—extracellular polymeric substances. Created with BioRender.com.