The symbiotic 'all-rounders': Partnerships between marine animals and chemosynthetic nitrogen-fixing bacteria

Abstract

Nitrogen fixation is a widespread metabolic trait in certain types of microorganisms called diazotrophs. Bioavailable nitrogen is limited in various habitats on land and in the sea, and accordingly, a range of plant, animal, and single-celled eukaryotes have evolved symbioses with diverse diazotrophic bacteria, with enormous economic and ecological benefits. Until recently, all known nitrogen-fixing symbionts were heterotrophs such as nodulating rhizobia, or photoautotrophs such as cyanobacteria. In 2016, the first chemoautotrophic nitrogen-fixing symbionts were discovered in a common family of marine clams, the Lucinidae. Chemosynthetic nitrogen-fixing symbionts use the chemical energy stored in reduced sulfur compounds to power carbon and nitrogen fixation, making them metabolic 'all-rounders' with multiple functions in the symbiosis. This distinguishes them from heterotrophic symbionts that require a source of carbon from their host, and their chemosynthetic metabolism distinguishes them from photoautotrophic symbionts that produce oxygen, a potent inhibitor of nitrogenase. In this review, we consider evolutionary aspects of this discovery, by comparing strategies that have evolved for hosting intracellular nitrogen-fixing symbionts in plants and animals. The symbiosis between lucinid clams and chemosynthetic nitrogen-fixing bacteria also has important ecological impacts, as they form a nested symbiosis with endangered marine seagrasses. Notably, nitrogen fixation by lucinid symbionts may help support seagrass health by providing a source of nitrogen in seagrass habitats. These discoveries were enabled by new techniques for understanding the activity of microbial populations in natural environments. However, an animal (or plant) host represents a diverse landscape of microbial niches due to its structural, chemical, immune and behavioural properties. In future, methods that resolve microbial activity at the single cell level will provide radical new insights into the regulation of nitrogen fixation in chemosynthetic symbionts, shedding new light on the evolution of nitrogen-fixing symbioses in contrasting hosts and environments.

FIG 1

Not all lucinid symbionts are capable of nitrogen fixation and may have lost this ability on multiple occasions throughout evolution. A maximum-likelihood phylogenetic tree of lucinid symbionts and their free-living and symbiotic relatives, based on 16S rRNA genes, is shown. Symbionts capable of diazotrophy are shown in blue; those with no evidence for diazotrophic ability are shown in in black. Asterisks denote organisms for which the evidence of diazotrophy comes from PCR amplification and sequencing of the nifH gene (or in the case of the symbionts of Epidulcina delphinae and Lucinoma borealis, lack of PCR amplification with nifH-specific primers). All others are based on screening draft genome sequences for nifH and associated diazotrophy genes. Free-living bacteria are indicated in boldface. The tree was calculated in IQ-TREE using the TIM+F+I+G4 nucleotide substitution model (119). The internal nodes show approximate likelihood-ratio test (aLRT) SH-like support values calculated from 10,000 bootstrap replicates.

FIG 2

Symbionts may be supplied with oxygen by diffusion from ambient seawater and host hemoglobins. This image is a model of hypothesized oxygen delivery systems to the symbionts of lucinid clams. (A) The image on the left shows two gill filaments, which are made up of a single layer of epithelial cells surrounding a lumen of circulatory fluid, the hemolymph. This epithelium is made up of symbiont-containing bacteriocyte cells, and symbiont-free cells with other functions (shaded in gray) such as ciliated cells at the outer edge, and intercalary cells (ic) that partially cover the bacteriocytes. Oxygen is expected to be depleted away from the ambient seawater (fully oxygenated) which first flows over the ciliated edges of the gill filaments (gradient shown in panel A). (B) Oxygen may also become depleted toward the hemolymph-facing side of the bacteriocytes as oxygen diffuses from the ambient seawater into the bacteriocytes and is consumed by the symbionts. Functional oxygen-binding hemoglobins have been found in lucinid hemolymph, and they also express intracellular hemoglobins. Thus far, this proposed model has not been experimentally tested.

FIG 3

Comparing carbon and nitrogen fluxes in intracellular nitrogen-fixing symbioses on land and in the sea using lucinid clams (left) and Medicago truncatula (right) as examples. Bacterial cells are housed in specialized host cells in both systems. Bacterial symbiont cells are polyploid and show morphological heterogeneity in both systems. The major difference is that in the M. truncatula root nodule example, the host provides organic carbon to the rhizobia symbionts and gains fixed nitrogen in exchange (arrows show direction of nutrient transfer, from host above to symbionts below). In the lucinid symbiosis, the bacterial symbionts provide organic carbon, and possibly also fixed nitrogen to the host, which provides a source of nitrogen to the seagrass sediments the hosts inhabit. Although not shown here, nitrogen fixation can occur in other seagrass-associated niches (see the main text).