Nitrogen fixation in a chemoautotrophic lucinid symbiosis

Abstract

The shallow water bivalve Codakia orbicularis lives in symbiotic association with a sulfur-oxidizing bacterium in its gills. The endosymbiont fixes CO 2 and thus generates organic carbon compounds, which support the host's growth. To investigate the uncultured symbiont's metabolism and symbiont-host interactions in detail we conducted a proteogenomic analysis of purified bacteria. Unexpectedly, our results reveal a hitherto completely unrecognized feature of the C. orbicularis symbiont's physiology: the symbiont's genome encodes all proteins necessary for biological nitrogen fixation (diazotrophy). Expression of the respective genes under standard ambient conditions was confirmed by proteomics. Nitrogenase activity in the symbiont was also verified by enzyme activity assays. Phylogenetic analysis of the bacterial nitrogenase reductase NifH revealed the symbiont's close relationship to free-living nitrogen-fixing Proteobacteria from the seagrass sediment. The C. orbicularis symbiont, here tentatively named 'Candidatus Thiodiazotropha endolucinida', may thus not only sustain the bivalve's carbon demands. C. orbicularis may also benefit from a steady supply of fixed nitrogen from its symbiont-a scenario that is unprecedented in comparable chemoautotrophic symbioses. Mutualistic associations between marine invertebrates and sulfur-oxidizing (thiotrophic) bacteria are a well-documented and widespread phenomenon in a variety of sulfidic habitats ranging from hydrothermal vents to shallow-water coastal ecosystems 1-3 . Thioautotrophic symbionts generate energy through sulfide oxidation and provide their hosts with organic carbon. In the Lucinidae, a diverse family of marine bivalves, all members are obligatorily dependent on their bacterial gill endosymbionts after larval development and metamorphosis 4 . The shallowwater lucinid Codakia orbicularis, which lives in the sediment beneath the tropical seagrass Thalassia testudinum along the Caribbean and Western Atlantic coast 5 , harbours a single species of endosymbionts in its gills 6 . The symbiont has been shown to be newly acquired by each clam generation 7,8 from a pool of freeliving symbiosis-competent bacteria in the environment 9 , rather than being inherited from clam parents. C. orbicularis appears not to release its endosymbionts, even under adverse conditions, but can digest them as a source of nutrition [10][11][12] . Moreover, bacterial cell division seems to be inhibited inside the host tissue. The majority of the symbiont population was shown to be polyploid (that is, containing multiple genome copies), while dividing symbiont cell stages are very rarely observed in host bacteriocytes 13 . The host undoubtedly benefits from the symbiont both by way of detoxification of its sulfidic environment and by supply of organic compounds through the bacterial Calvin-Benson cycle. It remains questionable, however, whether the symbiont gains any advantage from this association in evolutionary terms 11 .Biological nitrogen fixation (diazotrophy) is the conversion of ...

Metagenomic analysis suggests broad metabolic potential in extracellular symbionts of the bivalveThyasiracf.gouldi

“…Recent work has shown some sulfur-oxidizing chemosymbionts can also fix atmospheric nitrogen into bioavailable forms (18,48), however, we did not find any evidence of N fixation genes in the T. cf. gouldi symbiont.…”

“…The nitrite reduction (nir) pathway removes harmful nitrogen compounds by using them as an electron sink, but this process is not always coupled with carbon fixation (14). One approach to examining the metabolic potential of chemoautotrophic symbionts is to perform genomic, or metagenomic sequencing (15)(16)(17)(18). By identifying key genes in sequencing data, we can make inferences about the metabolic capabilities of the symbiont.…”

A Reverse TCA Cycle 2-Oxoacid:Ferredoxin Oxidoreductase that Makes C-C Bonds from CO2

“…MmFd2 (GenBank: CP000741.1, Mmc1_1207) and MmFd3 (GenBank: CP000741.1, Mmc1_1191) are in the nif-gene clusters known to be involved in FeMo cofactor biosynthesis and nitrogen fixation. [40][41][42] M. marinus MC-1 is able to fix nitrogen under both heterotrophic and autotrophic growth conditions, 27 but it is not clear if and how these Fds from the nif-gene clusters participate in the CO 2 fixation pathway. 43 whereas MmFd3 belongs to a type exemplified by the nif-specific FdIII ( Figure S6).…”

Metagenomic analysis suggests broad metabolic potential in extracellular symbionts of the bivalve Thyasira cf. gouldi

“…The nitrite reduction (nir) pathway removes harmful nitrogen compounds by using them as an electron sink, but this process is not always coupled with carbon fixation [14]. One approach to examining the metabolic potential of chemoautotrophic symbionts is to perform genomic, or metagenomic sequencing [15][16][17][18]. By identifying key genes in sequencing data, the metabolic capabilities of symbionts can be inferred.…”