Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling

Abstract

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N₂ fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N₂ fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems. Reduction of dinitrogen gas to biologically useful forms via nitrogen fixation is a key component of the biogeochemical cycle. In the marine environment, the cyanobacteria UCYN-A (Candidatus Atelocyanobacterium thalassa) has been found to be a primary contributor to biological nitrogen fixation at a global scale. UCYN-A exhibits a highly streamlined genome which lacks genes coding for essential cyanobacterial processes such as the energy-generating TCA cycle, oxygen-producing photosystem II, the carbon-fixing RuBisCo and de novo production pathways for numerous amino acids and nucleotides. Thus, it exists in a symbiosis with unicellular planktonic algae where it exchanges fixed nitrogen for fixed carbon with its host. However, both UCYN-A and its symbiotic partner remain uncultured under laboratory conditions. This necessitates implementing a computational approach to glean insights into UCYN-A’s unique physiology and metabolic processes governing the symbiotic association. To this end, we develop a constraints-based framework that infers all possible trophic scenarios consistent with the observed data. Possible mechanisms employed by UCYN-A to accommodate diazotrophy with daytime carbon fixation by the host (i.e., two mutually incompatible processes) are also elucidated. We envision that the developed framework using UCYN-A and its algal host will be used as a roadmap and motivate the study of similarly unique microbial systems in the future.