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

   https://doi.org/10.1371/journal.pcbi.1008983  

   Sarkar, Debolina; Landa, Marine; Bandyopadhyay, Anindita; Pakrasi, Himadri B.; Zehr, Jonathan P.; Maranas, Costas D. (May 2021, PLOS Computational Biology)

   Hatzimanikatis, Vassily (Ed.)

   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 N 2 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 N 2 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. 

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2. Lotus japonicus regulates root nodulation and nitrogen fixation dependent on the molecular form of nitrogen fertilizer

   https://doi.org/10.1007/s11104-022-05762-1  

   Ortiz-Barbosa, G. S.; Torres-Martínez, L.; Rothschild, J.; Sachs, J. L. (November 2022, Plant and Soil)

   Purpose Legumes form root nodules to gain fixed nitrogen from rhizobia and can also access nitrogen in soil. Data suggest that plants might discriminate among these sources to optimize growth, but recogni- tion of symbiotically fixed nitrogen and its regulation remain poorly understood. Methods A greenhouse inoculation study manipu- lated the molecular form and concentration of nitro- gen available using two Lotus japonicus genotypes and the nitrogen-fixing symbiont, Mesorhizobium loti. Plants were supplied with sole organic and inorganic nitrogen sources to simulate forms that plants might receive from symbiotic nitrogen fixation or from the soil. Host benefit from and regulation of symbiosis was investigated by quantifying symbiotic trait varia- tion and isotopic analysis of nitrogen fixation. Results Host growth varied in response to fertili- zation with alanine, aspartic acid, ammonium, and nitrate, suggesting differences in catabolism effi- ciency. Net benefits of nodulation were reduced or eliminated under all forms of extrinsic fertilization. However, even when symbiosis imposed significant costs, hosts did not reduce investment into nodulation or nitrogen fixation when exposed to aspartic acid, unlike with other nitrogen sources. Conclusions L. japonicus can adaptively down- regulate investment into symbiosis in the presence of some but not all nitrogen sources. Failure to down- regulate any aspect of symbiosis in the presence of aspartic acid suggests that it might be jamming the main signal used by L. japonicus to detect nitrogen fixation. 

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3. Transcriptional Activities of the Microbial Consortium Living with the Marine Nitrogen-Fixing Cyanobacterium Trichodesmium Reveal Potential Roles in Community-Level Nitrogen Cycling

   https://doi.org/10.1128/AEM.02026-17  

   Lee, Michael D.; Webb, Eric A.; Walworth, Nathan G.; Fu, Fei-Xue; Held, Noelle A.; Saito, Mak A.; Hutchins, David A.; Drake, Harold L. (December 2017, Applied and Environmental Microbiology)

   ABSTRACT Trichodesmium is a globally distributed cyanobacterium whose nitrogen-fixing capability fuels primary production in warm oligotrophic oceans. Like many photoautotrophs, Trichodesmium serves as a host to various other microorganisms, yet little is known about how this associated community modulates fluxes of environmentally relevant chemical species into and out of the supraorganismal structure. Here, we utilized metatranscriptomics to examine gene expression activities of microbial communities associated with Trichodesmium erythraeum (strain IMS101) using laboratory-maintained enrichment cultures that have previously been shown to harbor microbial communities similar to those of natural populations. In enrichments maintained under two distinct CO 2 concentrations for ∼8 years, the community transcriptional profiles were found to be specific to the treatment, demonstrating a restructuring of overall gene expression had occurred. Some of this restructuring involved significant increases in community respiration-related transcripts under elevated CO 2 , potentially facilitating the corresponding measured increases in host nitrogen fixation rates. Particularly of note, in both treatments, community transcripts involved in the reduction of nitrate, nitrite, and nitrous oxide were detected, suggesting the associated organisms may play a role in colony-level nitrogen cycling. Lastly, a taxon-specific analysis revealed distinct ecological niches of consistently cooccurring major taxa that may enable, or even encourage, the stable cohabitation of a diverse community within Trichodesmium consortia. IMPORTANCE Trichodesmium is a genus of globally distributed, nitrogen-fixing marine cyanobacteria. As a source of new nitrogen in otherwise nitrogen-deficient systems, these organisms help fuel carbon fixation carried out by other more abundant photoautotrophs and thereby have significant roles in global nitrogen and carbon cycling. Members of the Trichodesmium genus tend to form large macroscopic colonies that appear to perpetually host an association of diverse interacting microbes distinct from the surrounding seawater, potentially making the entire assemblage a unique miniature ecosystem. Since its first successful cultivation in the early 1990s, there have been questions about the potential interdependencies between Trichodesmium and its associated microbial community and whether the host's seemingly enigmatic nitrogen fixation schema somehow involved or benefited from its epibionts. Here, we revisit these old questions with new technology and investigate gene expression activities of microbial communities living in association with Trichodesmium . 

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4. Pangenome Evolution Reconciles Robustness and Instability of Rhizobial Symbiosis

   https://doi.org/10.1128/mbio.00074-22  

   Weisberg, Alexandra J.; Rahman, Arafat; Backus, Dakota; Tyavanagimatt, Parinita; Chang, Jeff H.; Sachs, Joel L. (June 2022, mBio)

   Cooper, Vaughn S. (Ed.)

   ABSTRACT Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume- Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings. 

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5. Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

   https://doi.org/10.1128/spectrum.02815-23  

   Rivier, Alex J.; Myers, Kevin S.; Garcia, Amanda K.; Sobol, Morgan S.; Kaçar, Betül (October 2023, Microbiology Spectrum)

   Youssef, Noha H. (Ed.)

   ABSTRACT Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome ofAzotobacter vinelandiiengineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCEAzotobacter vinelandiiis a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineeredA. vinelandiistrains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts. 

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