Biological nitrogen fixers perform an incredible feat – they break the strong triple N bond of N₂ gas and convert it into bioavailable ammonia. Humans learned to do this by the Haber-Bosch process only in the 20^th century, but microbes have been fixing nitrogen for millions of years. In the ocean, nitrogen fixation is important because it fertilizes the growth of photosynthetic organisms that in turn drawdown atmospheric CO_2. Trichodesmium is a keystone marine nitrogen fixer that contributes up to 80 Tg of fixed nitrogen to the surface ocean each year [1] (Picture 1). Like all nitrogen fixing organisms, Trichodesmium must juggle the high nutritional and energetic demands of the nitrogenase enzyme with other biological functions. 

Trichodesmium has a special place among nitrogen fixers because of its unique ability to fix nitrogen while simultaneously photosynthesizing during the daytime. While other organisms separate nitrogen fixation and photosynthesis spatially or temporally, Trichodesmium does not clearly use either strategy. This is confusing because the nitrogen fixing enzyme nitrogenase is susceptible to damage by molecular oxygen produced by photosynthesis. Why would Trichodesmium invest in the dangerous business of fixing nitrogen during the day? This question has puzzled scientists in fields ranging from marine microbiology to enzyme biochemistry, initiating a series of controversies over the years.

We took a new look at this question by analyzing the proteomes of cultured Trichodesmium throughout a simulated light and dark cycle. We used a global proteome approach that allowed us to observe molecular dynamics in an unbiased way. Coupling the proteomes with physiological data that revealed a surprisingly dynamic molecular profile, with wide-spread proteome changes occurring throughout the day and on time scales of just 2-3 hours.  These patterns were reproduced in another laboratory experiment, and in field populations. This was surprising, since the molecular biology of cyanobacteria is often thought characterized by day-night periodicity.

One thing that stood out in the proteomes was that the glycogen synthesis protein GlcG was strongly and negatively correlated with the photosystem proteins and nitrogenase enzyme. This suggested a special role for glycogen in Trichodesmium. In general, cyanobacteria use glycogen as a storage compound. For instance, Trichodesmium’s cousin Crocosphaera produces glycogen during the day and then breaks it down at night to fuel the energy-demanding nitrogenase enzyme [2]. Instead, our data indicated that in Trichodesmium energy captured by the photosystems is funneled directly to nitrogenase during the day, without involvement of glycogen.

This special link between the photosystems and nitrogenase reduces the glycogen content of Trichodesmium. This is important because Trichodesmium cells are constantly combating a tendency to sink out of the sunlight surface ocean. We used hydrodynamical modelling to demonstrate this problem, introducing a hypothetical case in which Trichodesmium fixed nitrogen at night time. In this case, the added mass of glycogen required to fuel nitrogenase activity at night would cause Trichodesmium to sink like a rock! 

And, speaking of rocks, another special behavior of Trichodesmium, particularly when they aggregated in the colonial form, is that they can capture and degrade dust particles that rain down from the atmosphere. Trichodesmium colonies can harvest iron from this dust to fuel metalloenzymes including nitrogenase [3,4]. The added mass of dust particles is non-trivial and our calculations indicate it would exacerbate sinking. Daytime nitrogen fixation counteracts this by reducing glycogen ballast in the cell and helping Trichodesmium to remain in the surface ocean where it is specially adapted to high light, high dust conditions.

In this way, the systems biology of Trichodesmium opens up an ecological sub-niche that goes beyond simply nitrogen fixation. This has implications for ocean biogeochemistry. Close coupling of nitrogen fixation and photosynthesis might help to balance the production of fixed C and N, as has been observed previously [5,6]. Additionally, daytime nitrogen fixation allows Trichodesmium colonies to act as “balloons” holding onto particulate iron-rich dust particles (Picture 2). In this way Trichodesmium retains particulate iron in the surface ocean and may facilitate Fe(III) mobilization by biotic and abiotic processes, including photoreduction to the soluble Fe(II) form.

One of the fun aspects of this study is that we used a combination of very new technology, like state of the art mass spectrometers, and old techniques like glycogen staining, working together as a multi-generational team of scientists. One highlight was the great experience of working directly from Freddy Valois’ 1985 lab notebook (Picture 3). For me, this study will serve as a reminder that as technology advances, nothing will replace the value of a carefully maintained lab notebook and time spent at a simple microscope!

Our finding is an example of how unbiased global proteome measurements can reveal new aspects of microbial systems biology, particularly in a non-model organism. Elucidating the inner workings of microbial cells is not easy, but can help us to understand interactions between life and the environment, and here it helped us to address unanswered questions in fields ranging from cyanobacterial physiology to marine biogeochemistry.   

References

1. Bergman, B., Sandh, G., Lin, S., Larsson, J. & Carpenter, E. J. Trichodesmium--a widespread marine cyanobacterium with unusual nitrogen fixation properties. FEMS Microbiol. Rev. 37, 286–302 (2013).
2. Saito, M. A. et al. Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii. Natl. Acad. Sci. U. S. A. 108, 2184–9 (2011).
3. Held, N. A. et al. Mechanisms and heterogeneity of in situ mineral processing by the marine nitrogen fixer Trichodesmium revealed by single-colony metaproteomics. ISME Commun. 1–9 doi:10.1038/s43705-021-00034-y (2021).
4. Rubin, M., Berman-Frank, I. & Shaked, Y. Dust-and mineral-iron utilization by the marine dinitrogen-fixer Trichodesmium. Geosci. 4, 529–534 (2011).
5. Rabouille, S., Staal, M., Stal, L. J. & Soetaert, K. Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp. Environ. Microbiol. 72, 3217–3227 (2006).
6. Breitbarth, E., Wohlers, J., Kläs, J., LaRoche, J. & Peeken, I. Nitrogen fixation and growth rates of Trichodesmium IMS-101 as a function of light intensity. Ecol. Prog. Ser. 359, 25–36 (2008).