As the sun goes down, these cells quit their day job of photosynthesis and unexpectedly begin to fix nitrogen, converting nitrogen gas (N2) into compounds that are useful for cell growth.

The study, published January 30 in the early online edition of the Proceedings of the National Academy of Sciences, is the first to document an organism that can juggle both metabolic tasks within a single cell at high temperatures, and also helps answer longstanding questions about how hot-spring microbial communities get essential nitrogen compounds.

Carnegie's Arthur Grossman, Devaki Bhaya, and Anne-Soisig Steunou, along with colleagues from four partner institutions*, are studying the tiny, single-celled cyanobacterium Synechococcus. Cyanobacteria evolved about three billion years ago, and are the oldest organisms on the planet that can turn solar energy and carbon dioxide into sugars and oxygen via photosynthesis. In fact, ancient cyanobacteria produced most of the oxygen that allows animals to survive on Earth.

Cyanobacteria such as Synechococcus are often found in the microbial mats that carpet hot springs, where life exists at near-boiling temperatures. These mats are highly organized communities where different organisms split up the work, with cyanobacteria serving as the main photosynthetic power plants.

Microbial mats in Yellowstone National Park's Octopus Spring contain Synechococcus that can grow in waters up to around 160�F, while other microbes in the hot spring can tolerate temperatures that exceed 175�F. But until now, it was unclear which organisms could fix nitrogen--especially in the hotter regions of the mat.

"The cyanobacteria are true multitaskers within the mat community," Grossman said. "We had assumed that the single-celled cyanobacteria growing at elevated temperatures were specialized for photosynthesis, but it looks like they have a more complicated metabolism than we initially suspected."

All cells require nitrogen for making proteins and nucleic acids, but N2 gas from the atmosphere cannot be directly used for this purpose; it must first be reduced or fixed into larger, carbon-containing compounds. N2 fixation is a problem for photosynthetic cells, since the oxygen produced during photosynthesis inhibits the nitrogenase complex--the enzyme factory that fixes N2.

Other organisms have found creative solutions to this dilemma. For example, plants rely on symbiotic N2-fixing bacteria that live in their roots, far away from the photosynthetic leaves. A different type of cyanobacteria grows in multicellular strands and makes specialized N2-fixing cells called heterocysts that are walled off from the photosynthetic cells.

Many researchers believed these filamentous cyanobacteria were the major N2 fixers in microbial mats. But they are not tolerant of extremely high temperatures, and only live at the cooler edges of the mat, raising the question of whether N2 fixation was critical for organisms in the hotter regions of the mat. Because heat tolerant, single-celled cyanobacteria like Synechococcus specialize in photosynthesis, many researchers had dismissed them as candidates for N2 fixation.

"Synechococcus cannot spatially separate photosynthesis and N2 fixation, as some photosynthetic organisms do," Bhaya explained. "Instead, they solve the problem by temporally separating the tasks."

Lead author Steunou and her collaborators tracked the activity of genes involved in photosynthesis and N2 fixation over a 24-hour period. They found that photosynthetic genes shut down shortly after nightfall, and N2-fixation genes switch on shortly thereafter. The nitrogenase enzyme complex snaps into action at about the same time, following the same pattern as the N2-fixation genes.

Fixing N2 requires a lot of energy, which raises another problem for Synechococcus. When photosynthesis shuts down at night, the mat becomes oxygen starved, making it difficult to perform respiration--an efficient energy-generating pathway that requires O2 to release the energy stored in sugars.

Instead, the cells must rely on fermentation--a less efficient energy-generating pathway that can proceed without oxygen. Steunou and colleagues found that at night, Synechococcus turns on genes for specific fermentation pathways that release energy from polyglucose, which probably powers N2 fixation.

"These results add to our understanding of microbial mats as complex, integrated communities that are exquisitely adapted to life in the tough hot spring environment," Grossman commented.

"There may be several different organisms living in a given mat, but it seems that they are engaging in community metabolism that changes depending on the time of day. Perhaps it is more correct to consider the mat as a single functional unit rather than as a group of individual organisms."

*In addition to Grossman, Bhaya, and Steunou, contributors to the study include Dave Ward and John Peters from Montana State University, Fred Cohan from Wesleyan University, Michael Kuhl from the University of Copenhagen, and John Heidelberg from The Institute for Genomic Research.

This work was supported by a grant from the National Science Foundation's Frontiers in Integrated Biology Program.

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