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TRANSUCYNA Report Summary

Project ID: 625188
Funded under: FP7-PEOPLE
Country: Spain

Periodic Report Summary 1 - TRANSUCYNA (Analyzing metabolism in an unusual nitrogen fixing symbiosis using metatranscriptomics)

- A summary description of the project objectives
One of the main aims of aquatic microbiology is to identify key organisms and to characterize their physiology in situ. Although metagenomics provides a “snapshot” of the gene content of microbial communities in a given environment, it cannot distinguish between expressed and non-expressed genes like transcriptomics and furthermore metagenomics cannot reveal the actual activities at a specific time and place, or how those activities change in response to environmental forces or biotic interactions.
For this reason, this project addresses the application of novel molecular approaches using transcriptomics to elucidate metabolic activities under different conditions in the ocean in cyanobacteria, which are important contributors to global primary production occupying a key position at the base of marine food webs.
In both phases of the project, we will investigate two globally abundant unicellular cyanobacteria: UCYN-A, a nitrogen-fixing cyanobacterium (diazotrophic) and Prochlorococcus (non-nitrogen-fixing).
We will study the metabolism of the major contributors to global primary productivity using approaches based on transcriptomics combined with detailed molecular analyses in the outgoing phase and approaches based on proteomics, transcriptomics and molecular analyses in the return phase. This will undoubtedly lead to novel insights into the functioning of nitrogen metabolic pathways and regulatory mechanisms.

- A description of the work performed since the beginning of the project
In the context of an effort to know whether UCYN-A has autonomous responses to environmental cues (such as light), independent of its symbiotic partner and how does the genome and metabolism function in comparison to other free-living cyanobacteria she developed the Project called TRANSUCYNA. Because this cyanobacterium is unusual in lacking the photosystem II, first protein complex in the light-dependent reactions of oxygenic photosynthesis, the main objective of this project has been trying to understand the function of PSI researching whether UCYN-A expresses PSI genes, and if PSI activity is linked to nitrogen fixation.
For that, during the first two years of the Project TRANSUCYNA, she carried out (not necessarily in chronological order):
1. The design of the microarray for the whole genome transcription of two strains of UCYN-A.
2. Sampling during two diel cycles in the Scripps Institute of Oceanography at the University of San Diego and the Station Aloha, Hawaii for transcriptomic analysis and microscope observation.
3. Setting up the protocol for this new technique using natural samples.
4. Development of all the molecular biology techniques.
5. Training using the R software.
6. Data analysis using the R software for the statistical computing and graphics.
7. Data Analysis.
8. Setting up a double CARD-FISH assay in the lab to observe under the microscope the different symbiosis with UCYN-A.
9. Study of the cell division in UCYN-A and the host during diel cycles.

- A description of the main results achieved so far
During the first year of the period (2014-2015) at the University of California in Santa Cruz with the Prof. Jonathan P. Zehr, a whole genome array for two different strains of UCYN-A was designed.
During this year, Dr. Muñoz set up the protocol for this new technique using natural samples. Apart from microarray data, she also measured the transcription of some genes by real-time qPCR to validate the microarray data.
Thanks to this methodology, Dr. M.C Muñoz got interesting results:
- PSI is transcribed and tightly regulated over the daily cycle, implying a function in cell energetics.
- Carbohydrate-selective porins (OprB) could be involved in nutrient exchanges between the host and the cyanobacterium.
- Because UCYN-A lacks photosystem II, the oxygen generated by this complex cannot inhibit the nitrogenase. Although is unknown how the nitrogenase is protected from the oxygen generated by the host during the photosynthesis. With the microarray we observed that the nickel ABC transporter and the Superoxide dismutase might be involved in the protection of the nitrogenase during the photosynthesis of the host.
- Furthermore, we observed that UCYN-A have different diel patterns from those of most cyanobacteria in this phylogenetic group.
On the other side she started a new line of the project at the same time observing UCYN-A under the microscope and how UCYN-A divide attached to the host. The transcription patterns and CARD-FISH suggested that the cyanobacteria grow and divide prior to the Prymnesiophyte cell division.
Based on the sequencing data, qPCR and morphology observed under the microscope, she could identify a big diversity of UCYN-A in the ocean.

- The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).

These results are of high ecological relevance because they give insight into the mechanics of the metabolism of UCYN-A, one of the most important nitrogen fixers in the ocean.
Our study is also innovative because is developed in the context of a large multidisciplinary research project, which will allow defining phytoplankton community activities in different oceanic environments, thus providing an ecological context to understand phytoplankton community metabolism and the basis for understanding how marine phytoplankton will be affected by climate change induced phenomena.
In the article that we submitted one month ago, and is currently under review, we summarize what is currently known about the geographical distribution of UCYN-A, as well as the environmental factors that govern this distribution, helping us to understand the substantial impact on the N budget in the ocean. A large reservoir of nitrogen on Earth resides in the air we breath: 80% of the atmosphere is nitrogen gas. Nitrogen gas, or dinitrogen (2 nitrogen atoms triple bonded together) is bioavailable to most organisms, in particular, the eukaryotic plants and animals, therefore this project has a global importance due to all life depending on combined forms of nitrogen that are usually limited in availability.

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Life Sciences
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