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Analyzing metabolism in an unusual nitrogen fixing symbiosis using metatranscriptomics

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

The TRANSUCYNA project coordinated by Prof. José Manuel García-Fernández from the University of Córdoba, has got a budget of about 255.243 euros for three years. In this project, Dr. Muñoz-Marín made a postdoctoral stay for 2 years with Prof. Jonathan Zehr from the University of Santa Cruz, California and one year more with Prof. García-Fernández in Córdoba. TRANSUCYNA addresses the application of novel molecular approaches using new OMICS techniques to elucidate metabolic activities under different conditions in the ocean in two different cyanobacteria, which are important contributors to global primary production occupying a key position at the base of marine food webs.
The marine cyanobacteria are the photosynthetic organisms more abundant in the marine phytoplankton and are essential for the evolution of the life in the Earth, because of the oxygen liberated by the photosynthesis. In fact, half the oxygen we breathe every day comes from green plants on land and the other half is bubbling out of all the tiny phytoplankton floating in the ocean. Moreover, the phytoplankton plays a fundamental role in the carbon cycle because take up CO2 from the atmosphere and produce around 25% of new organic matter in the ocean. Therefore, not only the cyanobacteria take an important place in the food chain, but also they play an important role against the global warming.

One of the main aims of marine microbiology is to identify key organisms and to characterize their physiology in situ. However, most bacterioplankton groups either defy cultivation (Giovanni et al., 1990, Amann, 1995) or grow poorly in laboratory culture (Rappé et al., 2002).
The metagenomic is a new field, in which the genome of the different microorganisms is obtained for the study of natural samples marine, cyanobacteria in this case, which takes part of a community, extracting and analyzing the total DNA. The possibility to sequence directly the genomes from environmental samples, without culturing, opens new possibilities in the marine microbiology.
Although metagenomics provide a “snapshot” of the gene content of microbial communities in a given environment, it cannot distinguish between expressed and non-expressed genes like metatranscriptomics (total RNA is extracted from a microbial community, converted into cDNA and sequenced) and furthermore metagenomics (based on the genomic analysis of microbial DNA that is extracted directly from communities in environmental samples and sequenced) cannot reveal the actual activities at a specific time and place, or how those activities change in response to environmental forces or biotic interactions (Moran, 2009).
During two years in the University of Santa Cruz (California), the project was focused in the study of the Candidatus Atelocyanobacterium thalassa (UCYN-A), the nitrogen fixing cyanobacterium (N2, molecular nitrogen obtained directly from the atmosphere) more abundant in the ocean (refs). For that, Dr. Muñoz-Marín performed field work at a number of institutions. During her postdoctoral appointment, she worked for two weeks at the Scripps Institution of Oceanography (SIO) Ellen Browning Scripps Memorial Pier in La Jolla, California, with the objective to obtain time-course samples collected throughout the day to examine diel cycles in the water near Scripps. Dr. Muñoz also participated in research cruises, including the KM14-04 cruise on the R/V Kilo Moana in order to obtain samples from the time-series station in the North Pacific Ocean (Station ALOHA). All of the samples were used for her research project on the cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A). This cyanobacteria has a symbiotic association with a unicellular Prymnesiophyte (Thompson et al., 2012) although it is still known whether the cyanobacterium is an endosymbiont or lives on the surface of the Prymnesiophyte. Furthermore was discovered that UCYN-A provides fixed nitrogen in exchange for fixed carbon from a symbiotic partner (Thompson et al., 2012).
In this study, we used a whole genome transcription array for UCYN-A to understand why UCYN-A, in contrast to its unicellular relatives Cyanothece and Crocosphaera, expresses N2 fixation genes and fixes N2 during the day. We compared UCYN-A diel gene transcription patterns to those of Trichodesmium erythraeum IMS101 (a filamentous N2-fixer), Prochlorococcus sp. MED4 (a non-N2-fixer), and Cyanothece sp. ATCC 51142 and Crocosphaera watsonii WH 8501 (unicellular N2-fixers). We found that UCYN-A has a daily gene expression pattern that is similar to daytime N2-fixers and non-N2-fixing cyanobacteria, rather than to its closest evolutionary relatives, the unicellular N2-fixing cyanobacteria. In addition, the daily cycle in UCYN-A is particularly intriguing since UCYN-A lacks two of the three clock genes that usually control daily rhythms in cyanobacteria.
These results have implications for understanding the ecological success of this cyanobacterium and its symbiotic partner in the global ocean. The impacts of this discovery are also significant for understanding how N2 fixation can be coupled to photosynthesis for aerobic N2 fixation, in natural or engineered crops. In addition, the approach was novel since it used whole genome microarrays to assay gene transcription in an uncultivated microorganism sampled directly from the sea.

Previously, phylogenetic analyses based on partial UCYN-A nifH gene sequences have shown that there are at least four distinct sublineages, UCYN-A1, UCYN-A2, UCYN-A3 and UCYN-A4. However, the only genomes that have been sequenced until now belong to UCYN-A1 and UCYN-A2.
In this study, DNA sequencing, qPCR and CARD-FISH assays were used revealing discrepancies in identification of sublineages and leading to new information on the third sublineage. Our studies show that the size of the UCYN-A3 cells (both cyanobacteria and host) and the number of cyanobacterial cells per host differ from that in the better characterized sublineages (UCYN-A1 and UCYN-A2). Additionally, thanks to the metagenomic analysis, we were able to detect and reconstruct about 13% of the UCYN-A3 expanding the known extent of UCYN-A diversity. Indeed, this is the first study reporting microscopic images of the UCYN-A3 sublineage, and a partial genome using a novel approach combining fragment recruitment and genome assembly techniques.
We believe our findings will be of interest since these results uncovers new nitrogen fixing microorganisms that are found in the global ocean.
Currently, during the third year of the project, we are studying the non-N2-fixing cyanobacterium Prochlorococcus in the Biochemistry and Molecular Biology Department at the University of Córdoba. During this year, Dr. Muñoz is using the techniques she learned during her stay in California.
During her P.hD at the same university, we discovered Prochlorococcus takes up glucose from the ocean beside synthetizing their own biomass by the photosynthesis. The combination of modes by which an organism can obtain its energy and carbon, such as the ability to switch between photoautotrophy and chemoheterotrophy, is called mixotrophy and it is being recognized as a larger component of the open ocean microbial assemblage (Eiler et al., 2006 and Béjà et al., 2000).
The aim of this second project was to measure how much carbon from the glucose Prochlorococcus takes up, comparing with thus during the photosynthesis. Thanks the collaborations she made during her stay in United States, she also participated in a research cruise on the R/V Kilo Moana in order to obtain samples from the time-series station in the North Pacific Ocean (Station ALOHA). This project has been successful thanks to the collaboration with the Prof. David Karl and the Dr. Karin Bjorkman from the University of Honolulu (Hawaii) and the Prof. Solange Duhamel from Columbia University (New York). During this project we are interested to study the possible energetic advantages that Prochlorococcus could have from environmental conditions against the competitors.
The more important characteristic that Prochlorococcus and UCYN-A have in common is the importance of both in the cycle of nitrogen. The nitrogen cycle of Earth is one of the most critical yet poorly understood biogeochemical cycles (Berman-Frank et al., 2003) and cyanobacteria play roles in the carbon cycle as in the nitrogen cycle of the biosphere. The diazotrophic cyanobacteria are of a particular importance in environments which are poor in combined nitrogen sources and the non-nitrogen fixing cyanobacteria are also able to utilize a variety of inorganic and organic combined nitrogen sources; therefore, the nitrogen budget of aquatic ecosystems is largely dominated by the cyanobacteria.
Moreover, both cyanobacteria have others things in common like the small size; Prochlorococcus is probably the non-nitrogen fixation cyanobacterium smallest and most abundant on Earth and UCYN-A is the nitrogen fixation smaller cyanobacterium with a size of <1 μm. Both cyanobacteria reveal genome reduction with a minimal inventory of protein coding regulatory genes. Other characteristics in common are that these cyanobacteria were found to be limited by the same nutrients (Moisander et al., 2012). Its significance in this field is strengthened by the ecological relevance and common occurrence of stress in marine environments.
TRANSUCYNA is a multidisciplinary research project, which allows to define 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.