Biosynthesis of phycoerythrins, photoysnthetic light harvesting proteins in cyanobacteria

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Intellectual Merit: Small unicellular cyanobacteria such as Synechococcus RS9916 are estimated to contribute 25-30% of the total marine productivity. Synechococcus is likely to be one of the most ubiquitous phytoplanktonic organisms in the world's oceans and numerically the second most abundant marine phototroph in this ecosystem. This ubiquity and abundance is in large part due to its remarkable phenotypic plasticity in its use of a diverse set of photosynthetic pigments, which is sustained by a complex set of genes and regulatory systems that can be laterally transferred between lineages. Much of this diversity occurs within the protein Phycoerythrin (PE), which has five phycoerythrobilin (PEB) or phycourobilin (PUB) chromophores attached at six cysteine residues. These posttranslational modifications are catalyzed by bilin lyase/isomerase enzymes. As blue light is the dominating wavelength present in the oligotrophic open ocean, strains capable of a process called Type IV chromatic acclimation (CA4) have adapted by increasing the PUB content on their PE to take advantage of the light filtering through the water by a novel mechanism: induction of a bilin lyase/isomerase. The research in this project will contribute to defining the function of these PE bilin lyase/isomerase enzymes that are critical to the success and adaptation of this diverse group of photosynthetic prokaryotes. A better understanding of this novel type of chromatic acclimation (CA4) will be achieved. The first objective is to characterize the biosynthesis of a "simple" PE in Fremyella diplosiphon. Six genes with similarity to known bilin lyase enzymes are upregulated with phycoerythrin structural genes. The second objective involves characterizing the function of bilin lyases specific for PEI and PEII in Synechococcus RS9916, focusing on the characterization of lyases responsible for CA4 changes. Analyses of PE produced in the bilin lyase knockout mutants will be performed, and recombinant protein expression in E. coli to test for bilin ligation and isomerization activity will also be used. Not only will these studies contribute to understanding how these ecologically important organisms acclimate to thrive in the ocean, they will also biochemically characterize enzymes which will have very important potential biotechnological and cell biology applications for the use of phycoerythrins as fluorescent tags.Broader impacts: The PI has a strong track record of training undergraduates and graduate students, especially members of under-represented groups in science, and these efforts will continue during this granting period. The PI will incorporate her research into a course called Biological Research Tools where students learn basic molecular cloning and protein expression/purification of phycobiliproteins in E. coli. At least two undergraduates (over the course of the grant) will be selected to go to the laboratory of Dr. David Kehoe at Indiana University (after working in the Schluchter lab for a year first) for 6-8 weeks in the summer in order to learn techniques to generate clean deletions and to study gene regulation related to the CA4 collaboration project. The student is likely to be a member of an under-represented group (50% of the PI's past undergraduates have been minorities), and the purpose of these exchanges is to expose these students to a larger, research university.