Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases. In this study, we characterize and explore the role of enzymes that are involved in the biosynthesis of phycoerythrin in cyanobacteria. Biochemical and molecular techniques were used in the characterization of these proteins to gain a better understanding of their roles in the post-translational modification of phycobiliprotein. In F. diplosipohon, the lyase activity of CpeT was characterized and studied using a heterologous, co-expression system in E. coli. It was determined that CpeT was able to ligate PEB to Cys-165 of CpeB in the presence of CpeZ, a chaperone-like protein. Next, the roles of three proteins, MpeY from RS9916 and MpeQ and MpeW from A15-62, were analyzed using a combination of gene-interruption mutants and recombinant protein expression techniques. The absence of mpeY resulted in the reduction of PEB chromophorylation of MpeA in green light conditions, and recombinant protein coexpression confirmed that MpeY was responsible for PEB attachment to Cys-83 of MpeA. The interruption of mpeQ in A15-62 resulted in a reduced PUB phenotype in MpeA in blue light. Recombinant protein expressions revealed that MpeQ was a lyase-isomerase responsible for the attachment of PUB to Cys-83 of MpeA. Two regulatory proteins located in two conserved configurations of a genomic island present in species that are able to change their phycobilin content in response to different light environments, known as Type-IV chromatic acclimation (CA4), were investigated. FciA and FciB from RS9916 were studied using gene interruption mutants from RS9916 and they were found to be responsible for the CA4 response in CA4-A containing species of Synechococcus.

abstract: Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS. / Dissertation/Thesis / Ph.D. Chemistry 2014