The role of the orfG gene in freshwater cyanobacterium Synechococcus elongatus

The goal of this research was to uncover the role and regulation of the orfG gene in freshwater Synechococcus elongatus. It was hypothesized that the orfG gene (1) functioned to optimize growth at favorable conditions and (2) was regulated upon the response of Synechococcus to stress conditions. The orfG gene in photoautotrophic freshwater cyanobacterium Synechococcus elongatus contains a 525 bp open-reading frame and is found in no other freshwater cyanobacterium but in five of the marine Synechococcus, five marine Prochlorococcus and one cyanophage, P-SSM4. The phylogeny of the OrfG amino acid sequence homologs did not cluster based on 16S-23S clades assigned to the marine strains from previous studies. The transcriptional levels of the orfG gene remained constitutive under all stress conditions and growth phases tested. However, OrfG protein levels were undetectable once Synechococcus cells entered chlorosis due to high-light stress, implying OrfG translational inhibition or protein degradation under increased irradiance. Synechococcus cells recovering from chlorosis due to low or depleted nitrogen conditions also reduced OrfG protein to undetectable levels once cell division resumed. Protein levels of OrfG were higher in the Synechococcus GNblR mutant, compared to wild-type Synechococcus during favorable growth conditions, implying that OrfG protein levels are partially repressed by NblR for Synechococcus cells to enter into chlorosis. OrfG protein levels were also undetectable in Synechococcus GNblR cells during high-light stress, once viability decreased after 6 hrs, indicating that the OrfG protein is controlled separately by the GNblR and high-light stress signaling pathways. Synechococcus GNblR cells prematurely removed OrfG protein associated with the thylakoid region. Removal of the orfG gene did not affect Synechococcus cell viability but overexpression of the orfG mRNA resulted in lower cell viability after 72 hrs in the presence of high-light stress. There were no stable OrfG protein-protein binding partners eluted through the yeast two-hybrid or coimmunoprecipitation assay, suggesting that the OrfG protein may function through transient interactions in the Synechococcus cell. Through these studies, we propose that the OrfG protein may play a role in light harvesting mechanisms in Synechococcus during favorable growth conditions.