Expression analysis of Synechocystis sp. PCC 6803 nblD mutant in nitrogen starvation and normal condition in contrast to wild type

Small proteins are an underinvestigated class of gene products in all domains of life. Here we describe the role of NsiR6/NblD, a cysteine-rich 66 amino acid small protein in the acclimation response of cyanobacteria to nitrogen starvation. Phycobilisomes, the macromolecular pigment-protein complexes for photosynthetic light harvesting, are rapidly degraded upon shift to low nitrogen. Deletion of nblD in Synechocystis sp. strain PCC 6803 prevents this degradation, indicated by the non-bleaching (nbl) phenotype. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type, while overexpression of NblD under nitrogen-replete conditions did not lead to any phenotypical effect, different from the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. However, transcriptome analysis revealed that nitrogen starvation induced nblA1/2 transcription in the ΔnblD strain, which excluded the possibility that the nbl phenotype was due to a possible NblD function as transcriptional co-regulator. In contrast, fractionation experiments indicated the presence of NblD in the phycobilisome fraction and pull-down experiments with NblD containing a triple FLAG tag identified the α and β phycocyanin subunits as the only two co-purifying proteins. Homologs of NblD exist in all cyanobacteria that use phycobilisomes but not in the genera Prochlorococcus and Acaryochloris which use alternative light-harvesting mechanisms. These data suggest that NblD plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. We performed a microarray, to examine the global expression pattern of wild type and ∆nblD isolated RNA after 0h and 3h past induction of nitrogen depletion (-N) to detect potential differences in the nitrogen acclimation process. Cultures were starved for nitrogen (-N) for 0 h (negative control), 3 h, 6 h and 24 h to induce nsiR6 nblD expression. After harvesting the culture by filtering through hydrophilic polyethersulfone filters (Pall Supor®-800, 0.8 μm) and immersion in PGTX (Pinto et al., 2009), samples were snap frozen in liquid nitrogen. RNA isolation was performed like stated before (Hein et al., 2013). For microarray analysis, the same procedure was used like described in before (Hernández-Prieto et al., 2012) using 5 µg of total RNA at time points 0 h and 3 h –N, for the array 500 ng Cy3-labeled RNA was applied. The microarray included a duplicate for each sample.