Discovery of a small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria

Abstract

Phycobilisomes are the major pigment–protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the ΔnblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the ΔnblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin β-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation.

AtpΘ is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria

“…Far western blotting was performed as described previously 44,73 with modifications. After electrophoresis, proteins were transferred onto a PVDF membrane.…”

“…Synthetic AtpQ peptide, anti-AtpQ antiserum and anti-rabbit IgG antiserum were used for incubation sequentially. Milk powder was omitted in the denaturing/renaturing steps of the blotted membrane as described by Krauspe et al 73 The membrane with renatured proteins was first blocked with 5% milk powder in TBST and then incubated with 3 mg/mL synthetic AtpQ peptide at 4 C overnight. Signals were detected with ECL start western blotting detection reagent (GE Healthcare) on a chemiluminescence imager system (Fusion SL, Vilber Lourmat).…”

In Living Color: Pigment-Based Microbial Ecology At the Mineral–Air Interface

“…A hallmark of chlorosis is the rapid proteolytic degradation of phycobiliproteins—the major light-harvesting pigments in red algae and most cyanobacteria—accompanied by cell color change from blue-green to yellow. It has been shown that phycobiliproteins degradation favors the release of soluble amino acids and reduces the intracellular reactive oxygen species level by decreasing the light absorption capability (Forchhammer and Schwarz 2019 , Krauspe et al 2021 ). The current understanding of the chlorotic cells’ regulatory processes depicts the intricate mechanism that modulates ribosomal proteins, RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase) components, central regulators, and metabolic enzymes, as well as hyperphosphorylation of nondegraded phycobiliproteins (Spät et al 2018 , Krauspe et al 2021 a).…”

“…It has been shown that phycobiliproteins degradation favors the release of soluble amino acids and reduces the intracellular reactive oxygen species level by decreasing the light absorption capability (Forchhammer and Schwarz 2019 , Krauspe et al 2021 ). The current understanding of the chlorotic cells’ regulatory processes depicts the intricate mechanism that modulates ribosomal proteins, RuBisCO (ribulose-1,5-bisphosphate carboxylase-oxygenase) components, central regulators, and metabolic enzymes, as well as hyperphosphorylation of nondegraded phycobiliproteins (Spät et al 2018 , Krauspe et al 2021 a). The degradation and resynthesis of phycobiliproteins were examined using hyperspectral confocal fluorescence microscopy coupled with single-cell analysis on the basis of the pigment content and localization in live cyanobacterial cells (Murton et al 2017 ).…”

Autofluorescence-based high-throughput isolation of nonbleaching Cyanidioschyzon merolae strains under nitrogen-depletion

“…The traditional method of identifying factors regulating photosynthetic pigments involves culturing many candidate strains on solid media, isolating those in which the pigments are not degraded, and identifying the causal genes ( Collier and Grossman, 1994 ; Karradt et al., 2008 ; Krauspe et al., 2021 ). Most knowledge about pigment degradation, obtained by studying cyanobacteria, has revealed the causative genes regulating phycobilisome levels in cyanobacteria.…”