The necessity of multi-parameter normalization in cyanobacterial research: A case study of the PsbU in Synechocystis sp. PCC 6803 using CRISPRi

Photosystem II (PSII) is a multiprotein complex and plays a central role in oxygenic photosynthesis. PsbU, a 12 kDa subunit of PSII, is associated with thermotolerance and structural stabilization of the oxygen-evolving complex in cyanobacteria. Corresponding knockout strains showed decreased oxygen evolution rates, although the growth was not impaired. In this study, we provide further insights into the consequences of PsbU perturbations and propose to revisit the impact of PsbU on cell physiology. We made use of CRISPRi to knock down the psbU gene in Synechocystis sp. PCC 6803, and assessed previously described effects referred to different biomass parameters including optical density, chlorophyll a content and cell number. After knocking down psbU, the growth rate was decreased by 15% based on counting the cell number, while this effect was not observed when monitoring optical density. Furthermore, the oxygen evolution rate per cell in the psbU knockdown strain did not show a significant difference compared to the control groups, which was probably due to its larger cell size and higher chlorophyll a content per cell. The decreased quantum efficiency of pigments was compensated by the increased pigment content on the single-cell level in the knockdown strain. Our results complement previous analyses and highlight the importance of evaluating cyanobacterial physiology based on different biomass quantitative units to avoid misinterpretation of the results.