Comparative Targeted Proteomics of the Central Metabolism and Photosystems in SigE Mutant Strains of Synechocystis sp. PCC 6803

Abstract

A targeted proteome analysis was conducted to investigate the SigE dependent-regulation of central metabolism in Synechocystis sp. PCC 6803 by directly comparing the protein abundance profiles among the wild type, a sigE deletion mutant (ΔsigE), and a sigE over-expression (sigEox) strains. Expression levels of 112 target proteins, including the central metabolism related-enzymes and the subunits of the photosystems, were determined by quantifying the tryptic peptides in the multiple reaction monitoring (MRM) mode of liquid-chromatography–triple quadrupole mass spectrometry (LC–MS/MS). Comparison with gene-expression data showed that although the abundance of Gnd protein was closely correlated with that of gnd mRNA, there were poor correlations for GdhA/gdhA and glycogen degradation-related genes such as GlgX/glgX and GlgP/glgP pairs. These results suggested that the regulation of protein translation and degradation played a role in regulating protein abundance. The protein abundance profile suggested that SigE overexpression reduced the proteins involved in photosynthesis and increased GdhA abundance, which is involved in the nitrogen assimilation pathway using NADPH. The results obtained in this study successfully demonstrated that targeted proteome analysis enables direct comparison of the abundance of central metabolism- and photosystem-related proteins.

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

“…S5 and S7). For example, it is likely that the reduction of carotenoid levels is related to the redirection of carbon skeletons to RPS overproduction, similar to what occurs in Synechocystis mutants overproducing other carbon reserves such as glycogen or polyhydroxybutyrate ‐ PHB (Antal et al ., ; Tokumaru et al ., ).…”

Assessment of Protein Content and Phosphorylation Level in Synechocystis sp. PCC 6803 under Various Growth Conditions Using Quantitative Phosphoproteomic Analysis

“…Proteins were extracted and digested with trypsin, and the tryptic peptide samples were subjected to nano-liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) assays. Using the MRM assays established in our previous study [18], 197 central metabolic and photosynthesis-related proteins were analyzed. In the present proteomic analysis, 128 proteins (58.0%) were successfully quantified (Table S2).…”

Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

“…In view of its universally recognized importance in photosynthesis research, Synechocystis has been a frequent target for mass spectrometry (MS)-based proteomic analysis, as reviewed by Gao et al ( 2015 ) and Battchikova et al ( 2018 ). A principal focus has been on the comparative quantification of protein abundance following adaptation to varying culture conditions (for example see Fulda et al 2000 ; Hong et al 2014 ; Angeleri et al 2019 ) and mutant strains have been used as tools for the dissection of adaptive and regulatory pathways (for example see Tokumaru et al 2018 ; Krynická et al 2019 ). This approach has also mapped proteins to their subcellular locations to track the development of thylakoid membranes (TMs), the specialized photosynthetic membranes that cyanobacteria, algae and plants all have in common (Kwon et al 2010 ; Pisareva et al 2011 ).…”