The rfb-associated gene cluster contributes to oxidative stress tolerance in Synechocystis sp. PCC 6803 through extracellular polysaccharide homeostasis.

Abstract

Paraquat (PQ; methyl viologen, MV) is a redox-cycling herbicide that causes oxidative injury through the continuous generation of reactive oxygen species (ROS). However, the genetic and extracellular mechanisms underlying cyanobacterial protection against MV-induced oxidative stress remain incompletely understood. Here, we investigated selected stress-responsive genes within the rfb-associated gene cluster spanning slr0978 to slr1611 in Synechocystis sp. PCC 6803. This cluster contains genes predicted to participate in sugar-nucleotide metabolism, polysaccharide assembly and modification, and ABC transporter-associated export, suggesting a potential role in maintaining cell-surface polysaccharide homeostasis. Deletion of slr0978, co-deletion of slr0978 and slr0980, or deletion of slr0983 markedly increased MV sensitivity, with the M0983 mutant showing the strongest phenotype. M0983 exhibited severe growth inhibition, reduced extracellular polysaccharide (EPS) accumulation, elevated ROS accumulation, and impaired photosynthetic efficiency. M0978 and M0978/0980 also displayed stress-sensitive phenotypes, consistent with partially overlapping contributions of these genes to MV stress tolerance. Targeted qPCR revealed local and gene-specific expression changes following gene deletion. Chlorophyll fluorescence kinetics, untargeted LC-MS-based metabolomic profiling, and protein-association analyses collectively indicated that Slr0983 may contribute to polysaccharide-associated homeostasis, redox buffering, and oxidative stress acclimation. Equal-volume supplementation of M0983 cultures with WT-derived EPS preparations partially restored growth under MV stress, supporting an extracellular protective contribution of EPS. Together, these findings indicate that selected genes within the rfb-associated gene cluster contribute to EPS-associated oxidative stress tolerance in Synechocystis, with slr0983 representing a functionally important component of this cluster.