An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium

Abstract

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth’s surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

The Glutathione System: A Journey from Cyanobacteria to Higher Eukaryotes

“…Singlet oxygens are unavoidably produced by the interaction of sunlight with photosynthetic pigments (chlorophyll a, carotenoids and phycobiliproteins) while superoxide anions, hydrogen peroxides and hydroxyl radicals are generated when the light-driven electron transport exceeds what is needed for nutrients assimilation [ 8 , 10 , 43 ]. Cyanobacteria are also strongly exposed to solar UV radiations (UVR) that also generate ROS [ 20 , 44 ]. Consequently, cyanobacteria represent a major source of ROS in aquatic environments [ 45 ].…”

“…To cope with ROS and environmental stresses, cyanobacteria have evolved the glutathione system [ 5 , 59 , 60 ], which is crucial to their photoautotrophic lifestyle [ 4 , 44 , 61 ] and has been conserved during evolution [ 5 , 6 , 59 , 60 , 62 ]. The glutathione system comprises the glutathione tripeptide itself (γ-L-glutamyl-L-cysteinyl-glycine, GSH) and its cysteine-less homologs (ophthalmate and norophthalmate); in humans, these serve as biomarkers of diseases (See below), as well as numerous GSH-dependent enzymes, such as glutaredoxins and glutathione-S-transferases [ 18 , 63 ], which have been conserved during evolution [ 11 , 26 , 27 , 62 , 64 , 65 ].…”

“…Furthermore, cyanobacteria have no pepT orthologous genes. In the cyanobacterium Synechococcus elongatus PCC 7942, the inactivation of a gene encoding a leucyl aminopeptidase (LAP) with cysteinyl-glycinase activity was found to decrease the tolerance to UV [ 44 ].…”

A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century

“…This enzyme is also involved in UV tolerance in a cyanobacterium where it exhibits cysteinyl‐glycinase activity. This involves glutathione turnover in E. coli and connects the enzyme to protection against and repair of UV damage (Weiss, Fang, et al, 2022 ). Because there is no glutathione in B. subtilis the details of an analogous pathway should be further investigated.…”