Protein stable isotope probing with H218O differentiated cold stress response at permissive temperatures from general growth at optimal conditions in Escherichia coli K12

RATIONALE Tracing isotopically labeled water into proteins allows for the detection of species-specific metabolic activity in complex communities. However, a stress response may alter the newly synthesized proteins. METHODS We traced 18-oxygen from heavy water into proteins of Escherichia coli K12 grown from permissive to retardant temperatures. All samples were analyzed using UPLC/Orbitrap Q-Exactive-MS/MS operating in positive electrospray ionization mode. RESULTS We found that warmer temperatures resulted in significantly (P-value < 0.05) higher incorporation of 18-oxygen as seen by both substrate utilization as relative isotope abundance (RIA) and growth as labeling ratio (LR). However, the absolute number of peptides with incorporation of 18-oxygen showed no significant correlation to temperature, potentially caused by the synthesis of different proteins at low temperatures, namely, proteins related to cold stress response. CONCLUSIONS Our results unveil the species-specific cold stress response of E. coli K12 that could be misinterpreted as general growth; this is why the quantity as RIA and LR but also the quality as absolute number of peptides with incorporation (relative abundance, RA) and their function must be considered to fully understand the activity of microbial communities.