Synergistic interactions between glycogen and trehalose mediate adaptation to the stationary phase in E. coli

Glycogen and trehalose are multifunctional carbohydrates that contribute to bacterial adaptability, stress tolerance, and energy homeostasis, yet the mechanisms underlying these characteristics are not fully elucidated. In Escherichia coli, we reveal a metabolic and regulatory coupling between glycogen and trehalose with the global flux-sensing regulators cAMP-CRP and Cra during the stationary phase. Using mutants lacking glycogen synthase (glgA), trehalose-6-phosphate synthase (otsA), or both, we measured carbon storage, competitive fitness, stress sensitivity, expression of flux-sensing target genes, and concentrations of ATP, reactive oxygen species, and advanced glycation endproducts. Single mutants showed reduced fitness, while the glgA otsA double mutant exhibited transient competitive advantages in early stationary phase but severe stress sensitivity and ultimate fitness collapse. The double mutant had the lowest ATP, yet elevated redox activity, indicative of uncoupled metabolism. Transcriptional analysis revealed upregulated pfkA and ptsG and downregulated pykF and rpoS, consistent with Cra inactivation and cAMP-CRP activation. Wild-type cells increased glycogen and trehalose storage in response to glucose stress, while the glgA otsA mutant showed severe glucose stress toxicity, demonstrating that both compounds act as glucose buffers; the antioxidant carnosine partially rescued the fitness of all strains. These results suggest that glycogen prevents fructose-1,6-bisphosphate accumulation that inappropriately inactivates Cra, while trehalose recycling maintains PTS flux, preventing aberrant cAMP-CRP activation. Loss of both causes dysregulated flux-sensing where transcriptional programs misalign with metabolic state, leading to overflow metabolism, methylglyoxal accumulation, and fitness collapse.IMPORTANCEBacteria in nature endure prolonged energy limitation interspersed with nutrient influxes- termed the "feast-famine" lifestyle. To cope with environmental scarcities and fluctuations, bacteria manage limited energy reserves and coordinate metabolic programming with external conditions and intracellular demands. This study reveals how Escherichia coli manages carbon storage during starvation through glycogen and trehalose, working in a functionally specialized yet synergistic partnership. Critically, these pathways are interconnected with global transcriptional regulatory systems-cAMP-CRP and Cra-coordinating nutrient scavenging, stress responses, and energy metabolism. Disrupting both pathways dysregulates these regulators, causing bacteria to initially outcompete wild-type cells through enhanced resource acquisition, but ultimately compromising long-term survival as stress resistance is impaired. Understanding stationary phase physiology is essential because this growth-arrested state characterizes many natural habitats.