Modulation of peptidoglycan crosslink network in Escherichia coli enhances water-responsive actuation

Peptidoglycan (PG), the primary load-bearing component of bacterial cell envelopes, is a bio-derived material whose mechanical and hydration properties are central to microbial viability and their environmental responsiveness. PG has been increasingly recognized as a scalable water-responsive (WR) material, capable of converting chemical potential gradients from ambient changes in relative humidity (RH) directly into mechanical work. In this study, we leverage stress-induced PG remodeling pathways in a BB-3 Escherichia coli (E. coli) strain to modulate the architecture of its PG sacculus for enhanced WR performance. Under arabinose-deprived conditions (-Ara), BB-3 PG exhibits a twofold increase in the ratio of crosslinked-to-linear muropeptide stems and a threefold rise in the relative abundance of ③-③ (mDap-mDap) linkages relative to an arabinose-rich (+Ara) control. When responding to RH changes between 10 % and 90 % RH, these molecular-level modifications translated into a fivefold increase in WR actuation energy density (623.0 kJ/m³) with rapid response times on the order of seconds (τ_d: 1.8 s, τ_h: 0.7 s). The remodeled PG also displays a significant increase in stiffness (E: 8.9 GPa at 10 % RH) and an 8 % greater uptake of water, driving the PG matrix to generate twofold higher WR strain (ε: 30.2 %). The observed increases in water retention and WR behavior of E. coli PG may also carry intriguing evolutionary implications, reflecting adaptive strategies microbes take to restructure PG layers and survive under microenvironmental nutrient scarcity. STATEMENT OF SIGNIFICANCE: Current testing of water responsive (WR) biomaterials has focused on a narrow subset of microbial species, leaving Gram-negative bacteria largely unexplored. Our work addresses this gap by demonstrating that the peptidoglycan (PG) sacculus of E. coli also exhibits robust WR properties. Through leveraging genetic and environmental stresses during the organism's growth phase, we demonstrate that the topology, mechanical stiffness, and water partitioning behavior of PG networks can be rationally improved to yield enhanced actuator materials. By establishing a connection between PG crosslinking architecture and emergent mechanical and hydration dynamics, we present a promising approach to engineer dynamic and sustainable WR materials from living systems that can be employed as active components in high-performance actuators.