bioRxiv2025
Encapsulation of microbes in natural or synthetic matrices is a key aspect of engineered living materials, although the influence of such confinement on microbial behavior is poorly understood. A few recent studies have shown that spatial confinement and mechanical properties of the encapsulating material significantly influence microbial behavior, including growth, metabolism, and gene expression. While such effects have been shown to elicit various responses in a few micro-organisms like E. coli, yeast, and cyanobacteria, systematic comparative studies between different organisms in the same confinement conditions are missing. Thus, in this study, we report the adaptive responses exhibited by rod-shaped gram-negative and gram-positive probiotic bacteria that are of great interest for developing therapeutic engineered living materials. Accordingly, gram-negative E. coli Nissle 1917 and gram-positive L. plantarum WCFS1 were encapsulated in hydrogel matrices and their growth, metabolic activity, and recombinant gene expression were investigated. By varying the polymer concentration and degree of chemical cross-linking in the hydrogels, it was possible to modulate their stiffness and study how the bacteria adapted to these different confinement conditions. In accordance with previous reports, both bacteria grow from single cells into confined colonies but more interestingly, in E. coli gels, mechanical properties influenced colony growth, size, and morphology, whereas this did not occur in L. plantarum gels. However, with both bacteria, increased matrix stiffness led to higher levels of recombinant protein production within the colonies. By measuring metabolic heat generated in the bacterial gels using a novel isothermal microcalorimetry technique, it was inferred that E. coli adapts to the mechanical restrictions through multiple metabolic transitions and is significantly affected by the different hydrogel properties. Contrastingly, both these aspects were not observed with L. plantarum. These results revealed that despite both these bacteria being gut-adapted probiotics with similar geometries, mechanical confinement affects them considerably differently. The weaker influence of matrix stiffness on L. plantarum is attributed to its slower growth and thicker cell wall possibly enabling the generation of higher turgor pressures to overcome restrictive forces under confinement. By providing fundamental insights into the interplay between mechanical forces and bacterial physiology, this work advances our understanding of how matrix properties shape bacterial behavior. The implications of these findings will aid the design of engineered living materials for therapeutic applications.