Integrative genome and metabolome analysis reveal the potential mechanism of osmotic stress tolerance in Bifidobacterium bifidum

Abstract

Osmotic stress affects the viability of probiotics, especially Bifidobacteria, which are less resistant to environmental stress. In this study, an adaptive evolution technique was applied to Bifidobacterium bifidum to select tolerant strains against high osmotic stress. B. bifidum CCFM16 was subjected to a gradually increasing penetration environment for 1000 generations. The generation time of the hyperosmotic tolerance mutant CCFM16m is 1/3 of its ancestor at an osmotic stress of 1300 mOsm/kg. Whole-genome comparison, candidate gene expression, and 3D protein structure simulation were performed and the results indicated that critical mutations were related to glutamate synthesis and transport. Untargeted metabolome analysis showed that compared to parent strain, the difference was mainly reflected in the increased accumulation of amino acids, especially proline, while the accumulation of central carbon decreased. Increase in the key enzyme F6PPK in the “bifidus” pathway indicated that cells transfer more energy from basal metabolism to resist osmotic stress. Since there was no proline transporter protein, excessive accumulation of proline showed that under long-term high osmotic stress, B. bifidum CCFM16 evolved a protective mechanism to synthesize proline from glutamic acid, an osmotic protection system with proline as the main compatible solute was formed.