Diamide-based screening method for isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries

The bacterium Bacillus subtilis is of high importance both as a model organism for Gram-positive bacteria and as an industrial work horse in the production of biomolecules. In recent years, advancements have been made to engineer the bacterium even further towards industrial applications. In this study, we present a novel screening method for mutant libraries using diamide, an oxidising agent that binds free thiols and creates disulfide bonds between them, thereby causing a so-called ‘disulfide stress’ in bacteria. The method shows promise to selectively identify phenotypes in B. subtilis with improved tolerance towards oxidative and disulfide-associated stress. Phenotypes initially identified by transposon mutagenesis were recreated through targeted gene deletions. Among the resulting deletion mutants, the largest difference in diamide tolerance compared to the parental strain was observed for pfkA and ribT deletion strains. A proteomics analysis showed that that diamide tolerance can be achieved through different routes involving increased expression of stress management proteins and reduced availability or activity of the RNA degradosome. We conclude that our screening method allows the facile identification of Bacillus strains with improved oxidative stress tolerance phenotypes