Four superoxide dismutases of Bacillus cereus 0-9 are non-redundant and perform different functions in diverse living conditions.

Superoxide dismutases (SODs) have been shown to exhibit high levels of conservation and exist in almost all aerobic organisms and even many strict anaerobes. There are four SODs in Bacillus cereus 0-9, and this coexistence of multiple homologous enzymes is of great significance in the evolution of bacteria. We hypothesized that the four sod genes in B. cereus 0-9 constituted non-redundant protection against oxidative damage in vivo and played unique roles in the pathogenicity of B. cereus 0-9 during different phases or growth environments. To test this hypothesis, we constructed four single-knockout mutants (∆sodA1, ∆sodA2, ∆sodS, and ∆sodC) and a mutant lacking all four sod genes (∆sod-4) of B. cereus 0-9 and assessed their various phenotypes. Our results indicated that sodA1 plays a major role in tolerance to intracellular oxidative stress and spore formation. The ∆sodA1 and ∆sod-4 mutants were very sensitive to oxidants. The spore formation of the ∆sodA1 mutant was dramatically delayed, and the ∆sod-4 mutant did not form any spores under our experimental conditions. The sodA2 gene may play an important role in negative regulation of swarming motility, pathogenicity, and phospholipase and haemolytic activity of B. cereus but also a role in positive regulation of biofilm formation under our experimental conditions. The other two genes, sodS and sodC, were key to the pathogenicity of B. cereus. The lethal rates of Helicoverpa armigera infected by the ∆sodS and ∆sodC mutants were only 26.67%, while wild-type B. cereus 0-9 caused lethality in up to 86.67% of the insects at 24 h after injection. Moreover, the ∆sod-4 mutant caused a reduced death rate of H. armigera of 46.70%, which was slightly higher than that caused by the ∆sodS and ∆sodC strains. Thus, these four sod genes were non-redundant for oxidative stress and may play different additional roles in B. cereus 0-9. These results can help us to further understand the biocontrol characteristics of B. cereus 0-9 and lay a theoretical foundation for further research.