Abstract
Transcriptional factors (TFs) play important roles in the responses to oxidative, acid, and other environmental stresses in Gram-positive bacteria, but the regulatory mechanism of TFs involved in oxidative stress remains unknown in lactic acid bacteria. In the present work, homologous overexpression strains with 43 TFs were constructed in the Lactobacillus plantarum CAUH2 parent strain. The strain overexpressing CopR displayed the highest sensitivity and a 110-fold decrease in survival rate under H2O2 challenge. The importance of CopR in the response to H2O2 stress was further confirmed by a 10.8-fold increase in the survival of a copR insertion mutant. In silico analysis of the genes flanking copR revealed putative CopR-binding “cop box” sequences in the promoter region of the adjacent gene copB encoding a Cu2+-exporting ATPase. Electrophoretic mobility shift assay (EMSA) analysis demonstrated the specific binding of CopR with copB in vitro, suggesting copB is a target gene of CopR in L. plantarum. The role of CopB involved in oxidative stress was verified by the significantly decreased survival in the copB mutant. Furthermore, a growth defect in copper-containing medium demonstrated that CopB functions as an export ATPase for copper ions. Furthermore, EMSAs revealed that CopR functions as a regulator that negatively regulates copB gene and Cu2+ serves as inducer of CopR to activate the expression of CopB in response to H2O2 stress in L. plantarum CAUH2. Our findings indicated that CopR plays an important role in enhancing oxidative resistance by regulating copB to modulate copper homeostasis.
Highlights
Lactobacillus plantarum is an aerotolerant Gram-positive bacteria belonging to the facultatively heterofermentative group of lactobacilli (Kleerebezem et al, 2003)
L. plantarum CAUH2 cultures were incubated at 37◦C in De Man Rogosa Sharp (MRS) medium, and L. lactis NZ9000 was grown at 30◦C in GM17 (M17 broth supplemented with 0.5% w/v D-glucose)
45 Transcriptional factors (TFs) were detected in the genome of CAUH2, only 43 TF genes were successfully inserted into the vector pslpA8148
Summary
Lactobacillus plantarum is an aerotolerant Gram-positive bacteria belonging to the facultatively heterofermentative group of lactobacilli (Kleerebezem et al, 2003). L. plantarum is widely used as a starter culture in fermented food and feed production, such as dairy, meat, and many vegetables and plants (Leroy and De Vuyst, 2004). L. plantarum strains have been declared to have healthpromoting effects, such as increasing iron absorption and enhancing immunity, and can function as probiotic active ingredients in fermented foods (Hoppe et al, 2015; Hulst et al, 2015). L. plantarum suffers from unavoidable oxidative stress caused by reactive oxygen species (ROS) during manufacturing. L. plantarum has developed a variety of adaptive mechanism to cope with oxidative stress
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