Cloning, deletion and complementation of catB

The full length (1,524 bp) of catB including ribosome binding site (gene ID: PXO_02830) was amplified by polymerase chain reaction (PCR) using the primer pairs P1/P2 (Additional file 5: Table S2). A right fragment (489 bp) and a left one (582 bp) were amplified by PCR using the primer pairs catBrF/catBrR and catBlF/catBlR (Additional file 5: Table S2), respectively. The PCR fragments were gel purified and cloned to the middle vector pMD18-T (Takara, Tokyo, Japan), resulting in constructs pMDcatB, pMDcatBr, and pMDcatBl, which were verified by DNA sequencing (Beijing Genomics Institute, Beijing, China).

The gene deletion mutant ΔcatB derived from PXO99^A was constructed by the homologous recombination as described previously by using the suicide vector pK18mobSacB [44]. The vector pMDcatBl with the left fragment and the vector pMDcatBr with the right fragment were digested with corresponding restriction enzymes and ligated to pK18-mobsacB. A gentamicin resistance gene (Gm ^r) at 855 bp was then inserted into the intermediate region between left and right fragment carried by pK18mobsacB, resulting in plasmids pKcatB, and then introduced into PXO99^A by electroporation. The deletion mutants were screened on PSA plates containing gentamicin and 10 % sucrose. For the complementation experiment, the vector pMDcatB with the catB gene including ribosome binding site was digested by enzymes and cloned into pBBR1MCS-4, generating pBBR-catB, and then transferred into ∆catB by electroporation and screened on PSA plates containing ampicillin.