Restoring virulence to mutants lacking subunits of multiprotein machines: functional complementation of a Brucella virB5 mutant

Complementation for virulence of a non-polar virB5 mutant in Brucella suis 1330 was not possible using a pBBR-based plasmid but was with low copy vector pGL10. Presence of the pBBR-based replicon in wildtype B. suis had a dominant negative effect, leading to complete attenuation in J774 macrophages. This was due to pleiotropic effects on VirB protein expression due to multiple copies of the virB promoter region and over expression of VirB5. Functional complementation of mutants in individual components of multiprotein complexes such as bacterial secretion systems, are often problematic; this study highlights the importance of using a low copy vector.

▸ A Brucella suis virB5 mutant is attenuated in J774 macrophages. ▸ VirB5 over expression and/or multiple virB promoter copies attenuate WT B. suis. ▸ VirB5 over expression and/or multiple virB promoter copies affect other VirB proteins. ▸ Expressing virB5 from a low copy number plasmid fully complements a virB5 mutant.