Improvement of electroporation-mediated transformation efficiency for a Bifidobacterium strain to a reproducibly high...

Bifidobacteria are representative probiotics which are defined as live microorganisms that confer a health benefit on the host. Because of their safety and healthfulness when applied to humans, bifidobacteria are suitable as genetically engineered bacteria for applications to benefit human physiology and pathology. However, molecular biological studies of bifidobacteria have been limited due to insufficient genetic tools including effective transformation methods. The aim of this study is to improve the electroporation-mediated transformation efficiency of bifidobacteria to a reproducibly high level. The crucial factors that determine electroporation efficiency are the restriction-modification system, together with the cell wall and cell membrane structure of the bacteria. We optimized the bifidobacterial electroporation conditions by focusing on these factors as well as the amount of plasmid DNA used, the electrical parameters and the bacterial growth phase. As a result, the electroporation efficiency of B. bifidum BGN4 drastically and consistently increased from 10³ to 10⁵ CFU / μg DNA. The most significant factor for increasing the electroporation efficiency was the cell wall weakening mediated by NaCl, which improved the electroporation frequency by 20 times. Because the optimized electrotransformation conditions reported here should be widely applicable to other Bifidobacterium species, these could promote the extensive genetic manipulation of the various Bifidobacterium species in future studies.