Activation of the chromosomally encoded mazEFBif locus of Bifidobacterium longum under acid stress

• A functional type II toxin–antitoxin module-mazEF (mazEFBif) was identified in Bifidobacterium longum.
• The mazEFBif was induced under acid stress.
• The mRNA levels of ClpP1XBif and ClpP2XBif were both increased under acid stress.
• Protease ClpP1XBif and ClpP2XBif were involved in MazEBif degradation.
• MazEFBif was activated through the hydrolysis of MazEBif by ClpP1XBif and ClpP2XBif in B. longum JDM301 under acid stress.

Toxin–antitoxin (TA) systems are distributed within the genomes of almost all free-living bacteria. Although the roles of chromosomally encoded TA systems are still under debate, they are suspected to be involved in various stress responses. Here, we provide the first report of a type II TA system in the probiotic bacterium Bifidobacterium longum. Bioinformatic analysis of the B. longum JDM301 genome identified a pair of linked genes encoding a MazEF-like TA system at the locus BLJ_811–BLJ_812. Our results showed that B. longum mazEFBif genes form a bicistronic operon. The over-expression of MazFBif was toxic to Escherichia coli and could be neutralized by the co-expression of its cognate antitoxin MazEBif. We demonstrated that MazEFBif was activated during acid stress, which would most likely be encountered in the gastrointestinal tract. In addition, we found that the protease ClpPXBif, in addition to MazEFBif, was induced under acid stress. Furthermore, we examined antitoxin levels over time for MazEFBif and observed that the antitoxin MazEBif was degraded by ClpPXBif, which suggested that MazEFBif was activated through the hydrolysis of MazEBif by ClpP1XBif and ClpP2XBif under acid stress. Our results suggest that the MazEFBif TA module may play an important role in cell physiology and may represent a cell growth modulator that helps bacteria to cope with acid stress in the gastrointestinal tract and environment.