Functional analysis of the type II toxin–antitoxin systems of the MazEF and RelBE families in Bifidobacterium longum subsp. infantis ATCC 15697

• The Bifidobacterium longum subsp. infantis ATCC 15697 genome harbors six type II TA gene modules.
• The five MazF and RelE toxins are toxic when expressed in Escherichia coli.
• At least two of the toxins, RelE2 and VapC, possess direct ribonuclease activity.
• All of the studied TA genes are transcribed in B. longum ATCC 15697.
• The RelE toxins are linked to the regulation of growth.

Analysis of the Bifidobacterium longum subsp. infantis ATCC 15697 genome sequence for the presence of toxin-antitoxin genes revealed two relBE-like operons, three relB-mazF-like operons, one relB-vapC-like operon, one solitary gene coding for the MazF toxin and one gene coding for the RelB antitoxin. An attempt to clone the selected relE and mazF toxin genes from B. longum subsp. infantis ATCC 15697 revealed their toxic effects on Escherichia coli, which could be neutralized by coexpression of these toxins with their cognate antitoxins. The only two toxin proteins, RelE and VapC, that were found to be non-toxic to E. coli, were overproduced and purified. Electrophoretic assays showed that both RelE and VapC possessed direct endoribonuclease activity. The expression levels of toxin genes in B. longum subsp. infantis ATCC 15697 increased during the nutrient starvation and entry into the late stationary phase. The two relBE bicistronic operons relE2-relB1 and relE1-relB4 from B. longum subsp. infantis ATCC 15697 were cloned and overexpressed in B. longum subsp. longum NCC2705 strain. The strain B. longum NCC2705 [pCESH80::relE1-relB4] showed a significantly decreased growth rate with later onset of the log phase and decreased cells density in the stationary phase.