The twin-arginine translocation (Tat) system, which is used for folded protein secretion, is rare in lactic acid bacteria (LAB). Previously, a Tat system composed of TatA(S) and TatC(S) subunits (the subscript S denotes a Streptococcus thermophilus origin) was identified in S. thermophilus CGMCC 7.179. In the present study, the tatA (S) and tatC (S) genes were cloned and functionally analyzed in Escherichia coli DE3 tat-deficient mutants. The E. coli tatABCDE-deficient mutant complemented with tatC (S) A (S) exhibited shortened cellular chains, but its ability to grow in the presence of sodium dodecyl sulfate (SDS) was not restored, suggesting that the S. thermophilus Tat system could partially replace that of E. coli. Surprisingly, the E. coli tatABE-deficient mutant complemented with tatA (S) and the E. coli tatC-deficient mutant complemented with tatC (S) displayed relatively normal cellular morphology and enhanced tolerance to SDS. These results suggest that TatA(S) and TatC(S) could serve as active protein translocases in E. coli DE3 tat-deficient mutants. Moreover, TatA(S) acted as a bifunctional subunit to fulfill the roles of both TatA and TatB of E. coli DE3. Thus, this minimal Tat system would be a promising candidate to translocate recombinant proteins in LAB.