Homologous expression and characterization of gassericin T and gassericin S, a novel class IIb bacteriocin produced by Lactobacillus gasseri LA327

Lactobacillus gasseri LA327, isolated from the large intestine tissue in humans, is a bacteriocinogenic strain with two kinds of class IIb bacteriocin structural genes, i.e., those for gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the genes for GT and Acd LF221A from L. gasseri LA327 revealed that the amino acid sequences for GT corresponded with those for GT genes, except for GatK (histidine kinase). However, Acd LF221A genes had analogues which differed in at least one amino acid residue, to encode a class IIb bacteriocin designated gassericin S (GS). The LA327 strain retained antimicrobial activity after the deletion of the GT structural genes (gatAX); however, both GS and GT activities were lost by deletion of the putative ABC transporter gene (gatT). This indicates that the LA327 strain produces GS and GT and that GS secretion is performed via GT genes with the inclusion of gatT. Homologous expression using deletion mutants of GS and GT, each containing a single peptide, elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as class IIb bacteriocins and that no synergistic activity was observed between GS and GT peptides. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active class IIb bacteriocin. Furthermore, the stability of expressed GS to pH, heat, and protease was determined.