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. IMPORTANCE Bacteriocins are regarded as potential alternatives for antibiotics in the absence of highly resistant bacteria. In particular, two-peptide (class IIb) bacteriocins exhibit the maximum activity through the synergy of two components, and their antimicrobial spectra are known to be relatively wide. However, there are few reports of synergistic activity of class IIb bacteriocins determined by isolation and purification of individual peptides. Our results clarified the interaction of each class IIb component peptide for GT and GS via the construction of homologous mutants, which were not dependent on the purification. These data may contribute to understanding the mechanisms of action by which class IIb bacteriocins exhibit wide antibacterial spectra.

23Lactobacillus gasseri LA327 isolated from the large intestine tissue in humans is a 24 bacteriocinogenic strain and is predicted to produce two kinds of class IIb bacteriocins, i.e. 25 gassericin T (GT) and acidocin LF221A (Acd LF221A). In this study, DNA sequencing of the 26 genes for GT and Acd LF221A on Lb. gasseri LA327 revealed that the amino acid sequences for 27 GT completely corresponded with those of gat except for GatK (histidine kinase). However, 28 those for the Acd LF221A had analogues which differed in at least one amino acid residue to be a 29 putative class IIb bacteriocin designated as gassericin S (GS). By deletion test of GT structural 30 genes (gatAX), the LA327 strain retained the bacteriocin activity, and the LA327 mutant strain 31 lacking the ABC-type transporter gene (gatT) completely lost the bacteriocin activity. This 32 indicates that LA327 strain is a GS producer, and GS production is performed via gat with the 33 inclusion of gatT. Homologous expression using deletion mutants for GS and GT containing 34 each single peptide elucidated that GS (GasAX) and GT (GatAX) showed synergistic activity as 35 class IIb bacteriocins, respectively, and no synergistic activity was observed between each 36 peptide of GS and GT. The molecular mass of GS was estimated to be theoretical ca. 5,400 Da 37 by in situ activity assay after SDS-PAGE, clarifying that GS was actually expressed as an active 38 class IIb bacteriocin. Furthermore, stability of GS expressed against pH, heat and protease was 39 determined. 40 41 Importance 42We determined the complete DNA sequence for GS, a novel class IIb bacteriocin of Lb. 43 gasseri, and succeeded to express GS as active bacteriocins. Our results clarified the interaction 44 3 of each class IIb component peptide for GT in addition to GS via construction of homologous 45 mutants which were not dependent on the purification. These data may demonstrate the 46 characteristics of class IIb bacteriocins for Lb. gasseri. 47 48 Lactic acid bacteria (LAB), one of the potent candidates which are generally recognized 50 as safe (GRAS) microorganisms, have a long history of consumption by humans, and inhabiting 51 various ecosystems, including gastrointestinal (GI) tracts of humans and animals, and other 52 environments (for instance, vegetables, milks, and meats). In addition, lactobacilli are recognized 53 as important members of beneficial GI microbiota of humans and animals, along with 54 bifidobacteria 1 . Especially, Lactobacillus gasseri is one of the predominant species in 55 lactobacilli of human small intestines, and has been isolated not only from the GI tracts and the 56 feces, but also the oral and the vaginal cavities and mammary areola, and is considered one of 57 the representative probiotics 2-5 . 58 Many researchers have reported the beneficial effects of Lb. gasseri on the hosts, 59including the immunoregulation, the alleviation of allergic symptoms, the prevention of bacterial 60 and viral infections, the antitumor effect, and the inhibition of lip...