Homologous Expression and Characterization of Gassericin T and Gassericin S, a Novel Class IIb Bacteriocin Produced by Lactobacillus gasseri LA327

Abstract

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.

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk

“…1a). Bact_1 and Bact_2 show 100% amino (Kasuga et al 2019), while the surrounding genes do not resemble any other genes associated with bacteriocin production. The putative immunity protein showed 97% amino acid homology to those of the acidocin LF221A and gassericin S clusters (Kasuga et al 2019;Majhenič et al 2004).…”

“…The gassericin E operon was identified in L. gasseri EV1461, isolated from the vagina of a healthy woman; it encodes both antimicrobial gassericin E, with high similarity to gassericin T, and gene gaeX, whose product is identical to GatX and gassericin K7 B (Maldonado-Barragán et al 2016). Recently, genes encoding gassericin T (GatA and GatX) and the novel gassericin S, with similarity to acidocin LF221A (GasA and GasX), were found in the genome of L. gasseri LA327, isolated from the colon of a human adult (Kasuga et al 2019). This study demonstrated synergistic activity between the two components of gassericin T, and those of gassericin S, but not between gassericin S and gassericin T (Kasuga et al 2019).…”

“…Recently, genes encoding gassericin T (GatA and GatX) and the novel gassericin S, with similarity to acidocin LF221A (GasA and GasX), were found in the genome of L. gasseri LA327, isolated from the colon of a human adult (Kasuga et al 2019). This study demonstrated synergistic activity between the two components of gassericin T, and those of gassericin S, but not between gassericin S and gassericin T (Kasuga et al 2019).…”

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, byLactobacillus gasseriLM19, a strain isolated from human milk

“…Bact_2), respectively (Kasuga et al, 2019). While Bact_1 and Bact_2 showed synergistic activity, the 509…”

“…Finally, genes encoding gassericin T (GatA and GatX) and the novel 95 gassericin S, with similarity to acidocin LF221A (GasA and GasX), were all found in the genome of L. 96 gasseri LA327, isolated from human large intestine tissue (Kasuga et al, 2019). Kasuga et al 97 demonstrated the synergistic activity between the two components of gassericin T, and those of 98 gassericin S (Kasuga et al, 2019). However, they could not demonstrate synergistic activity between 99 gassericin S and gassericin T when they mixed the four peptides together.…”