Functional Analysis of Four Bile Salt Hydrolase and Penicillin Acylase Family Members in Lactobacillus plantarum WCFS1

Abstract

Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.

Genetic Characterization and Gene Expression of Bile Salt Hydrolase (bsh ) from Lactobacillus reuteri CRL 1098, a Probiotic Strain

“…lactis NZ9000 cells, expressing the bsh gene showed activity against all BA tested, being the enzyme in both cases more active on GDCA. The highest affinity observed by the glycoconjugated substrates is consistent with our previous results [39,38], as well as for numerous choloylglycine hydrolase enzymes described in the literature [19], which could be related to the higher toxicity of glycine-conjugated BA respect to taurine-conjugated BA [6]. Although the precise mechanism is unknown, the capability to hydrolyze BA may contribute to the survival and persistence of bacterial strains in the intestinal tract, as was previously suggested by many authors [19,39,1].…”

“…lactis NZ9000. Electrocompetent cells prepared as previously described [6] were exposed to a single electrical pulse delivered by a Gene-Pulser (Bio-Rad Laboratories, Richmond, California) set at 25 µF and normally at 2.0 kV. Recombinant cells of Lac.…”

“…Plasmid DNA isolation and electroporation technique used for Lac. lactis NZ9000 were performed as described by Lambert et al [6]. Purification of DNA fragments from agarose gels was performed using the PCR AccuPrep ® Purification Kit (Bionner, Korea).…”

“…[11], Listeria [12], and Enterococcus [13]. Additionally, a number of bsh genes from Lactobacillus plantarum [14,6], Lactobacillus johnsonii [2], Lactobacillus fermentum [3], Lactobacillus casei [15] and Bifidobacterium [16][17][18] strains have been cloned and characterized. Interestingly, the presence and genetic organization of bsh genes are highly variable and also, several strains possess more than one BSH homologo, which in most cases are not identical [19].…”

“…BSH activity has been described among a wide variety of genus, including Lactobacillus [2][3][4][5][6][7], Bifidobacterium [8][9][10], Clostridium (C). [11], Listeria [12], and Enterococcus [13].…”