Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance

Abstract

. 2000. Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N H -nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The speci®c growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDpH) was involved in Lactobacillus bile salt resistance.

Consequences of bile salt biotransformations by intestinal bacteria

“…BSH mutant strains of Lactobacillus amylovorus, 44 and Listeria monocytogenes 45,46 were shown to be significantly more sensitive to conjugated bile salts than their wild-type counterparts. Indeed, glycine-conjugated BA was found to be significantly more inhibitory than taurine-conjugates, particularly at low environmental pH.…”

Bile salt biotransformations by human intestinal bacteria

“…The presence of a functional BSH results in the intracellular accumulation of free bile acids, which become protonated in a stoichiometric manner, decreasing energydependent H 1 -ATPase-driven proton efflux. BSHs from human intestinal lactobacilli generally have higher affinity for glycine conjugates (26)(27)(28)(29). This observation may lend weight to the hypothesis of De Smet et al (26), or the higher affinity of BSHs for glycine conjugates may have evolved because glycine conjugates are generally higher in proportion (3:1) than taurine conjugates in human bile (6).…”

Allelic Variation of Bile Salt Hydrolase Genes in Lactobacillus salivarius Does Not Determine Bile Resistance Levels

“…Disruption and deletion of bsh in lactobacilli caused loss of corresponding activity against tauro/glyco-CBA (34,40). The resistance of bsh mutants of Lactobacillus amylovorus and Lactobacillus plantarum to bile acids/salts was reduced compared to the respective wild-type strains (13,16,24). However, no convincing in vivo experiments have so far demonstrated that bsh contributes to bile resistance in these or other probiotic bacteria.…”

“…However, there is also evidence that the production levels and enzymatic activity of Bsh are not directly related to overall bile resistance levels (24,41). In addition to bsh, other genes (pva and btlB) and the sigma factor B have been shown to contribute to bile resistance in L. monocytogenes EGDe (3).…”