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

Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrola se (CBSH) activity was examined as a function of culture growth. The CBSH a ctivity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wil d-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methy l-N'-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutan ts without CBSH activity. In resting cell experiments, the strain showed re duced activity. Differences between growth parameters determined for wild-t ype and mutant strains were not detected. Comparative native gel electropho resis 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 th e mutant. The specific growth rate of the mutant strain was affected more b y bile salts than the wild-type strain. Nevertheless, bile was more toxic f or the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity t han taurodeoxycholic acid in a pH-dependent manner. No difference was appar ent between the two strains. In the absence of nutrients, the wild-type str ain died after 2 h whereas no effect was observed for the mutant. The de-en ergization experiments performed using the ionophores nigericin and valinom ycin suggested that the chemical potential of protons (Z Delta pH) was invo lved in Lactobacillus bile salt resistance.