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 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'-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 specific 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 (ZDeltapH) was involved in Lactobacillus bile salt resistance.
Grill
J P
JP
Université Henri Poincaré-Nancy 1, Laboratoire de Biochimie des Bactéries Gram+, Vandoeuvre les Nancy, and CIRDC Danone, Le Plessis Robinson, France.
Cayuela
C
C
Antoine
J M
JM
Schneider
F
F
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D018533:Biomass; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006002:Glycodeoxycholic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007779:Lactobacillus acidophilus / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D016296:Mutagenesis / Q000187:drug effects; D009153:Mutagens / Q000494:pharmacology; D013552:Swine
D001647:Bile Acids and Salts; D009153:Mutagens; D008769:Methylnitronitrosoguanidine; D006002:Glycodeoxycholic Acid; D000581:Amidohydrolases; C023154:choloylglycine hydrolase