Gene disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity

• Published in: Molecular & general genetics Vol. 239; no. 1-2; p. 269
• Main Authors: Leer, R J, Christiaens, H, Verstraete, W, Peters, L, Posno, M, Pouwels, P H
• Format: Journal Article
• Language: English
• Published: Germany 01-05-1993
• Subjects: Amidohydrolases - biosynthesis; Amidohydrolases - genetics; Chloramphenicol Resistance - genetics; Genes, Bacterial; Lactobacillus - enzymology; Lactobacillus - genetics; Lactobacillus - isolation & purification; Mutagenesis, Site-Directed; Mutation; Recombination, Genetic; Restriction Mapping
• ISSN: 0026-8925
• DOI: 10.1007/BF00281627

A chloramphenicol-resistance gene (cml) was introduced into the Lactobacillus plantarum gene encoding conjugated bile acid hydrolase (cbh) on a ColE1 replicon. This plasmid which is nonreplicative in Lactobacillus was used to transform L. plantarum strain 80. A homologous double cross-over recombination event resulted in replacement of the chromosomal cbh gene by the cml-containing cbh gene. The transformants obtained were unable to synthesize active conjugated bile acid hydrolase (Cbh). The Cbh- CmlR phenotype was stably maintained for more than 100 generations under nonselective conditions.