Genetic diversity of bile salt hydrolases among human intestinal bifidobacteria

Abstract

This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.

Fig. 1

Alignment of partial bsh genes from various bifidobacteria and design of universal primers for PCR. Abbreviation for BSHs: B. adol B. adolescentis ATCC 15703 (AP009256.1), B. long B. longum SBT2928 (AF148138.1), B. bif B. bifidum ATCC 29521 (AY604517.1), B. cat B. catenulatum DSM 16992 (NZ_ABXY01000011.1), B. pscat B. pseudocatenulatum DSM 20438 (NZ_ABXX02000003.1), B. dent B. dentium Bd1 (CP001750.1), B. animalis subsp. lactis KL612 (AY530821.1). Asterisks indicate identical nucleotides. Four highly conserved regions of bsh genes used for primers design are underlined

Fig. 2

PCR screening of different Bifidobacterium strains with primers: Bif-bshA-1F and Bif-bshC-1R (a), Bif-bshA-1F and Bif-bshD-2R (b), Bif-bshB-2F and Bif-bshC-1R (c), and Bif-bshB-2F and Bif-bshD-2R (d). Lanes: 1 DNA molecular weight marker (in base pairs), 2 B. adolescentis DSM 20087, 3 B. animalis subps. lactis NRRL B-41405, 4 B. animalis subps. animalis NRRL B-41406, 5 B. bifidum DSM 20456, 6 B. breve DSM 20091, 7 B. breve NRRL B-41408, 8 B. catenulatum DSM 20224, 9 B. longum subsp. infantis ATCC 15697, 10 B. longum subsp. longum NRRL B-41409, 11 B. pseudocatenulatum DSM 20439, 12 B. pseudolongum DSM 20099, 13 B. longum subsp. suis NRRL B-41407, 14 B. asteroides DSM 20089, 15 B. coryneforme DSM 20216, 16 negative control

Fig. 3

Agarose gel electrophoresis of PCR products obtained with primers Bif-bshA-1F and Bif-bshD-2R for DNA samples extracted from feaces (a) and probiotic products (b). Lanes: a 1 DNA molecular weight marker (in base pairs), 2–6 genomic DNAs directly extracted from five stool samples, 7 negative control, b 1 DNA molecular weight marker, 2–7 genomic DNA directly extracted from six probiotic products, 8 negative control

Fig. 4

Phylogenetic trees based on partial sequences of bsh for bifidobacteria used in this study. Tree was constructed using the neighbor joining method from 1000 bootstrapping replicates