title: IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.
abstract: Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system.
authors: Molecular Biology Unit, Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001, India; Department of Microbiology and Immunology, National Institute of Nutrition, Hyderabad 500007, India; Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India; Ministry of Environment & Forests, Sector - 31 A, Chandigarh 160030, India. Electronic address: rajesh.mbu@gmail.com.
authors: Rajesh
authors: R
authors: Kumar
authors: Molecular Biology Unit, Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001, India.
authors: Sunita
authors: S
authors: Grover
authors: Animal Biotechnology Centre, National Dairy Research Institute, Karnal 132001, India.
authors: Jai K
authors: JK
authors: Kaushik
authors: Molecular Biology Unit, Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001, India. Electronic address: vkbatish@gmail.com.
authors: Virender Kumar
authors: VK
authors: Batish
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D005243:Feces / Q000382:microbiology; D020868:Gene Silencing; D006801:Humans; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D000581:Amidohydrolases; C023154:choloylglycine hydrolase
keywords: Bile salt hydrolase; Insertional inactivation; Lactobacillus plantarum; Transposon