Computational protocol: Identification of a mouse Lactobacillus johnsonii strain with deconjugase activity against the FXR antagonist T β MCA

Similar protocols

Protocol publication

[…] Isolated colonies were scraped into a PCR tube and microwaved for 5 mins to rupture the cells. PCR was performed using primers specific to Lactobacillus 16S rRNA using conditions described by Byun et al. []. Reaction products were visualized on a 2.0% agarose gel with SYBR-SAFE dye (Applied Biosystems) and treated with 10 units of exonuclease I (New England Biolabs), and 1 unit of Antarctic phosphatase (New England Biolabs) at 37°C for 45 minutes to remove unincorporated primers and dNTPs, then at 85°C for 15 minutes to inactivate the enzymes. Purified PCR amplicons were sequenced by the Penn State Genomics Core Facility by Sanger sequencing on an Applied Biosystems 3730XL. Individual sequences were assembled using the DNASTAR Lasergene 12 software suite (DNASTAR Inc.) and identified using NCBI’s BLAST database []. All isolates were also characterized phenotypically, measuring growth and acidification rate in MRS broth. Each isolate was inoculated in sterile MRS broth and incubated anaerobically at 37°C for 24 hours. OD600 and pH measurements were taken every four hours. All isolates were determined to be L. johnsonii by 16S rRNA analysis, and all grew in and acidified MRS at indistinguishable rates. Thus, one isolate was chosen, designated LB1, and used for further experiments. [...] L. johnsonii strains LB1 and NCK88, a well-studied L. johnsonii isolate, were each inoculated from frozen glycerol stocks into sterile MRS and incubated anaerobically at 37°C overnight. Cells were precipitated by centrifugation and genomic DNA was extracted using Promega’s Wizard DNA kit. Whole genome sequencing was performed by the Penn State Genomics Core Facility using an Illumina MiSeq instrument with 300 bp paired end reads. Sequencing data was assembled de novo with DNASTAR’s SeqMan NGen (DNASTAR Inc.), and contigs were mapped against the reference L. johnsonii NCC533 genome with progressiveMauve [,]. Genomes were compared with WebACT and dotplots of translated open reading frames were generated with PROmer in Galaxy [–]. Sequence data is available under BioProject ID PRJNA315676. [...] BSH sequences from each strain in our Lactobacillus collection with publically available genomes were identified using NCBI’s Gene database. BSH genes from the fully sequenced and annotated strain L. johnsonii NCC533 [] were used to identify BSH genes from L. johnsonii LB1 and L. johnsonii NCK88 contigs using BLASTp. All putative translated open reading frames were also screened to ensure no additional BSH genes were present in either LB1 or NCK88. Sequences were translated and aligned by ClustalW with MEGA6 []. Maximum Likelihood phylogenic reconstruction of the alignment was performed by bootstrapping with 100 replications based on the Le Gascuel 2008 model of amino acid substitution []. The amino acid substitution model was selected based on the lowest Bayesian Information Criterion (BIC) score of maximum likelihood fit from 56 different amino acid substitution models. Active site amino acids were identified with NCBI’s Conserved Domains Database search tool []. […]

Pipeline specifications

Software tools DNASTAR Lasergene, Mauve, WebACT, Galaxy, BLASTP, Clustal W, MEGA
Applications Phylogenetics, WGS analysis, Sanger sequencing, Nucleotide sequence alignment, Genome data visualization
Organisms Mus musculus, Lactobacillus johnsonii, Homo sapiens, Lactobacillus johnsonii NCC 533, Escherichia coli
Diseases Sprains and Strains
Chemicals Glucose