Genome-wide identification of genes required for mucin utilization in Akkermansia muciniphila

Aliquots of the library containing approximately 108 CFU from the mutant pool stored at −80°C were grown in 30 ml of mucin-free medium overnight at 37°C anaerobically. Subsequently, 1000 μL of overnight culture was inoculated into three 50 mL tubes containing 20 mL of BHI broth. 2 mL of bacteria from the three cultures in mucin-free medium were spun down to extract genomic DNA and for Tn-seq sequencing. The mutant library was treated in the same way in mucin medium.The bacterial genomic DNA was isolated by a genomic DNA extraction kit (Sigma Aldrich). One microgram of mutant library genomic DNA was digested for 1 hr at 37°C with MmeI. The digestion products were dephosphorylated with calf intestine alkaline phosphatase (Invitrogen) for 30 min at 50°C. The samples were barcoded using 6 bp barcodes, and DNA suitable for sequencing was generated by PCR as described previously. In this experiment, NEBNext High-Fidelity 2 PCR master mix (New England Biolabs) was used for PCR amplification. In Personalbio (China), Tn-seq libraries were sequenced, and then the 16-20 nucleotide fragment of each read that corresponded to the WT strain sequence was extracted and mapped to the WT strain genome using Bowtie2