Qubit 2.0 device

The genomic DNA was obtained from overnight cultures of the isolates in LB using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). The quality of gDNA samples was assessed using the Qubit dsDNA BR Assay Kit on a Qubit 2.0 device (Thermo Fisher Scientific) and by running the samples on 1% agarose gels with Midori Green (Nippon Genetics Co) staining to assess potential shearing. The gDNA was subsequently prepared for Illumina sequencing using either a TruSeq DNA PCR-Free kit (Illumina) or the NEBNext Ultra DNA Library Prep kit (New England Biolabs) and sequenced on an HiSeq 4000 machine (Illumina) using a paired-end approach (2*151 bp). Wild-type strains gDNA were also sequenced by long-read PacBio RSII technology to fully resolve the chromosome.

Microbial community profiles were assessed by sequencing the 16S rRNA gene. The first step was the amplification of V3 and V4 variable fragments of analyzed gene according to the protocol recommended by Illumina (San Diego, CA, USA). Primers with overhanging adapters compatible with Illumina indexes and sequencing adapters in paired-end sequencing technique were used. Kappa HiFi polymerase (Roche, Mannheim, Germany) was used to amplify fragment with an average 464 bp length. Next the specificity of obtained products was evaluated in an agarose gel and then purified on AMPure XP magnetic beads (Beckman Coulter, CA, USA). The indexing reactions were also carried out using the Kappa HiFi polymerase (Roche, Mannheim, Germany) with the Nextera XT dual-index set (Illumina, San Diego, CA, USA). The concentration of the obtained libraries was determined using the Qubit 2.0 device (Thermo Fisher Scientific, Waltham, MA, USA) and pooled in equal concentrations. The library thus prepared was sequenced on the Miseq platform (Illumina, San Diego, CA, USA) using the kit (MiSeq Reagent Kit v3, 600 cycles).

Microbial community profiles were assessed by sequencing the 16S rRNA gene. The first step was the amplification of the V3 and V4 variable fragments of the analyzed gene according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). Primers with overhanging adapters compatible with Illumina indexes and sequencing adapters in paired-end sequencing techniques were used. Kapa HiFi polymerase (Roche, Cape Town, South Africa) was used to amplify a fragment with a mean length of 464 bp. Next, the specificity of the obtained products was evaluated in an agarose gel and then purified on AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). The indexing reactions were also carried out using Kapa HiFi polymerase (Roche, Cape Town, South Africa) with the Nextera XT dual-index set (Illumina, San Diego, CA, USA). The concentration of the obtained libraries was determined using the Qubit 2.0 device (Thermo Fisher Scientific, Waltham, MA, USA) and pooled in equal concentrations. The resulting library was sequenced on the MiSeq platform (Illumina, San Diego, CA, USA) using a MiSeq Reagent Kit v3 (600 cycles).