Genomic DNA Isolation and Bioinformatics Analysis

PCR products or DNA fragments from agarose gel were purified with the Wizard SV gel and PCR clean-up system (Promega, USA). Plasmid and genomic DNA was isolated using a plasmid minikit and a genomic minikit (A&A Biotechnology, Poland). For DNA manipulation, restriction and modification enzymes purchased from Thermo Fisher Scientific (USA) were used. Samples for genome sequencing were prepared with the Nextera XT DNA sample preparation kit, the Nextera XT indexing kit, and the PhiX control V3 kit (Illumina, USA) according to the manufacturer’s instructions and sequenced on a MiSeq Sequencer (Illumina). The data were analyzed with CLC Genomics Workbench 8.5 (Qiagen, Germany). Nucleotide sequences were translated to corresponding peptide sequences using an online translation tool on the ExPasy server (61 (link)). LiaF and LiaS transmembrane helices were predicted using TMHMM Server v. 2.0 (62 (link)). Conserved domains (CD) and active sites of LiaFSR-X were identified using the CD search online tool at the NCBI Conserved Domain Database (63 (link)). Additionally, LiaX N- and C-terminal domains composed mainly of α-helices and β-strands (22 (link)), respectively, were predicted based on the structure modeled using the I-TASSER web service (64 (link)). Protein models were visualized using Protter (65 (link)).