Additional polysaccharides were removed following the protocol outlined in Wilson . FS ATCC43239 gDNA was isolated following the protocol described in Ausubel et al.  and FA UTEX1903 gDNA was extracted following a protocol described in JAK inhibitor Mustafa . Whole genome sequencing and bioinformatics High molecular weight gDNA from WI click here HT-29-1 and HW IC-52-3 was sent to BGI (Beijing Genome Institute, China) for genome sequencing via high throughput Illumina sequencing technology. BGI performed genome assembly and gene annotation using Glimmer v3.0. Extracted gDNA from FA UTEX1903 and FS ATCC43239 was submitted to Case Western Reserve Genomics Core Facility for whole genome sequencing. Paired end
DNA libraries were obtained by using Nextera DNA sample preparation kit and sequenced using the Illumina GAIIx platform. Raw reads quality was assessed using FastQC 0.10.1 (Babraham Bioinformatics) with default settings see more and trimmed with Seqyclean 1.3.12 (http://cores.ibest.uidaho.edu/software/seqyclean). Filtered
reads were assembled de novo using the velvet package (Version 1.2.08) and a kmer range between 55-63. The optimal assembly based on expected genome size, N50 and contig number was used for downstream annotation and analysis. Gene annotation was performed by BGI using Glimmer v3.2. A Basic Local Alignment Search Tool (BLAST) search was performed to identify the putative function of proteins based on sequence similarity . Nucleotide and protein sequences were organized and visualized using Monoiodotyrosine Geneious v6.1.7 created by Biomatters. Available from http://www.geneious.com/. Nucleotide alignments were performed using Geneious Alignment with default settings. For protein alignments, Clustal Omega (Version 1.2.1) was used with default settings, except order changed from aligned to input . For phylogenetic analysis, the sequences were first aligned using the Clustal W program built into Geneious. Phylogenetic trees were constructed using the Geneious Tree Builder program, which uses the neighbour-joining method . A 929 bp nucleotide fragment was
used for the phylogenetic analysis of 16S rDNA sequences, while a 315 amino acid sequence alignment was used for phylogenetic analysis of the prenyltransferase. The outgroup was constituted by the distantly related cyanobacterium Synechocystis sp. for 16S rDNA analysis. PCR and sequencing reactions A 50 μL PCR reaction mixture contained 10 pmol of specific forward and reverse primer (Additional file 11) (Geneworks, Australia), 1× PCR Buffer (KAPA Biosystems), 2.5 mM MgCl2, 1 pmol dNTPs (Fisher Biotec), 1 U of KapaTaq polymerase (KAPA Biosystems) and 50 ng of gDNA template. Pfu DNA polymerase (Sigma) was used in addition to KapaTaq at a ratio of 1:10 (v/v). Hotstart PCR was performed by first heating the samples to 95°C.