Jetflex kit

Genomic DNA was extracted from a culture of the strain WaF17.12 using a JetFlex kit (Genomed, Germany). Amplifications of EXG1 and EXG2 genes were obtained using specific oligonucleotides (described in Table S1) designed on the W. anomalus BS91 EXG1 and EXG2 sequences available in the data bases (accessions: JQ734563 and JQ734566). Reaction mixtures were prepared in 25 µl using: 0.6 U of Dream Taq Polymerase (Fermentas, Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA), 0.25 mMdNTPs, 1X Taq Polymerase Buffer, 0.2 µM each primer and 50 ng DNA. Reactions were run for 2 min at 95°C and cycled 30 times through 30 sec at 95°C, 30 sec at 55°C and 40 sec at 72°C. Finally, reactions were kept for 8 min at 72°C. PCR products were then resolved in 1% agarose gel stained with ethidium bromide. The fragments were cloned in T-Vector System following manufacture’s instructions (Promega, USA). The cloned fragments were sequenced after colony PCR using the plasmid primers SP6 (5′-atttaggtgacactatagaat-3′) and T7 (5′-aatacgactcactataggg-3′). The obtained sequences were firstly analysed by BLASTN. (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence alignment were then generated by ClustalW (http://www.genome.jp/tools/clustalw/). Sequences of WaF17.12 genes EXG1 and EXG2 were deposited through the EMBL-Bank.

Genomic DNA from P. pseudoalcaligenes AVO110 was extracted from bacterial cells grown overnight in LB medium supplemented with Nf at 25°C. DNA was extracted using a genomic DNA purification JetFlex kit (GenoMed GmbH, Löhne, Germany) according to the manufacturer’s instructions. The DNA sample was further purified by first extracting with phenol-chloroform-isoamyl alcohol (25:24:1) and then extracting with chloroform-isoamyl alcohol (24:1). DNA was precipitated with one-tenth volume of 3 M sodium acetate and two volumes of 100% ethanol and resuspended in Milli-Q water. NanoDrop measurements gave a concentration of 315 ng · μl⁻¹ (315 μg of DNA in total) with an A₂₆₀/A₂₈₀ of 1.79. The draft genome of P. pseudoalcaligenes AVO110 was sequenced using the Illumina HiSeq 2000 platform at BGI Tech Solutions Co., Ltd. (Hong Kong), and paired-end reads with insert sizes of 500 bp were assembled using SOAP de novo software (75 (link), 76 (link)). Statistics regarding the assembly results are summarized in Table S2. Automatic annotation of the draft genome was obtained using the RAST server (77 (link)).