Genelute dna extraction kit

DNA was extracted with the GenElute DNA extraction kit (Sigma-Aldrich, Milan, Italy). Sequence libraries were generated from extracted DNA as reported previously9 (link). Genomes were sequenced to high depth on the IlluminaMiSeq platform. Resulting reads were adapter trimmed with Trimmomatic26 (link), error corrected with Hammer27 (link), and assembled with SPAdes v3.128 (link). The read coverage across each contig was evaluated, and contigs of an anomalous coverage, due to read crossover in multiplexed runs, were manually removed. The assembly stats for each genome are shown in Supplementary Table S1. All assemblies and raw reads were deposited in public databases (accession numbers in Supplementary Table S1). Annotation was performed with the NCBI PGAP pipeline.

Paenarthrobacter nitrojuajacolis LG01 and Paenarthrobacter histidinolovorans LG02 were isolated from enriched culture, obtained from granular soil sampled from the A horizon, Royal Park (Melbourne, Australia). There was no apparent fire history of the sampled soil.
Approximately 1 g of soil samples were suspended in 5 mL of sterilized LG growth media containing 5 mM of LG as sole carbon source. The culture was incubated at 28 ℃ for 5 days with continuous shaking at 250 rpm. A subsample of 50 µL was supplemented into freshly prepared levoglucosan media and grown for a further 3 days. This step was repeated 5 times and after the 5th transfer, cells were plated on LB agar plates consisting of 10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast, 15 g/L agar and incubated overnight at 28 ℃ in the dark. Single colonies were picked and used to inoculate 2 mL of fresh LG growth media and incubated at 28 ℃ with continuous shaking at 250 rpm. Then, cells were plated and re-inoculated into fresh liquid LG growth media 2 more times. Stock cultures were prepared by addition of 10% glycerol and stored at − 80 ℃. Genomic DNA of LG degrading species was extracted using the GenElute DNA extraction kit (Sigma) with inclusion of lysozyme and RNAase.

DNA was extracted from the saliva samples by means of the Genelute DNA extraction kit (Sigma-Aldrich), and 16S rRNA genes were amplified by polymerase chain reaction (PCR) with primers 27F (with the YM modification) and 519R.^{22 (link), 23} The primers incorporated a unique barcode and Roche 454 adapters. Polymerase chain reaction amplicons were purified, sized, quantified, and pooled in equimolar proportions. Emulsion PCR and unidirectional sequencing of the libraries were performed using the Lib-L kit and Roche 454 GS-FLX Titanium sequencer.