2.1.3. 16S rRNA Gene Sequencing of Colonies

Colonies were identified from agar plates by partial 16S rRNA gene sequencing (universal primers [44] covering variable regions 3 and 4 in the 16S rRNA gene (V3 and V4) directly on colonies using a variation of a yeast colony PCR method as described before [45,46]. Amplification was performed using 0.25 µmol/l (each) primer, 5 µL of 5 Prime HotMastermix (AH Diagnostics, Oslo, Norway) to a total volume of 25 µL. The cycling conditions were: 94 °C 2 min, then 30 cycles of denaturing at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 7 min. The PCR products were purified before sequencing, using ExoSap-IT (USB Corp., Cleveland, OH, USA). Sequencing PCR was carried out using the Big Dye^® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Dublin, Ireland) according to the manufacturer’s instructions with the forward universal 16S rRNA gene primer. The sequencing reactions were carried out in 25 cycles of 96 °C for 15 s and 60 °C for 4 min. A BigDye XTerminator Purification Kit (Applied Biosystems) was used according to the manufacturer’s recommendations to clean up the sequencing reactions and sequencing was performed on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems). The taxonomy was identified using the RDP (Ribosomal Database Project) SeqMatch (http://rdp.cme.msu.edu/, accessed on 25 June 2014, 21 October 2015, 16 March 2016). The thresholds used in the RDP search were as follows: both type and non-type strains, both uncultured and isolates, only good sequences >1200 nt and KNN = 1.