Sanger sequencing

Nucleotide sequences were obtained from PCR products using Sanger sequencing (Beckman Coulter Genomic, Essex, UK). When PCR amplification with the external primers was successful in recovering an amount of PCR product that was sufficient for sequencing, we sequenced this longer fragment. However, when it was unsuccessful, the shorter fragment obtained from the nested PCR was used. For DNA fragments longer than 1000 bp, sequencing was performed on both strands. Base calls were checked manually in ChromasLite version 2.1, and IUPAC codes were used to indicate ambiguous states. DNA sequences were aligned using the “Clustal Omega” algorithm in SeaView version 4. The sequences analyzed in this study are available in Genbank under the accession numbers KJ832158-KJ833031.

Genomic DNA of isolates which produced clear zones of inhibition was extracted from culture cell pellets using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich; Co. Wicklow, Ireland). Extraction of DNA was confirmed by agarose gel electrophoresis and subsequently the 16S rRNA gene was amplified using the following 16S eubacterial primers CO1; 5’-AGTTTGATCCTCCTGGCTCAG-3’ and CO2; 5’-TACCTTGTTACGACTT-3’.^{55 (link)} The DNA was amplified with Invitrogen Platinum PCR Supermix (ThermoFisher Scientific, Dublin, Ireland) and PCR reactions performed on the Applied Biosystems 2720 Thermocycler (ThermoFisher Scientific, Dublin, Ireland). The amplification cycle used was as follows: 94°C for 2 min, and 30 cycles of the following: 94°C for 30 s, 50°C for 30 s and 72°C for 1.5 min. The purity and quantity of DNA present was checked on the NanoDrop 1000 (ThermoFisher Scientific, Dublin, Ireland) and the PCR product was then purified using the QIAquick™ PCR Purification Kit (Qiagen; Manchester, UK). The complete sequence of the 16S rRNA gene was determined by Sanger sequencing (Beckman Coulter, Essex, UK). The species was putatively identified by comparing the resulting sequence with deposited species in the NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with a high percentage nucleotide identity (>98%).

The Macherey-Nagel Nucleobond PC20 kit (MN, Hoerdt, France) was used to isolate fosmidic DNA using the manufacturer’s protocol. DNA was quantified using a NanoDrop N-1000 spectrophotometer (NanoDrop Technologies) and quality assessment was performed with 0.6% (w/v) ethidium bromide-stained agarose electrophoresis.
Transposon mutagenesis was carried out on fosmidic DNA using the EZ-Tn5 Insertion Kit and Transformax electro-competent E. coli EC100-T1^r cells (Epicentre Technologies, Madison, USA) following the manufacturer’s protocol. Inactivated clones for polysaccharidic activity were then identified by plating the kanamycin-resistant transposon insertion clones on LB-Cm-Kan agar plates supplemented with the appropriate polysaccharide. Sanger sequencing (Beckman Coulter Genomics, United Kingdom) was performed to localize the transposon insertion site in metagenomic insert DNA of inactivated clones, using the FP-1 and/or RP-1 primers supplied in the kit.

The yeast strains used in this study are listed in Supplemental Material, Table S2. Strains were constructed using standard techniques, and standard yeast growth medium including 2% (w/v) of the indicated carbon source (Sherman 2002 (link)). Yeast plasmids were created using the gap-repair cloning technique, which combines a linearized plasmid with PCR products using in vivo recombination. All PCR products were generated using primers from Sigma Life Science and PfuII Ultra proof reading polymerase (Agilent Technologies, UK). All plasmid constructs (listed in Table S3) were validated using Sanger sequencing (Beckman Coulter Genomics, UK). Selective ploidy ablation (SPA) screening followed the established protocol using the donor strain W8164-2B (Reid et al. 2011 (link)), and utilizing a ROTOR pinning robot (Singer Instruments, UK).