Phire polymerase

PCR amplification was attempted for the two main types of autotrophic CO₂ fixation mechanisms, RubisCO (Calvin-Benson-Bassham cycle) and ATP citrate lyase (reverse TCA cycle) to determine the possible presence of different carbon fixation pathways (Table 1). Each PCR reaction contained 1 U of Phire polymerase (Thermo Fisher Scientific, Waltham, MA), 1 X PCR Buffer, 200 μM dNTP, 2 μM each primer, 5 μg BSA, and 1 ng/μl environmental cave DNA. Cycling conditions began with a 2 min hot start at 98°C, followed by 30 cycles of 98°C for 10 s, 60°C for 20 s, and 72°C for 30 s and completed with a 2 min extension at 72°C. PCR product from positive reactions were excised from a 1.5% agarose gel and purified using the GeneJET extraction kit (Thermo Fisher Scientific). Purified PCR products were then ligated into the pJET1.2 vector (Thermo Fisher Scientific) at a 3:1 ratio and transformed into chemically competent cells (Active Motif, Carlsbad, CA). Both strands of inserts from 48 clones were sequenced from each clone library using primers Pjet1.2F and Pjet1.2R. The sequences were then translated and aligned with representative sequences from described isolates and uncultured clones. Phylogenetic trees were calculated using RaxML v7.0.3 (Stamatakis, 2006 (link)) using the Whelan and Goldman model of amino acid substitution (Whelan and Goldman, 2001 (link)).

All PCR reactions were performed using Phire polymerase (Thermo Fischer) according to the manufacturer’s manual. All primers are listed in Table 2 and were obtained from Sigma-Aldrich. PCR reactions were visualized and analyzed by gel electrophoresis on 1% (w/v) TBE agarose gels containing 5 mg L^–1 ethidium bromide in an electric field (110 V, 0.5× TBE running buffer).

To sequence the farA gene in JR11.1 derived mutants, the farA gene was amplified using Phire polymerase (high fidelity DNA polymerase, Thermo Fisher Scientific, Carlsbad, CA, USA) from genomic DNA of the respective mutant using primers FarAP1 and FarAP18 (Supplementary Table 1). The amplified 5.6- kb farA gene including ~1-kb flanking regions was sequenced using primers listed in Supplementary Table 1. Sequence reads were aligned to the farA gene of NRRL3 retrieved from gb.fungalgenomics.ca/portal/and analyzed in DNAman.

All PCRs for analysing the presence of insertion sequences were performed using Phire polymerase (ThermoFisher, Waltham, MA, USA), and for sequence analysis, Phusion polymerase (ThermoFisher, Waltham, MA, USA) was used according to the manufacturers’ manual. All primers were purchased from Sigma‐Aldrich (St. Louis, MO, USA) and are listed in Table S1. All PCRs were analysed by size specific gel electrophoresis on 1% (w/v) TAE agarose gels containing 5 μl ethidium bromide per 100 ml agarose solution in an electric field (90V, 0.5× TAE running buffer). The PCR fragments were isolated from the agarose gel using the High Pure PCR Preparation Kit (ThermoFisher) and were either cloned into pJET1.2 (ThermoFisher) before sequencing or directly sequenced at Macrogen (Amsterdam, the Netherlands). P_up, P_{up_soxR} and P_down sequences were amplified by nested PCR using primers listed in Table S1 from gDNA of P. putida S12. These sequences were subsequently cloned into mini‐Tn7 probe vector using PacI and AvrII restriction sites (Zobel et al., 2015), and integration into the genome of S12 was verified by PCR. The promoter activities were calculated based on the GFP intensity measurements during the growth at the log phase using the biolector (m2p‐labs), and induction of the promoters was done with H₂O₂ at early exponential phase.