Dntp mixture

Total RNA was isolated from HH10 chick embryos using a Trizol kit (Invitrogen, USA) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized to a final volume of 25 μl using SuperScript RIII first-strand (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed as described previously^{29 (link),30 (link)}. The sets of primers used for RT-PCR are described in the Supplementary Fig. 1. The PCR reactions were performed in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The final reaction volume was 50 μl, comprising 1 μl of first-strand cDNA, 25 μM forward primer, 25 μM reverse primer, 10 μl PrimeSTARTM Buffer (Mg2+ plus), 4 μl dNTP Mixture (TaKaRa, Japan), 0.5 μl PrimeSTARTM HS DNA Polymerase (2.5 U/μl TaKaRa, Japan) and RNase-free water. cDNA was amplified for 30 cycles. One round of amplification was performed at 94 °C for 30 s and then 30 s at 58 °C and 30 s at 72 °C. The PCR products (20 μl) were resolved using 1% agarose gels (Biowest, Spain) in 1× TAE buffer (0.04 M Trisacetate and 0.001 M EDTA) and 10,000× GeneGreen Nucleic Acid Dye (TIANGEN, China) solution. The resolved products were visualized using a transilluminator (SYNGENE, UK), and photographs captured using a computer-assisted gel documentation system (SYNGENE). Each of these experiments was replicated at least three times.

Plasmid DNA from the P. ethanolidurans CUPV141 strain was used to determine the 1,704 bp nucleotide sequence of the gtf gene by the dideoxy method at Secugen as previously described for the gtf gene of P. parvulus strains (Garai-Ibabe et al., 2010b (link)).
For detection of the priming-GTF coding gene (p-gtf), degenerate primers (Provencher et al., 2003 (link)) and genomic DNA from the two P. ethanolidurans strains were used for DNA amplification. The 20 μL reaction mixtures for each sample contained: 1 U of Taq DNA polymerase (Sigma), 1X PCR Buffer (Sigma), 2.5 mM MgCl₂ (Sigma), 0.1 mM dNTP mixture (TaKaRa), 6.25 mM of each primer, and 200 ng of total template DNA. Conditions for the PCR were as follows. First, an incubation at 94°C for 9 min. Then, 5 cycles at 94°C for 30 s, 62°C for 31 s and 72°C for 32 s. Finally, 40 cycles at 94°C for 30 s, 52°C for 31 s, and 72°C for 32 s.
After fractionation in 2.5% agarose gels, the amplicons were purified using the ‘NucleoSpin^® Gel and PCR Clean-up’ kit, according to the manufacturer’s instructions, and the nucleotide sequence determined at Secugen.

Genomic DNA was extracted from B. bronchiseptica HH0809 through a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) and was used as the template DNA for PCR assays. The upstream and downstream fragments of the bscN gene was amplified by PCR assays performed in a 50 µL reaction volume containing 5 µL of 10 × PCR Buffer (TAKARA, Kyoto, Japan), 4 µL of dNTP mixture (TAKARA, Japan), 4 µL of DMSO, 2 µL of template DNA, 0.5 µL of r-Taq polymerase (TAKARA, Japan), 30.5 µL of nuclease-free water (TAKARA, Kyoto, Japan), and 2 µL each of the forward primers and reverse primers listed in Table 1. The cycling conditions were 95 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min, with a final extension 72 °C for 10 min. PCR amplification of fragments were displayed by electrophoresis on a 1% agarose gel and then purified by using a TIANgel Midi Purification Kit (TIANGEN, Beijing, China). The upstream and downstream fragments of bscN were cloned into pBluescript II KS/SK (+) to generate an intermediary plasmid pSKΔbscN, double digested by KpnI/SacI. Finally, the fragment containing the upstream and downstream fragments of bscN from pSKΔbscN was cloned into the suicide plasmid pRE112 (CmR) to generate the transforming plasmid pRE∆bscN, which was then transformed into E. coli X7213 for the conjugation experiment.