Multiplex RT-PCR and sequencing of pilY1, pilW, and pilV

All primers for RT-PCR and alignment analysis were designed using the At. thiooxidans ATCC 19377 genome (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AFOH01000001","term_id":"348617446"}}AFOH01000001) originally described by Valdés et al. [7]. Accordingly, derived annotated protein sequences for PilY1, PilV and PilW were also taken from GenBank under the accession numbers {"type":"entrez-protein","attrs":{"text":"WP_010638975.1","term_id":"498324819"}}WP_010638975.1, WP_010638981.1 and {"type":"entrez-protein","attrs":{"text":"WP_010638979.1","term_id":"498324823"}}WP_010638979.1, respectively. Likewise, derived-specific gene sequences for pilY1 (ATHIO_RS0106065), pilW (ATHIO_RS0106075) and pilV (ATHIO_RS0106080) were also taken from the GenBank database. Primers were designed using the free internet-based software Primer3 [31] and confirmed using Vector NTI (InforMax-Invitrogen, USA). As a positive control for each RT-PCR reaction, a specific pair of primers to amplify the expression of the 16S rRNA ({"type":"entrez-nucleotide","attrs":{"text":"AJ459803","term_id":"20799098"}}AJ459803) was designed.

Total RNA was extracted from 5, 15 and 21 days cultures. Briefly, cells were collected by centrifugation at 0.08 x g for 1 minute to separate the cells from S⁰. Cells were then concentrated and washed by centrifugation at 21.1 x g for 1 min using saline phosphate buffer (PBS; in g/L: 8 NaCl, 1.44 Na₂HPO₄, 0.24 KH₂PO₄ (J.T. Baker, USA), and 0.2 KCl (Sigma-Aldrich, USA); pH 7.4).

RNA extraction was performed using TRIzol reagent (Invitrogen, USA) according the manufacturer's protocol. After quantification using a NanoDrop 2000c (Thermo Scientific, USA), cDNA synthesis was performed according to the manufacturer’s recommendation, using 200U M-MLV (Invitrogen, USA) in a 20 μL total volume reaction. The amplification products were purified using the QIAquick kit (Qiagen, USA) following the recommended procedure, quantified and sequenced. The products were sequenced by the Sanger method at LANBAMA (National Laboratory of Biotechnology, San Luis Potosí, Mexico) and LANGEBIO (National Laboratory of Genomic for the Biodiversity, Guanajuato, México) facilities. Each sequence was confirmed at least five times by analyzing amplification products obtained from different culture replicates. The generated sequences were compared with the annotated sequences of each corresponding gene using the Multalin software [32].