Cultures inoculated from single colonies (2 ml, three replicates; strains IE01 and DB01) were grown overnight in LB medium at 37 °C and 170 rpm. Cultures were then diluted 1:200 in M9 minimal medium, and were grown for 2 h at the same conditions. Cells were pelleted by centrifuging 2 min at 13,000 rpm, and resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Cells were broken with phenol-chloroform and vortexing thoroughly, recovering the RNA in the aqueous phase. Samples were then passed through a silica-based, DNA-clean column (Zymo), and were eluted in TE buffer with application of DNase I (Fermentas). Total RNA eluted was quantified in a NanoDrop.
One-step SYBR PrimerScript RT-PCR Kit II (Takara) was used for detection, following the Kit protocol for preparing the reaction volumes. 16S ribosomal RNA (rRNA) was used as housekeeping gene to normalize RNA quantity in each reaction. The primer sequences for amplifying marA and 16S rRNA were taken from previous work.53 (link),54 (link) Reactions in triplicate were carried out using a Step One Plus Real-Time PCR System (Applied Biosystems). The thermal cycling program for amplification was 5 min at 42 °C, 10 s at 95 °C, and 40 cycles of 5 s at 95 °C and 34 s at 60 °C (Shuttle PCR), followed by default melting curves.
One-step SYBR PrimerScript RT-PCR Kit II (Takara) was used for detection, following the Kit protocol for preparing the reaction volumes. 16S ribosomal RNA (rRNA) was used as housekeeping gene to normalize RNA quantity in each reaction. The primer sequences for amplifying marA and 16S rRNA were taken from previous work.53 (link),54 (link) Reactions in triplicate were carried out using a Step One Plus Real-Time PCR System (Applied Biosystems). The thermal cycling program for amplification was 5 min at 42 °C, 10 s at 95 °C, and 40 cycles of 5 s at 95 °C and 34 s at 60 °C (Shuttle PCR), followed by default melting curves.
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