qRT-PCR

cDNAs were synthesized by reverse transcription using a PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Bio). Total RNA (1 µg) was reverse transcribed using PrimeScript reverse transcriptase with random primers (6-mers). For each sample, a reverse transcriptase negative control was also prepared to verify no genomic DNA contamination had occurred. The synthesized cDNA was purified using a NucleoSpin Gel and PCR Clean-up Kit (Takara Bio) and eluted with 30 µl 5 mM Tris-HCl buffer (pH 8.5). qRT-PCR analyses were performed as previously described³³, with specific primers listed in Table ^S4. For amplification of ligM, desA, and desR, specific primers were designed to amplify 5′-regions of these genes that remained in SME021, SME021, and SME047, respectively. The amounts (mol/µg total RNA) of each mRNA and 16S rRNA were measured using standard DNAs. To normalize the amount of mRNA in each sample, 16S rRNA was used as an internal standard (Table ^S5).