FIG 1.

Functional characterization of SaraC. (a) Phylogenetic analysis of the SaraC homologous proteins. A phylogenetic tree was generated using the maximum likelihood method in MEGA X. Bootstrap values based on 1000 replicates are shown at the branching points. (b) SDS-PAGE analysis of the heterologous expression and purification of SaraC. Lane M: protein molecular weight marker; lane 1: soluble protein; lane 2: cell extract of total protein after induction with 0.1 mM IPTG at 16°C for 28 h; lane 3: purified fusion SaraC. (c) SPR analysis of the interaction of oligonucleotide (Oligo1309, Oligo5564, and Oligo7291 as analytes) with SaraC protein (activated with S-adenosylmethionine, as a capture). Different-colored traces indicate increasing analyte concentrations (ranging from 0.25–4 μM). (d) Chromatogram profiles of subtracts and products of oligonucleotides. In Oligo7291 reaction system, SIM1338.2386 shows the specific ion of Oligo7291 SIM peak at m/z 1338.2 [M–2H]^2–; SIM1331.2339 shows the specific ion of product SIM peak at m/z 1331.2339 [M–2H]^2–. SIM1609.5300 and SIM1392.2377 are the chromatograms of the Oligo 1309 and Oligo 5564 substrates, respectively; and the below chromatograms are the profiles of the corresponding de-monomethyl and de-dimethyl products, respectively. (e) PCR confirmation of the 2 transformants. Lane M, DL2000 DNA marker; Lanes 1 and 2: plasmid and WT as positive and negative controls, respectively; Lanes 3 and 4: the two transformants SA4 and SA6, respectively. (f) Relative transcription levels of SaraC quantified using RT-qPCR at 10 and 15 days. CK (β-tubulin) was used as the standard for statistical analysis of the transcription level of SaraC in the transformants relative to that in the WT strain under a given condition; error bars indicate the standard deviations.