Fig. 2

Identification of genome-wide binding sites of WatR. A Binding consensus sequence of WatR, discovered by ChIP-seq analysis. B Confirmation of WatR binding to the promoters of four selected target genes identified by ChIP-seq analysis. Binding of WatR-Flag to the target promoters in JHM80WF strain was detected by ChIP with anti-Flag antibody and indicated as fold enrichment relative to the binding to a negative control (glgA ORF). Each value represents the average ± standard deviations from three independent experiments. C Expression levels of watR operon genes (watR and smtM) in wild-type and JHM80 strains. The mRNA levels detected by qRT-PCR were normalized to that of mxaF gene and indicated as relative expression levels compared with those of wild type. The glgA gene was used as a negative control. Each value represents the average ± standard deviations from two independent experiments. Significant difference from wild-type strain is shown as *p < 0.1; **p < 0.05. D Binding of WatR to the watR promoter detected through in vitro EMSA assay. EMSA assay was performed by incubating GST-WatR protein with biotin-labeled watR promoter probes with or without TT deletion. WatR-binding sites (arrows), a putative -10 box, and transcription start site (TSS) are indicated. The deleted TT nucleotides in JHM80 are shown in red. E Autoregulation of watR expression. In wild type, WatR binds to its own promoter, repressing the expression. In the JHM80 strain, the TT deletion in the promoter prevents WatR binding, resulting in derepression of the operon. F Repression of gltA1 by WatR. Expression levels of gltA1 in the wild-type, JHM80, and JHM80 ΔwatR (JHM82) strains were detected by qRT-PCR. The relative mRNA levels are indicated compared with those of JHM80. Each value represents the average ± standard deviations from two independent experiments