Reporter Assays in E. coli and P. aeruginosa

For reporter assay in E. coli, lacZ fusions were constructed as described (Cheng et al., 2008 (link)). Briefly, DNA fragments were amplified by PCR using specific primers (Table S3) and inserted upstream of the promoter-less lacZ gene in pACYC184 (New England Biolabs, MA, USA). BL21(DE3) strain containing pET-28a(+) encoding rRruR, or pET-28a(+) alone (negative control) was transformed with the lacZ fusion constructs. After inducing the recombinant protein with 0.1 mM IPTG at 37°C for 2 h, β-galactosidase activity was measured as described previously (Wang et al., 2007 (link)). Recombinant protein expression was confirmed by Western blot analysis using the anti-His-tag antibody (Sigma, MO, USA).
For reporter assay in P. aeruginosa, DNA fragment was amplified by PCR with specific primers (Table S3) and inserted upstream of the promoter-less lacZ gene in pDN19lacZΩ. P. aeruginosa strains were transformed with the lacZ fusion construct or pDN19lacZΩ vector (negative control). β-galactosidase activity was measured as described (Weng et al., 2016 (link)).