Figure 6.

NepR2 is a negative feedback regulator of the GSR. (A) β-Galactosidase activity of the EcfG-dependent nhaA2p-lacZ reporter in S. melonis Fr1 wild type (WT) and a ΔnepR2 mutant in low stress TYE medium after overnight overexpression of NepR2 from the vanillate-inducible pVH vector with 250 µM vanillate. pVH was used as empty vector control. Values are given as mean ± SD of three independent experiments. (B) Salt sensitivity assay with S. melonis Fr1 wild type (WT), a ΔecfG, a ΔnepR2 mutant, and the latter overexpressing NepR2 from the vanillate-inducible pVH vector with 250 µM vanillate. pVH was used as an empty vector control. The respective strains were grown in NB medium. OD₆₀₀ was normalized to 1 prior to spotting 10-fold serial dilutions of each culture on NB agar plates with or without 300 mM sodium chloride. The pictures were taken 2-8 days after incubation at 28 °C and are representative of three independent biological replicates. (C) Co-immunoprecipitation using lysates originating from wild type either overexpressing NepR-Flag or NepR2-Flag from pVH or carrying the corresponding empty vector control. To allow binding of the Flag-tagged proteins, the lysates were incubated with the ANTI-FLAG M2 affinity gel. Samples were taken prior to incubation with the resin (sample I), from the supernatant after incubation (sample II), from the last wash of the resin (sample III), and from the ANTI-FLAG M2 eluate (sample IV) prior to analysis by non-reducing SDS-PAGE and Western blot analysis with a PhyR-antiserum (exposure time: 10 s) as well as an anti-Flag antibody (exposure time: 7 s). Results are representative of three independent biological replicates. Full-length blots are shown in Fig. S6.