Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR
Figure 6
Catalytic Activity and Oligomerization of WspR in Cells
(A) Congo Red (CR) assay monitoring WspR-catalyzed c-di-GMP production in cells. E. coli BL21 were transformed with plasmids encoding wild-type or mutant variants of WspR. Cells were grown to mid-log phase at 37 °C, and 2.5 μl of the culture was spotted onto a CR-containing LB plate with or without IPTG and incubated for 24 h at 30 °C. Leaky expression in the absence of IPTG and IPTG-induced WspR expression was visually assayed for a red colony phenotype (rdar morphotype). Cells expressing an untagged version of WspRwt behave similarly to cells expressing WspR with a C-terminal hexahistidine tag.
(B) Loss of CR staining correlates with high WspR expression levels. Cultures were grown for 16 h at 25 °C in the absence or presence of IPTG. Lysates from cells expressing hexahistidine-tagged wild-type and mutant variants of WspR (see above) were prepared by sonication and analyzed by western blotting using a hexahistidine tag-specific antibody to detect recombinant protein. Samples were normalized to total protein amount prior to SDS-PAGE and blotting. Western blot detection of the native E. coli protein LexA was used as a control. LexA levels were slightly lower in some samples due to high WspR expression levels obscuring total protein normalization.
(C) Gel filtration profile of WspR in cell lysates. Cell lysates were subjected to SEC in gel filtration buffer. Fractions (0.1 ml) were collected, and hexahistidine-tagged proteins in the fractions were detected by western blotting. Elution profiles were compared to profiles obtained for purified proteins (c-di-GMP–bound WsrRwt or nucleotide-free WspRGGAAF) under identical conditions.