FIG 1.

Constructs and proof of principle experiments to assess the activity and stability of VirB fusion proteins. (A) Constructs producing VirB fused to superfolder GFP (sfGFP) at the N terminus using one of two linkers. (B) β-Galactosidase assay used to assess the regulatory activity of GFP-VirB fusions with either a SGGGG (49) or “12AA” linker (48) at the VirB-dependent icsP promoter (PicsP-lacZ; pAFW04a). Student’s t tests were used to measure statistical significance, *, P < 0.05. Note that β-galactosidase activities generated in the presence of VirB (pJNS04) and GFP-VirB (pJNS12) were equivalent to those achieved with native VirB levels in previous work (35). (C) Western blots to assess GFP-VirB protein stability, probed with an anti-VirB antibody (i) or anti-GFP antibody (ii). For these analyses, phenylmethylsulfonyl fluoride (PMSF) was added during sample preparation. (D) Densitometry of Western blots shown in panel C (subpanels i and ii, respectively). Lanes are labeled accordingly.