FIG 3.

Assembly of the Lpt complex by Pa-LptC and LptC chimeras. Dodecyl β-d-maltoside-solubilized total membranes from AMM04 strains harboring pGS100 derivatives expressing the His-tagged proteins of interest were affinity purified using a Talon metal affinity resin, as described in Materials and Methods. Proteins were then fractionated by SDS-PAGE and immunoblotted with suitable antibodies to detect the corresponding proteins in the fraction. (A) Assembly of the Lpt IM complex. Samples were prepared from AMM04 harboring the pGS100 derivatives pGS108 (expressing Ec-LptC-H, CCC), pGS200 (expressing Pa-LptC-H, PPP), or those expressing His-tagged LptC chimeras, namely, pGS201H (CPP-H), pGS202H (PCC-H), pGS203H (CPC-H), pGS204H (PCP-H), pGS206H (CCP-H), and pGS207H (PPC-H). Immunoblotting was performed with anti-LptF and anti-His antibodies to detect LptF and the different His-tagged LptC forms, respectively, which display different electrophoretic mobility on SDS-PAGE. (B) Assembly of the transenvelope Lpt complex. Samples were prepared from AM604 harboring pGS108 (Ec-LptC-H, CCC), pGS200 (Pa-LptC-H, PPP), the His-tagged LptC CCP chimera (pGS206H), or pGS100 expressing the His tag (−) as a negative control. Immunoblotting was performed with the antibodies anti-His (to detect the different LptC forms) and anti-LptA, anti-LptB, anti-LptF, and anti-LptD.