Bacterial two-hybrid assays map the interacting regions of OutC, OutL and OutM.

(A), the fusions of T18(T25) to either the full-length proteins (top left), or the transmembrane segments (top right), or the periplasmic regions (bottom) were generated and used in two-hybrid assays as shown here. The fusions of the full-length proteins (B), the periplasmic regions (C and D) or the transmembrane segments (F) of OutC, OutL and OutM were co-expressed in E. coli DHP1 cyaA strain. The values of β-galactosidase observed with each pair (T18-fusion/T25-fusion) are expressed as percentage of that with the full-length OutC/OutC, considered as 100% and indicated as C/C (B and C) or *Cf/Cf (F). In (F), the triple T18(T25)-TMS-BlaM fusions are denoted as C, L and M, for an easy reading. All assays were performed from triplicate culture on three to four different transformants, standard deviations are indicated. (E), to check the integrity of the triple T25-TMS-BlaM fusions, E. coli cells expressing these fusions (indicated at the top) were probed by immunoblotting with BlaM-antibodies (left panel). To assess the correct insertion of these fusions into the inner membrane, as shown in (A), E. coli cells expressing the fusions were converted into spheroplasts and treated with trypsin for the indicated time periods (right panel). In contrast to the T25-BlaM fusion that remained in the cytoplasm, the triple T25-TMS-BlaM fusions were exposed in the periplasm since they were degraded by trypsin.