Extended Data Fig. 5: OmpM1 is located in the outer membrane of V. parvula.
Comparison of WT SKV38 strain of V. parvula containing pRPF185 (empty vector), pJW34, a vector expressing untagged OmpM1, and pJW35, a vector expressing HA-tagged OmpM1 both under the control of tet promoter. All cultures were induced overnight with 250 µg.l–1 anhydrotetracycline. In (a), we used an anti HA primary and antibodies coupled to horse radish peroxidase (HRP) to detect the expression of OmpM1-HA (arrow) from pJW35 by Western blot. M corresponds to SeeBlue Plus Prestained Protein Standard (Invitrogen). In (b), we performed immunofluorescence microscopy of WT cells expressing either OmpM1-HA (pJW35) or native OmpM1 (pJW34) using an anti-HA antibody coupled to fluorescein. Cells were non-permeabilized. While native OmpM1 was not detectable, OmpM1-HA that contains the HA tag in one of the predicted outer loops of its beta-barrel could be detected as all around the cell, confirming that it located in the outer membrane and is surface exposed. Surface exposition of tagged OmpM1 expressed from pJW35 was confirmed using Scanning Electron Microscopy in (c). We used anti-HA primary antibodies and secondary antibodies coupled to 20 nm gold particles on non-permeabilized cells. Images were obtained using a JEOL JSM 6700 F field emission scanning electron microscope and with a backscattered electrons detector (BSE) to detect the gold labelling and with a secondary electron detector (SEI) to image the surface of the sample. The acquisitions were performed once.
