Fig. 5.

Dual membrane-spanning 1 (Dma1) has the properties to span both the inner and outer membranes of a gram-negative bacterium. (A) AlphaFold predicted stucture of Dma1. (B) Proposed orientation of Dma1 in the membrane of Bt (Created with https://BioRender.com). (C) Western blots were performed using anti-polyHis against cells constitutively expressing either 1. Dma1 and Das1 together, 2. Das1 alone, and 3. empty vector control. Bands adjacent to stars (*) are nonspecific bands. This demonstrates that full-length Dma1 is present in WCs, and the state of the protein is growth-phase dependent. (D) Western blots of WCs, TMs, and OMVs collected from Bt strain constitutively expressing Dma1 containing a C-terminal His tag and an N-terminal Flag tag (Flag-Dma1-His). The green channel is anti-polyHis, while the red channel is anti-Flag. Full-length Dma1 is present solely in the WC and TM fraction, while only C-terminal fragments are detected in the OMVs.