Fig 3.

Candidate MOMPs localize to the detergent extractable cell envelope fraction. (A to C) Immunoblots were probed with antisera recognizing FtsH, MMAR_0752, or the HA epitope. (A) Subcellular fractions of M. marinum expressing HA-tagged versions of the control proteins and the candidate MOMPs. Total lysate (T), soluble lysate (cyt), detergent pellet (DP), and supernatant (DS) obtained after treatment of cell envelopes with PBS or 1% OBG were loaded in a 1:1:5:5:5:5 ratio. (B to C) Heat modifiability and protease accessibility of putative MOMPs. (B) Cell envelope fractions were denatured in 2% SDS at 95°C (+) or incubated in 0.2% SDS at 37°C (−) and loaded on an SDS-free PAGE gel. (C) MOMPs were treated with different concentrations of proteinase K (protK) or trypsin, without or after detergent extraction with OBG. (D) Representative Tokuyasu cryosections of M. marinum expressing MMAR_0617-HA or MMAR_5387-HA probed with HA antiserum followed by goat anti-mouse conjugate labeled with 10-nm gold particles. Scale bar is 100 nm. Particles are marked with color coding similar to that described in panel E. (E) Quantification of gold particles assigned to different subcellular compartments (see Material & Methods for definitions).