RB formation evolved through the expansion of the outer membrane and folding of the protoplasmic cylinder. (a) Stepwise demonstration of RB development using TEM images of H2O and HS RBs. Presented from left to right: parental spirochaete; spirochaete with initial membrane expansion; bleb where folding of the protoplasmic cylinder inside the outer membrane is initiated; bleb transitioning to the RB formation with folded protoplasmic cylinder under the expanded outer membrane. Bars, 500 nm. (b) Cross-sections of completed RBs and the organization of folded protoplasmic cylinder are visualized with TEM micrographs. RB is displayed from the side, from the front and from the top, respectively, from left to right. Bars, 200 nm, 200 nm and 500 nm, respectively.