Coordinated Membrane Deformation Driven by a Minimal Set of Spiroplasma MreB Isoforms

Abstract Spiroplasma are wall-less helical bacteria that swim by switching the handedness of their helices. MreB, a bacterial actin, is widely conserved among bacteria and typically functions in cell shape maintenance and cell wall synthesis. Like other members of the Mollicutes, Spiroplasma eriocheiris lacks a cell wall but encodes five MreB isoforms (SpeMreBs). Swimming requires a pair of isoforms, SpeMreB5 together with SpeMreB4 (or the related isoform SpeMreB1) (Kiyama et al. Sci. Adv. 2022). Yet how these MreBs generate force and membrane deformation remains unclear. To examine the membrane-deforming activities of SpeMreBs, we employed two complementary liposome-based reconstitution systems using lysates from the synthetic bacterium JCVI-syn3B expressing SpeMreBs and, separately, purified SpeMreBs expressed in Escherichia coli. Lysates of syn3B expressing SpeMreB5 deformed liposomes in a concentration-dependent manner. In contrast, lysates co-expressing both SpeMreB5 and SpeMreB4 showed reduced membrane deformation at higher concentrations, suggesting that SpeMreB4 suppresses membrane deformation driven by SpeMreB5. Deformed liposomes exhibited either fluctuating or stable behaviors. ATP depletion changed both the frequency and behavior of deformation, indicating that membrane deformation depends on the nucleotide state of SpeMreBs. Reconstitution with purified SpeMreBs confirmed that SpeMreB5 possesses an intrinsic membrane-deforming activity, whereas SpeMreB1, a member of the same class as SpeMreB4, suppresses deformation. These results suggest that membrane shape in Spiroplasma is determined by antagonistic interactions among SpeMreB isoforms and their nucleotide-dependent assembly states.