BamA self-insertion drives inner-to-outer membrane remodelling in diderm Firmicutes

Exclusive to Firmicutes, endospore formation is a complex process that results in the release of a mature spore after the mother cell lyses. The spore is protected by multiple layers, including inner and outer spore membranes (IsM and OsM), both originating from the mother cell's inner membrane (IM). While well understood in monoderm bacteria like Bacillus subtilis, less is known about diderm sporulators such as Acetonema longum, which retain and remodel their OsM into a functional outer membrane (OM) during germination. Here, we demonstrate that outer membrane proteins (OMPs), including the essential BAM complex component BamA and the LPS translocon protein LptD, are present in vegetative and germinating cells but absent from mature spores, indicating that OM biogenesis occurs without pre-existing OMPs. Growth-stage proteomics and expression profiling identify two previously uncharacterized proteins, SonA and SonB, co-expressed with BamA and in a conserved operon among diderm Firmicutes. SonA is a ß-barrel OMP with three POTRA domains, while SonB is a predicted OM lipoprotein. In vitro, BamA and SonA spontaneously fold and insert into liposomes, supporting a model where BamA and/or SonA initiate self-insertion into the OsM, driving its stepwise transformation into a fully functional OM. Overall design: RNAseq profiling of Acetonema longum APO-1 (DSM 6540) across discrete stages of the sporulation cycle: vegetative growth (3 day subcultures in exponential phase), sporulation (5 day subcultures in stationary phase) and germination (pure germinating spores at 16 hours post-inoculation).