The Mla system of the diderm Firmicute Veillonella parvula reveals an ancestral envelope-spanning core for phospholipid trafficking
Despite extensive characterisation of envelope biogenesis systems in diderm (Gram-negative) bacteria, glycerophospholipid (GPL) trafficking remains poorly understood, and has only been studied in a handful of model species. In classical model diderms (Proteobacteria), the Mla system has been proposed to facilitate retrograde GPL trafficking and is composed of six proteins, MlaA-F. GPLs are extracted from the outer leaflet of the outer membrane (OM) by the lipoprotein MlaA which associates with porin trimers, then shipped through the periplasmic space by the periplasmic chaperone MlaC, which delivers GPLs to the inner membrane (IM) complex formed by MlaBDEF. Here, we investigate GPL trafficking in Veillonella parvula, a diderm member of the Firmicutes which encodes an Mla system devoid of MlaA and MlaC. V. parvula ∆mla mutants display phenotypes characteristic of disrupted lipid asymmetry such as hypervesiculation and detergent hypersensitivity. Lipid content analysis from outer membrane vesicles (OMVs) reveals an enrichment for the major lipid component phosphatidylethanolamine (PE). Interestingly, suppressor analysis identifies mutations in tamB that rescue detergent hypersensitivity of ∆mla strains, supporting the involvement of these two systems in antagonistic GPL trafficking functions across diverse bacterial lineages. A combination of structural modeling and subcellular localisation assays shows that MlaDVp is longer than in classical diderm models and forms a transenvelope bridge, encoding both an IM-localised MCE domain and an OM ß-barrel. These results strongly suggest that V. parvula possesses a minimal Mla system for GPL trafficking replacing the need for chaperones and OM lipoproteins by directly connecting the two membranes. Finally, phylogenomic analysis indicates that this MlaEFD self-contained architecture is widely distributed in diderm bacteria and most likely represents the ancestral functional core of the Mla system, which subsequently increased in complexity in Proteobacteria and closely related phyla following the emergence of MlaABC. Our work broadens the diversity of current models of GPL trafficking in diderm bacteria, challenging the paradigm set by classical models and shedding light on the evolution of a crucial system in the biogenesis and maintenance of the bacterial outer membrane.