The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Anchors the Peptidoglycan Layer by Spanning It with All Subunits.

Gram-negative bacteria possess a three-layered envelope composed of an inner membrane, surrounded by a peptidoglycan (PG) layer, enclosed by an outer membrane. The envelope ensures protection against diverse hostile milieus and offers an effective barrier against antibiotics. The layers are connected to each other through many protein interactions. Bacteria evolved sophisticated machineries that maintain the integrity and the functionality of each layer. The β-barrel assembly machinery (BAM), for example, is responsible for the insertion of the outer membrane integral proteins including the lipopolysaccharide transport machinery protein LptD. Labelling bacterial cells with BAM-specific fluorescent antibodies revealed the spatial arrangement between the machinery and the PG layer. The antibody detection of each BAM subunit required the enzymatic digestion of the PG layer. Enhancing the spacing between the outer membrane and PG does not abolish this prerequisite. This suggests that BAM locally sets the distance between OM and the PG layer. Our results shed new light on the local organization of the envelope.