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

Abstract

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.

The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Crosstalks with the Divisome

“…In this study, we revealed the spatiotemporal topography of the BAM complex in E. coli cells, and its dependence on the presence of the Z-ring. The BAM complex localizes all along the cell periphery in distinct foci or clusters, as has been reported before [19][20][21][22]. Here, new insight was gained into the localization of BAM, by studying its cellular distribution during the cell division cycle.…”

“…Analysis of the amount of fluorescence of the labelled BAM per cell divided by the calculated cell volume assuming the cell to have a rod-shape provides the BAM concentration per µm 3 . The BAM complex is continuously produced as its concentration remained quite constant at all cell ages when grown in minimal medium (Figure 1C), as well as in rich medium [22]. Strikingly, the fluorescence demographic map of cells sorted by their length revealed a BAM complex enriched at the site of constriction, in the second half of the cell division cycle (Figure 1B).…”

“…In order to observe the endogenous expression of the BAM complex, a polyclonal antiserum raised against the whole BamA-E complex, which recognizes all subunits (αBAM), was used for the immunolabeling of wild type E. coli K12 cells [22]. The immunofluorescence, performed on cells grown to steady state, revealed that the BAM complex displays a distribution along the cell surface in bright distinct foci (Figure 1A,B), consistent with previous studies [20][21][22]. Steady-state growth allows the correlation of cell length to cell age [41], enabling observation of the protein expression and localization over the course of the cell cycle.…”

“…Although much is known about the structure of the BAM complex, its in vivo cellular distribution has been studied only partially [20,21]. We have recently used an antiserum against the whole BAM complex to investigate BAM localization by whole-cell immunofluorescence [22] and showed that the PG layer is anchored by BAM. In the present work, we show that the BAM complex localizes in the lateral wall and at midcell in constricting cells.…”

Midcell localization of PBP4 ofEscherichia colimodulates the timing of divisome assembly

“…1C). The OM pore inserting BAM complex is also not accessible without lysozyme treatment [64]. This suggests that PBP4 resides between the OM and the PG layer.…”