Spatiotemporal organization of BamA governs the pattern of outer membrane protein distribution in growingEscherichia colicells

Abstract

The outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on β-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the β-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/μm2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth.SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the β-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cell’s growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.

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

“…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.…”

“…In conclusion, the topography of BAM in the cells is specific, as other OM proteins such as NlpI and OmpA clearly have a different distribution. OMPs are reported to be inserted by BAM and therefore likely to be also present at midcell, as was reported previously [21]. To verify whether this is indeed the case, the OM β-barrel protein OmpA was immunolabelled.…”

“…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.…”

“…Aztreonam inhibits PBP3, thus blocking septal PG biogenesis, without affecting divisome assembly [44,45]. Importantly, it was already shown that the aztreonam does not affect the overall level of BAM in the OM [21]. Incubating the bacteria with 1 µg•mL −1 of aztreonam, for 1, 2 or 3 generations, causes the cells to elongate without division.…”

“…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.…”