A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria

Abstract

Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM–PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans . Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

Interdigitated immunoglobulin arrays form the hyperstable surface layer of the extremophilic bacterium Deinococcus radiodurans

“…The analysis of cryo-ET data of FIB-milled cells further show that HPI is the sole protein making up the S-layer. We show that no densities are present for the highly abundant OM SlpA protein (37), which belongs to the OmpM superfamily of PG-OM tethering proteins (38), in line with previous reports (8, 9, 11–13, 20–22). Further, our data from FIB-milled cells indicates that the S-layer is positioned ∼18 nm away from the OM, in close agreement with recent cryo-EM studies (10, 37).…”

“…We show that no densities are present for the highly abundant OM SlpA protein (37), which belongs to the OmpM superfamily of PG-OM tethering proteins (38), in line with previous reports (8, 9, 11–13, 20–22). Further, our data from FIB-milled cells indicates that the S-layer is positioned ∼18 nm away from the OM, in close agreement with recent cryo-EM studies (10, 37). Our atomic structure of the S-layer and the fit of this structure into the subtomogram averaging map of the cellular S-layer show that this lattice coats D. radiodurans cells as a highly interconnected sheet with exceptional stability (Figure 5).…”

“…Although limited in resolution, our subtomogram averaging agrees with the atomic structure of the HPI S-layer, showing that both are organized in a hexagonal array with the same lattice spacing (Figure 4B-C). The S-layer is seen as large patches coating the cell surface, punctuated with discontinuities, as reported previously (10,37). The multilayered arrangement of the S-layer was also confirmed by our subtomogram average, with densities for both the canopy (HPI3-7) and the base (HPI2) of the S-layer observed on cells (Figure 4C).…”

“…HPI3-7 form the S-layer lattice farthest away from the cell, while HPI1-2 are closer to and probably tethered to the OM. An abundant protein called SlpA protein is buried in the OM, connecting it to the PG layer via long coiled coils and an N-terminal SLH domain, as shown previously (37, 39). Our results place previous studies into context, and our updated model of the D. radiodurans cell envelope will serve as a framework for understanding the cell surface organization of phylogenetically deep-branching bacteria.…”

“…The cell envelope of D. radiodurans has been studied for over five decades because of its hyperstability and uniqueness compared to other bacteria (17, 22, 37, 39). Numerous studies have employed a variety of approaches to obtain structural and molecular information on the D. radiodurans S-layer, its composition (13, 20), symmetry (9, 17, 22), and overall arrangement (11, 12).…”