After transport across the cytoplasmic membrane, bacterial outer membrane proteins are assembled into the outer membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various outer membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of outer membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in outer membrane protein assembly.

The cell envelope of gram-negative bacteria consists of two membranes, the inner and the outer membrane, that are separated by the periplasm. The outer membrane consists of phospholipids, lipopolysaccharides, integral membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner membrane and have to be transported across the inner membrane and through the periplasm to assemble eventually in the correct membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.

Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Lipopolysaccharide (LPS), also known as endotoxin due to its severe pathophysiological effects in infected subjects, is an essential component of the outer membrane (OM) of most Gramnegative bacteria. LPS is synthesized in the bacterial inner membrane, a process that is now well understood. In contrast, the mechanism of its transport to the outer leaflet of the OM has remained enigmatic. We demonstrate here that the OM protein, known as increased membrane permeability (Imp) or organic solvent tolerance protein, is involved in this process. An Impdeficient mutant of Neisseria meningitidis was viable and produced severely reduced amounts of LPS. The limited amount of LPS that was still produced was not accessible to LPS-modifying enzymes expressed in the OM or added to the extracellular medium. We conclude therefore that Imp mediates the transport of LPS to the cell surface. The role of Imp in LPS biogenesis and its high conservation among Gram-negative bacteria make it an excellent target for the development of novel antibacterial compounds. G ram-negative bacteria are enclosed by a cell envelope consisting of an inner membrane (IM) and an outer membrane (OM), separated by the periplasm. The IM is a phospholipid bilayer, whereas the OM is an asymmetrical bilayer, containing phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS consists of a hydrophobic membrane anchor, lipid A, substituted with a nonrepeating oligosaccharide, the core region. In many bacteria, the core region is extended with the O antigen, a repeating oligosaccharide. The lipid A-core region and the O antigen are synthesized as separate units at the cytoplasmic leaflet of the IM. Almost all of the enzymes involved in their biosynthesis have been identified in Escherichia coli (1, 2). The transport of the lipid A-core moiety to the periplasmic side of the IM is mediated by the MsbA protein, an ATP-binding cassette family transporter (3), whereas flipping of O antigen units over the IM can be facilitated by several distinct mechanisms (1). At the periplasmic side of the IM, the O antigen is ligated to the lipid A-core region. The next step, transport of the fully assembled LPS through the periplasm and across the OM, remains an entirely elusive aspect of LPS biogenesis (1, 2). Recently, Omp85, an essential OMP, was suggested to be involved in this process (4). However, we found a severe OMP assembly defect in a Neisserial Omp85 mutant (5). This phenotype, together with the affinity of Omp85 for OMPs (5) and the presence of omp85 homologs in Gram-negative bacteria lacking LPS, are much more consistent with a role of this protein in OMP assembly, with only an indirect role in LPS transport. Braun and Silhavy (6) identified another essential OMP in E. coli, depletion of which resulted in the formation of aberrant membranes. Missense mutations in the gene encoding this 87-kDa OMP, called increased membrane permeability (Imp) or organic solvent tolerance protein, were already known to affect OM permeability (7,8). He...

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts harbor ␤-barrel proteins. The signals that allow precursors of such proteins to be targeted to mitochondria were not characterized so far. To better understand the mechanism by which ␤-barrel precursor proteins are recognized and sorted within eukaryotic cells, we expressed the bacterial ␤-barrel proteins PhoE, OmpA, Omp85, and OmpC in Saccharomyces cerevisiae and demonstrated that they were imported into mitochondria. A detailed investigation of the import pathway of PhoE revealed that it is shared with mitochondrial ␤-barrel proteins. PhoE interacts initially with surface import receptors, and its further sorting depends on components of the TOB/SAM complex. The bacterial Omp85 and PhoE integrated into the mitochondrial outer membrane as nativelike oligomers. For the latter protein this assembly depended on the C-terminal Phe residue, which is important also for the correct assembly of PhoE into the bacterial outer membrane. Collectively, it appears that mitochondrial ␤-barrel proteins have not evolved eukaryotic-specific signals to ensure their import into mitochondria. Furthermore, the signal for assembly of ␤-barrel proteins into the bacterial outer membrane is functional in mitochondria.outer membrane ͉ PhoE ͉ protein import ͉ TOB complex ͉ TOM complex

A peek inside: Dynamic nuclear polarization (DNP) enhances the spectroscopic sensitivity of solid‐state NMR measurements of uniformly (13C,15N)‐labeled preparations of Escherichia coli cells by more than an order of magnitude (see picture; MW=microwaves, ε=enhancement factor). The major molecular components in the cells can be characterized in this way.