Martine P. Bos

Assembly factor Omp85 recognizes its outer membrane protein substrates by a species-specific C-terminal motif.

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.

Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization†

Solid-state NMR (ssNMR) spectroscopy offers increasing possibilities to study complex biomolecules at the atomic level. An important target area concerns membrane-associated proteins, which can be investigated by ssNMR methods after reconstitution in synthetic bilayers. While such preparations allow examination of functional aspects of the protein of interest, the influence of the native cellular environment on protein structure and function cannot be monitored. Very recently, we introduced a general approach aimed at determining complex molecular structures, including integral membrane proteins, in their native cellular environment by ssNMR under magic-angle-spinning (MAS) conditions. Using dedicated sample-preparation routes, we demonstrated that high-resolution ssNMR spectra can be obtained on uniformly C,N-labeled preparations of Escherichia coli whole cells (WC) and cell envelopes (CE). Both CE and WC morphology are preserved under standard ssNMR experimental conditions and the corresponding C and N crosspolarization (CP-MAS) spectra are invariant over time. However, with increasing levels of molecular complexity, especially in the case of WC preparations, spectroscopic sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has developed into a routine tool to increase the sensitivity of multidimensional ssNMR. DNP enhancements of up to 148fold have been obtained on micro/nanocrystalline biomolecular samples, including an amyloidogenic peptide and a deuterated protein, 6] while enhancements between 18and 46fold have been reported for membrane-embedded polypeptides, purple membrane preparations, and bacteriophages. Here, we investigated the use of DNP to conduct ssNMR studies on C,N-labeled preparations of E. coli WC overproducing the integral outer membrane protein PagL. In Figure 1, we compared C and N CP-MAS spectra of uniformly C,N-labeled WC with the CE isolated from PagL-overproducing E. coli cells, recorded in the presence and absence of microwave irradiation. At higher temperatures (271 K), ssNMR spectra of the E. coli CE had previously revealed atomic details of PagL as well as endogenous membrane-associated macromolecules, including the major lipoprotein Lpp and non-proteinaceous components such as lipopolysaccharides (LPS), peptidoglycans (PG), and phospholipids. Under low-temperature (LT) DNP conditions, we observed significant DNP enhancement factors for both preparations in spectral regions characteristic for protein signals (aliphatic C resonances: d = 50–55 ppm, amide N backbone and side-chain resonances at about 120 and 80–30 ppm) as well as for C signals of endogenous

Cellular solid-state nuclear magnetic resonance spectroscopy

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 well-established 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.

Signals in bacterial β-barrel proteins are functional in eukaryotic cells for targeting to and assembly in mitochondria

The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts harbor β-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 β-barrel precursor proteins are recognized and sorted within eukaryotic cells, we expressed the bacterial β-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 β-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 native-like 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 β-barrel proteins have not evolved eukaryotic-specific signals to ensure their import into mitochondria. Furthermore, the signal for assembly of β-barrel proteins into the bacterial outer membrane is functional in mitochondria.

Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain

β‐Barrel proteins are present in the outer membranes of Gram‐negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino‐terminal periplasmic extension containing five polypeptide‐transport‐associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy‐terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.

Additive and Synergistic Bactericidal Activity of Antibodies Directed against Minor Outer Membrane Proteins of Neisseria meningitidis

ABSTRACT Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking childrens PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection.

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

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 beta-barrel outer membrane protein assembly complex of Neisseria meningitidis.

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the beta-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.

Viability of a Capsule- and Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis

ABSTRACT Neisseria meningitidis is the only lipopolysaccharide (LPS)-producing gram-negative bacterial species shown to be viable also without LPS. It was thought that the presence of capsular polysaccharide is necessary for this unusual feature. However, we show now that no part of the capsule gene cluster is required for maintaining LPS deficiency in N. meningitidis.

Role of the periplasmic chaperones Skp, SurA and DegQ in outer membrane protein biogenesis in Neisseria meningitidis

The periplasmic chaperones Skp, SurA, and DegP are implicated in the biogenesis of outer membrane proteins (OMPs) in Escherichia coli. Here, we investigated whether these chaperones exert similar functions in Neisseria meningitidis. Although N. meningitidis does not contain a homolog of the protease/chaperone DegP, it does possess a homolog of another E. coli protein, DegQ, which can functionally replace DegP when overproduced. Hence, we examined whether in N. meningitidis, DegQ acts as a functional homolog of DegP. Single skp, surA, and degQ mutants were easily obtained, showing that none of these chaperones is essential in N. meningitidis. Furthermore, all combinations of double mutants were generated and no synthetic lethality was observed. The absence of SurA or DegQ did not affect OMP biogenesis. In contrast, the absence of Skp resulted in severely lower levels of the porins PorA and PorB but not of other OMPs. These decreased levels were not due to proteolytic activity of DegQ, since porin levels remained low in a skp degQ double mutant, indicating that neisserial DegQ is not a functional homolog of E. coli DegP. The absence of Skp resulted in lower expression of the porB gene, as shown by using a P(porB)-lacZ fusion. We found no cross-species complementation when Skp of E. coli or N. meningitidis was heterologously expressed in skp mutants, indicating that Skp functions in a species-specific manner. Our results demonstrate an important role for Skp but not for SurA or DegQ in OMP biogenesis in N. meningitidis.