Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane ␤-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.The widespread type II secretion systems (T2SS) 3 of Gram-negative bacteria allow the secretion of hydrolytic enzymes (lipases, amylases) or virulence factors, collectively referred to as exoproteins, into the external medium (1, 2). These exoproteins are first translocated by the Sec (3) or Tat (4) translocons into the periplasm. They are then specifically transported through the outer membrane by an ATP and proton-motive force-dependent machinery (the secreton) (5, 6) composed of 12-15 proteins (1, 2). The secreton components include several integral inner membrane proteins, pseudopilins (proteins with structural features similar to those of type IV pilins (7)) and an integral outer membrane protein called secretin. Besides their role in protein secretion by the T2SS (e.g. Klebsiella oxytoca protein PulD (8) and Pseudomonas aeruginosa protein XcpQ (9, 10)) and the type III secretion system (e.g. Yersinia enterocolitica protein YscC (10)), secretins are also required for filamentous bacteriophage secretion (e.g. bacteriophage f1 protein pIV (11)) and type IV pilus assembly (e.g. Neisseria meningitidis and P. aeruginosa PilQ (9, 12)).According to electron microscopy, 12-14 identical secretins form ring-like complexes with an internal channel (estimated diameters range from 5 nm (PilQ, YscC) to 10 nm (XcpQ) (9, 13, 14)), large enough to accommodate their substrates (9,10,15,16). Negative stain analysis of PulD (17) and cryoelectron microscopy of pIV (18) revealed a central channel plug. Incorporation of secretins into the Escheri...

ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergentinduced oligomeric complex at 12 Å resolution by combining cryo-and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.

SummaryDivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like a a a a -helical coiledcoil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6-to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, twodimensional lattices in a time-dependent manner.

The iron-limitation-inducible protein FrpB of Neisseria meningitidis is an outer-membrane-localized siderophore receptor. Because of its abundance and its capacity to elicit bactericidal antibodies, it is considered a vaccine candidate. Bactericidal antibodies against FrpB are, however, type-specific. Hence, an FrpB-based vaccine should comprise several FrpB variants to be capable of providing broad protection. To facilitate the development of a meningococcal subunit vaccine, we have established a procedure to obtain large quantities of the protein in a native-like conformation. The protein was expressed without its signal sequence in Escherichia coli, where it accumulated in inclusion bodies. After in vitro folding, the protein was biochemically, biophysically and biologically characterised. Our results show that in vitro folded FrpB assembles into oligomers, presumably dimers, and that it induces high levels of bactericidal antibodies in laboratory animals.

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