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...

Pathogenic Yersinia spp. can be subdivided into highly pathogenic (high-pathogenicity) and low-pathogenicity strains. Several genes specific for the high-pathogenicity strains are clustered on a chromosomal fragment designated a "high-pathogenicity island" (HPI). In the present work, the HPI of biotype 1B strain Ye 8081 of Y. enterocolitica was characterized. We demonstrate important differences from the HPI of Y. pestis. The HPI of Y. enterocolitica is smaller (45 kb) and is not flanked by insertion sequences. A copy of the gene coding for the tRNA-Asn is present at one extremity of the HPI and may, as in uropathogenic Escherichia coli, participate in the excision of the island. In addition to the genes encoding the yersiniabactin-pesticin receptor and the high-molecular-weight protein 2, four repeated sequences are present on the HPI of Y. enterocolitica. At least two of them are insertion elements: previously described IS1328 and newly characterized IS1400. Comparison of the HPI of strain Ye 8081 with that of other Y. enterocolitica strains of biotype 1B indicates that most of the island is conserved, apart from 15 kb at the left-hand end which is variable, especially in the region where three repeated sequences are clustered.

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C‐terminal half of the polypeptide (PulD‐CS). In the absence of its cognate chaperone PulS, PulD‐CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD‐CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD‐CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD‐CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD‐CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.

We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/ selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.calorimetry ͉ intrabody ͉ ribosome display ͉ Sac7d ͉ type II secretion system