The YscC secretin is a major component of the type III protein secretion system of Yersinia enterocolitica and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in trans in a Y. enterocolitica strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ϳ30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.

As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root-colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded.

The pid4 gene of Escherichia coli encodes an outer membrane phospholipase A. A strain carrying the most commonly used mutant pkld allele appeared to express a correctly assembled PldA protein in the outer membrane. Nucleotide sequence analysis revealed that the only difference between the wild type and the mutant is the replacement of the serine residue in position 152 by phenylalanine. Since mutants that lack thepld4 gene were normally viable under laboratory conditions and had no apparent phenotype except for the lack of outer membrane phospholipase activity, the exact role of the enzyme remains unknown. Nevertheless, the enzyme seems to be important for the bacteria, since Western blotting (immunoblotting) and enzyme assays showed that it is widely spread among species of the family Enterobacteriaceae. To characterize the PldA protein further, the pid4 genes of Salmonella typhimurium, Klebsiella pneumoniae, and Proteus vulgaris were cloned and sequenced. The cloned genes were expressed in E. coli, and their gene products were enzymatically active. Comparison of the predicted PldA primary structures with that of E. coli PldA revealed a high degree of homology, with 79%o of the amino acid residues being identical in all four proteins. Implications of the sequence comparison for the structure and the structure-function relationship of PldA protein are discussed.Most bacterial outer membrane proteins are involved in the transport of nutrients across this membrane by forming pores or receptors. In addition, these membranes contain a few enzymes, e.g., the detergent-resistant outer membrane phospholipase A or PldA protein of Escherichia coli. Several activities reside in this enzyme, i.e., those of phospholipases Al and A2 and of 1-acyl and 2-acyl lysophospholipase and lipase, with the phospholipase A1 activity being six times greater than the phospholipase A2 activity (24). The PIdA protein is encoded by the pldA gene, the nucleotide sequence of which has been determined (23). This gene codes for a 30-kDa mature protein of 269 amino acid residues preceded by a signal sequence of 20 amino acid residues. The three-dimensional structure of the enzyme is unknown. Like other outer membrane proteins, PldA protein lacks hydrophobic sequences long enough to span the lipid bilayer. Therefore, its structure might be comparable with those of the porins which have recently been determined (10,54,55). In these outer membrane proteins, the polypeptide chain traverses the outer membrane repeatedly as antiparallel 3-strands. A comparison of the outer membrane protein PhoE among three species of the family Enterobacteriaceae has revealed that during evolution some parts of the polypeptide have undergone more extensive divergence than others (49). For PhoE, these variable regions correspond to cell surface-exposed segments. A similar finding was reported for the OmpA protein (7). Thus, sequence comparisons can be helpful in predicting the topology of outer membrane proteins.The exact function of the PldA protein is unknown. The pro...

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YscC is the integral outer membrane component of the type III protein secretion machinery of Yersinia enterocolitica and belongs to the family of secretins. This group of proteins forms stable ring-like oligomers in the outer membrane, which are thought to function as transport channels for macromolecules. The YscC oligomer was purified after solubilization from the membrane with a nonionic detergent. Sodium dodecyl sulfate did not dissociate the oligomer, but it caused a change in electrophoretic mobility and an increase in protease susceptibility, indicating partial denaturation of the subunits within the oligomer. The mass of the homo-oligomer, as determined by scanning transmission electron microscopy, was approximately 1 MDa. Analysis of the angular power spectrum from averaged top views of negatively stained YscC oligomers revealed a 13-fold angular order, suggesting that the oligomer consists of 13 subunits. Reconstituted in planar lipid bilayers, the YscC oligomer displayed a constant voltage-independent conductance of approximately 3 nS, thus forming a stable pore. However, in vivo, the expression of YscC did not lead to an increased permeability of the outer membrane. Electron microscopy revealed that the YscC oligomer is composed of three domains, two stacked rings attached to a conical domain. This structure is consistent with the notion that the secretin forms the upper part of the basal body of the needle structure of the type III secreton.The outer membrane of gram-negative bacteria not only protects the cell against harmful compounds in the extracellular environment, such as antibiotics, detergents, and digestive enzymes, but also forms a barrier to the secretion of proteins and uptake of nutrients. The passage of small hydrophilic molecules across the outer membrane is facilitated by the presence of a large number of proteinaceous channels, formed by general and substrate-specific porins and TonB-dependent receptors (31, 41). Gram-negative bacteria have evolved several specialized pathways for the secretion of proteins. These pathways all require one or more integral outer membrane proteins, which in the cases of the type II and III protein secretion systems are related and belong to the family of secretins (19).