title: Three homologues, including two membrane-bound proteins, of the disulfide oxidoreductase DsbA in Neisseria meningitidis: effects on bacterial growth and biogenesis of functional type IV pili.
abstract: Many proteins, especially membrane and exported proteins, are stabilized by intramolecular disulfide bridges between cysteine residues without which they fail to attain their native functional conformation. The formation of these bonds is catalyzed in Gram-negative bacteria by enzymes of the Dsb system. Thus, the activity of DsbA has been shown to be necessary for many phenotypes dependent on exported proteins, including adhesion, invasion, and intracellular survival of various pathogens. The Dsb system in Neisseria meningitidis, the causative agent of cerebrospinal meningitis, has not, however, been studied. In a previous work where genes specific to N. meningitidis and not present in the other pathogenic Neisseria were isolated, a meningococcus-specific dsbA gene was brought to light (Tinsley, C. R., and Nassif, X. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11109-11114). Inactivation of this gene, however, did not result in deficits in the phenotypes commonly associated with DsbA. A search of available genome data revealed that the meningococcus contains three dsbA genes encoding proteins with different predicted subcellular locations, i.e. a soluble periplasmic enzyme and two membrane-bound lipoproteins. Cell fractionation experiments confirmed the localization in the inner membrane of the latter two, which include the previously identified meningococcus-specific enzyme. Mutational analysis demonstrated that the deletion of any single enzyme was compensated by the action of the remaining two on bacterial growth, whereas the triple mutant was unable to grow at 37 degrees C. Remarkably, however, the combined absence of the two membrane-bound enzymes led to a phenotype of sensitivity to reducing agents and loss of functionality of the pili. Although in many species a single periplasmic DsbA is sufficient for the correct folding of various proteins, in the meningococcus a membrane-associated DsbA is required for a wild type DsbA+ phenotype even in the presence of a functional periplasmic DsbA.
authors.affiliation: INSERM U570, Faculte de Medecine Necker, 156 Rue de Vaugirard, 75730 Paris CEDEX 15, France.
authors.forename: Colin R
authors.initials: CR
authors.lastname: Tinsley
authors.forename: Romé
authors.initials: R
authors.lastname: Voulhoux
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Beretti
authors.forename: Jan
authors.initials: J
authors.lastname: Tommassen
authors.forename: Xavier
authors.initials: X
authors.lastname: Nassif
mesh_terms: D000595:Amino Acid Sequence; D018407:Bacterial Physiological Phenomena; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology; D010861:Fimbriae, Bacterial / Q000201:enzymology / Q000502:physiology*; D017353:Gene Deletion; D005978:Glutathione / Q000633:toxicity; D006090:Gram-Negative Bacteria / Q000201:enzymology / Q000235:genetics; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology*; D010641:Phenotype; D017354:Point Mutation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases; D005978:Glutathione