title: Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.
abstract: The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded beta-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.
authors.affiliation: Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nagano
authors.forename: Erik K
authors.initials: EK
authors.lastname: Read
authors.forename: Yukitaka
authors.initials: Y
authors.lastname: Murakami
authors.forename: Takashi
authors.initials: T
authors.lastname: Masuda
authors.forename: Toshihide
authors.initials: T
authors.lastname: Noguchi
authors.forename: Fuminobu
authors.initials: F
authors.lastname: Yoshimura
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016254:Mutagenesis, Insertional; D010518:Periodontitis / Q000382:microbiology; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D017384:Sequence Deletion
chemical_list: D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D046912:Multiprotein Complexes; C496834:Pgm6 protein, Porphyromonas gingivalis; C496835:Pgm7 protein, Porphyromonas gingivalis; C078167:OMPA outer membrane proteins