Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis.
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.
Nagano
Keiji
K
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan.
Read
Erik K
EK
Murakami
Yukitaka
Y
Masuda
Takashi
T
Noguchi
Toshihide
T
Yoshimura
Fuminobu
F
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
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