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.

FIG. 1.

Construction of pg0695- and pg0694-deletion mutants from P. gingivalis ATCC 33277. (A) Gene arrangement in the chromosome and procedure for construction of allele-exchange gene deletion and location of primers. (B) Restriction sites and predicted lengths of the DNA regions in each genotype. Sizes of the DNA regions are shown as base pairs under the names and lines. (C) Verification of the mutants by gel electrophoresis of PCR products. After amplification by PCR with primers AGU-40 and AGU-41 shown at the top of panel B as 40 and 41, respectively, the PCR products were digested with the restriction enzymes described below and subjected to electrophoresis. Dotted arrows, pg0695; diagonally striped arrows, pg0694; gray arrows, cat; small arrows, primers; wavy line, overlap regions on cat; underlined numbers, primer designation in a simplified manner (see Table 1 for details); U, undigested; E, digested with EcoRI; H, digested with HpaI; B, digested with BanII.

FIG. 2.

Typical protein patterns and Western blots of envelope fractions. Bacterial envelopes were denatured in SDS with or without 2-ME at 100°C for 5 min and then loaded onto SDS-polyacrylamide gels. The gels were stained with CBB (A) and subjected to Western blot analysis with anti-Pgm6/7 serum (B). Each 50-μg protein was applied to each lane of the gel. Arrows show the predicted monomers and trimers that consisted of Pg0695 (Pgm6 monomer, 41 kDa) and/or Pg0694 (Pgm7 monomer, 40 kDa). Monomers of Pgm6 and Pgm7 were not discriminated from each other in SDS-PAGE assays.

FIG. 3.

2-D, diagonal SDS-PAGE and Western blotting of P. gingivalis wild-type envelope fraction. Envelopes (100 μg) were denatured in SDS at 100°C for 5 min under nonreducing conditions before the first-dimension electrophoresis. A gel strip was cut out from the first gel and placed on a well of the second gel. Then the strip in the well was overlaid with a buffer containing 2-ME. After the second electrophoresis, the gel was stained with CBB (A) or subjected to Western blotting with the ant-Pgm6/7 serum (B). The top and left parts of the figure show protein patterns in one dimension of the sample treated without and with 2-ME, respectively, to more easily elucidate the result in the central panel. The asterisk and double asterisks show the 120 (predicted trimer)- and 40 (monomer)-kDa bands, respectively.

FIG. 4.

Cross-linking of P. gingivalis wild-type Pgm6/7 proteins. The samples were treated with disuccinimidyl suberate as a cross-linker for 1 or 2 h. The cross-linked samples, denatured in SDS with or without 2-ME, were loaded onto SDS-polyacrylamide gels, and gels were stained with CBB.

FIG. 5.

Growth curves in various media. P. gingivalis ATCC 33277 (wild type), Δ695, Δ694, and Δ695-694 were cultured in sTSB, CDM, CDM supplemented with 0.15 M NaCl or 0.2 M NaCl, and DMEM supplemented with 1% BSA (FV, fraction V) or 1% BSA (HG, high grade purified).

FIG. 6.

Permeability of outer membranes in P. gingivalis wild type and the mutants. Permeability of the outer membranes was estimated by hydrolysis of p-nitrophenylphosphate with alkaline phosphatase. The released p-nitrophenol was determined by measuring the OD₄₀₀. Values are means of duplicate determinations. Similar results were obtained in three independent assays. Open bar, intact cell suspension; dotted bar, sonicated broken cells; closed bar, supernatant obtained from intact cell suspension.