Development of a gene inactivation system for Bacteroides forsythus: construction and characterization of a BspA mutant

Abstract

Bacteroides forsythus is a gram-negative anaerobic bacterium associated with periodontitis. The bspA gene encoding a cell surface associated leucine-rich repeat protein (BspA) involved in adhesion to fibronectin and fibrinogen was recently cloned from this bacterium in our laboratory. We now describe the construction of a BspA-defective mutant of B. forsythus. This is the first report describing the generation of a specific gene knockout mutant of B. forsythus, and this procedure should be useful in establishing the identity of virulence-associated factors in these organisms.

FIG. 1

Schematics of B. forsythus suicide vector construction.

FIG. 2

(A) Schematics of the integration of suicide plasmid pBFS-57 into the bspA locus of the B. forsythus genome. (B) Southern hybridization. Genomic DNA isolated from B. forsythus wild-type (ATCC 43037, lanes 1, 4, and 7), mutant (BFM-571, lanes 2, 5, and 8), and suicide plasmid DNA (pBFS-57, lanes 3, 6, and 9) digested with restriction enzymes shown on top were hybridized with the bspA probe. Numbers on the left indicate sizes in kilobases.

FIG. 3

Western immunoblot analysis of wild-type and mutant B. forsythus. Proteins were transferred onto nitrocellulose membranes after separation on an SDS–10% PAGE gel. Membranes were probed with anti-rBspA antibody (16). Lanes: 1, total cell lysate of wild-type B. forsythus ATCC 43037; 2, total cell lysate of mutant BFM-571; 3, recombinant BspA protein (70-kDa rBsp70). The positions of molecular mass standards (in kilodaltons) are shown on the left.

FIG. 4

Binding curves of wild-type B. forsythus ATCC 43037 and BFM-571 mutant to immobilized fibronectin (A) and fibrinogen (B). Fibonectin- or fibrinogen-coated microtiter wells were incubated for 1 h with different numbers of ³H-labeled wild-type and mutant bacteria (1.25 × 10⁷ to 2 × 10⁸ cells of each). Both strains showed similar specific activities (9,200 ± 900 cpm/10⁸ cells). After the nonadherent cells were washed, the numbers of cells bound per well were calculated from the bound radioactivity. The results presented are the mean values of duplicate samples representative of two independent experiments.