Characteristics of Bacteroides fragilis lacking the major outer membrane protein, OmpA.

OmpA1 is the major outer membrane protein of the Gram-negative anaerobic pathogen Bacteroides fragilis. We identified three additional conserved ompA homologues (ompA2-ompA4) and three less homologous ompA-like genes (ompAs 5, 6 and 7) in B. fragilis. We constructed an ompA1 disruption mutant in B. fragilis 638R (WAL6 OmegaompA1) using insertion-mediated mutagenesis. WAL6 OmegaompA1 formed much smaller colonies and had smaller, rounder forms on Gram stain analysis than the parental strain or other unrelated disruption mutants. SDS-PAGE and Western blot analysis (with anti-OmpA1 IgY) of the OMP patterns of WAL6 OmegaompA1 grown in both high- and low-salt media did not reveal any other OmpA proteins even under osmotic stress. An ompA1 deletant (WAL186DeltaompA1) was constructed using a two-step double-crossover technique, and an ompA 'reinsertant', WAL360+ompA1, was constructed by reinserting the ompA gene into WAL186DeltaompA1. WAL186DeltaompA1 was significantly more sensitive to exposure to SDS, high salt and oxygen than the parental (WAL108) or reinsertant (WAL360+ompA1) strain. No significant change was seen in MICs of a variety of antimicrobials for either WAL6 OmegaompA1 or WAL186DeltaompA1 compared to WAL108. RT-PCR revealed that all of the ompA genes are transcribed in the parental strain and in the disruption mutant, but, as expected, ompA1 is not transcribed in WAL186DeltaompA1. Unexpectedly, ompA4 is also not transcribed in WAL186DeltaompA1. A predicted structure indicated that among the four OmpA homologues, the barrel portion is more conserved than the loops, except for specific conserved patches on loop 1 and loop 3. The presence of multiple copies of such similar genes in one organism would suggest a critical role for this protein in B. fragilis.