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

Abstract

OmpA1 is the major outer membrane protein of the Gram-negative anaerobic pathogenBacteroides fragilis. We identified three additional conservedompAhomologues (ompA2–ompA4) and three less homologousompA-like genes (ompAs5,6and7) inB. fragilis. We constructed anompA1disruption mutant inB. fragilis638R (WAL6 ΩompA1) using insertion-mediated mutagenesis. WAL6 ΩompA1formed 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 ΩompA1grown in both high- and low-salt media did not reveal any other OmpA proteins even under osmotic stress. AnompA1deletant (WAL186ΔompA1) was constructed using a two-step double-crossover technique, and anompA‘reinsertant’, WAL360+ompA1, was constructed by reinserting theompAgene into WAL186ΔompA1. WAL186ΔompA1was 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 ΩompA1or WAL186ΔompA1compared to WAL108. RT-PCR revealed that all of theompAgenes are transcribed in the parental strain and in the disruption mutant, but, as expected,ompA1is not transcribed in WAL186ΔompA1. Unexpectedly,ompA4is also not transcribed in WAL186ΔompA1. 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 inB. fragilis.