1887

Abstract

OmpA1 is the major outer membrane protein of the Gram-negative anaerobic pathogen . We identified three additional conserved homologues () and three less homologous like genes (s , and ) in . We constructed an disruption mutant in 638R (WAL6 Ω) using insertion-mediated mutagenesis. WAL6 Ω 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 Ω grown in both high- and low-salt media did not reveal any other OmpA proteins even under osmotic stress. An deletant (WAL186Δ) was constructed using a two-step double-crossover technique, and an ‘reinsertant’, WAL360+, was constructed by reinserting the gene into WAL186Δ. WAL186Δ was significantly more sensitive to exposure to SDS, high salt and oxygen than the parental (WAL108) or reinsertant (WAL360+) strain. No significant change was seen in MICs of a variety of antimicrobials for either WAL6 Ω or WAL186Δ compared to WAL108. RT-PCR revealed that all of the genes are transcribed in the parental strain and in the disruption mutant, but, as expected, is not transcribed in WAL186Δ. Unexpectedly, is also not transcribed in WAL186Δ. 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 .

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2009-08-01
2020-01-21
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