Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953.

Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Microbial Pathogenesis (Impact Factor: 2). 12/1996; 21(5):331-42. DOI: 10.1006/mpat.1996.0066
Source: PubMed

ABSTRACT The major outer membrane protein, FomA, of the Gram-negative human oral pathogen Fusobacterium nucleatum functions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such as Streptococcus sanguis and Porphyromonas gingivalis. We describe here the cloning of fomA from F. nucleatum in E. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of the fomA gene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the whole fomA gene. The ATCC 10953 fomA gene was cloned under the phosphate limitation-inducible phoE promoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.

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    ABSTRACT: FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a β-barrel consisting of 16 transmembrane segments with eight surface-exposed loops. Several conserved charged residues are proposed to extend from the β-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of β-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic β-lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.
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