The Fusobacterium nucleatum porin FomA possesses the general topology of the non-specific porins.
FomA is a major non-specific porin of Fusobacterium nucleatum with no sequence similarity to other known porins. According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surface-exposed loops and seven periplasmic turns. In this study, the insertion mutagenesis approach was applied to probe the topology model. A Semliki Forest Virus (SFV) epitope was successfully inserted at 11 different sites of the FomA protein and a 6-aa insertion was successfully inserted at two different sites. Correct folding of the mutant proteins and proper incorporation into the outer membrane were assessed by heat modifiability and by an in vivo porin activity assay. Immunofluorescence microscopy analysis of intact cells, using mAbs directed against the inserted SFV epitope, revealed that three of the eight putative extracellular loops are indeed surface-exposed. Trypsin accessibility experiments confirmed the cell surface exposure of two additional loops. The results support the proposed topology model, showing that FomA possesses the general beta-barrel topology of the non-specific porins, with the interesting exception that the third loop does not seem to fulfil the role of a constriction loop.
Puntervoll
Pål
P
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
Kleivdal
Hans
H
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
Dahl
Karl Ole
KO
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
Bitter
Wilbert
W
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2.
Tommassen
Jan
J
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2.
Jensen
Harald B
HB
Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1.
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005673:Fusobacterium / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D018272:Porins / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012672:Semliki forest virus / Q000235:genetics / Q000276:immunology
D001425:Bacterial Outer Membrane Proteins; D004274:DNA, Recombinant; D000939:Epitopes; D018272:Porins; D011994:Recombinant Proteins; C083760:fomA protein, Fusobacterium