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 seque nce similarity to other known porins, According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surfa ce-exposed loops and seven periplasmic turns. In this study, the insertion mutagenesis approach was applied to probe the topology model, A Semliki For est Virus (SFV) epitope was successfully inserted at 11 different sites of the FomA protein and a 6-aa insertion was successfully inserted at two diff erent sites. Correct folding of the mutant proteins and proper incorporatio n 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, show ing that FomA possesses the general beta-barrel topology of the non-specifi c porins, with the interesting exception that the third loop does not seem to fulfil the role of a constriction loop.