THE FUSOBACTERIUM-NUCLEATUM MAJOR OUTER-MEMBRANE PROTEIN (FOMA) FORMSTRIMERIC, WATER-FILLED CHANNELS IN LIPID BILAYER-MEMBRANES

The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, A TCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate a nd lithium chloride and had an apparent molecular mass of about 40 kDa . When solubilized at low temperatures, the protein ran with an appare nt molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experimen ts and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were s usceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were stu died in reconstitution experiments with black lipid bilayer membranes. The F: nucleatum porins formed channels with single-channel conductan ces in the range 0.66-1.30 nS in 1 M KCl. The single-channel conductan ce was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms gen eral diffusion channels since (a) the conductance showed a linear depe ndence on the salt concentration, (b) the ion selectivity was small an d varied for the three strains, and (c) the channels did not exhibit a ny binding site for maltotriose or triglycine. The water-filled channe l was voltage dependent, and conductance decrements were observed at t ransmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.