TROMP1, A PUTATIVE RARE OUTER-MEMBRANE PROTEIN, IS ANCHORED BY AN UNCLEAVED SIGNAL SEQUENCE TO THE TREPONEMA-PALLIDUM CYTOPLASMIC MEMBRANE

Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensiv e sequence homolog with substrate-binding proteins of ATP-binding cass ette transporters, Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify , Tromp1's physicochemical properties and cellular location in T. pall idum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel elec trophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed tha t the native protein is not processed, Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the a bility to form aqueous channels in liposomes which permit the influx o f small hydrophilic solutes, (ii) an extensive beta-sheet secondary st ructure, and (iii) amphiphilicity. Subsurface localization of native T romp1 was demonstrated hv immunofluorescence analysis of treponemes en capsulated in gel microdroplets, while opsonization assays failed to d etect surface-exposed Tromp1. Incubation of motile treponemes with rif luoromethyl)-3-(m-[I-125]iodophenyl)-diazarine, a photoactivatable, li pophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein, Fina lly, analysis of fractionated treponemes revealed that native Tromp1 i s associated predominantly with cell cylinders, These findings compris e a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.