PORIN ACTIVITY AND SEQUENCE-ANALYSIS OF A 31-KILODALTON TREPONEMA-PALLIDUM SUBSP PALLIDUM RARE OUTER-MEMBRANE PROTEIN (TROMP1)

We have recently reported the isolation and purification of the Trepon ema pallidum outer membrane and the identification of its rare protein constituents, including a 31-kDa protein markedly enriched in the out er membrane preparation (D, R, Blanco, K. Reimann, J. Skare, C, I. Cha mpion, D, Foley, M. M, Exner, R. E. W. Hancock, J. N. Miller, and R I. A. Lovett, J, Bacteriol. 176:6088-6099, 1994), In this study, we repo rt the cloning, sequencing, and expression of the structural gene whic h encodes the 31-kDa outer membrane protein, designated Tromp1, The de duced amino acid sequence from the tromp1 gene sequence encodes a 318- amino-acid polypeptide with a putative 40-amino-acid signal peptide, P rocessing of Tromp1 results in a mature protein with a predicted molec ular mass of 30,415 Da and a calculated pi of 6.6, Secondary-structure predictions identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topol ogical model of Tromp1 containing 14 transmembrane segments is propose d. Specific antiserum against a recombinant Tromp1 fusion protein was generated and was used to identify native Tromp1 in cellular fractiona tion, Upon Triton X-114 extraction and phase separation of T. pallidum , the 31-kDa Tromp1 protein was detected in the detergent-phase fracti on but not in the protoplasmic cylinder or aqueous-phase fractions, co nsistent with a hydrophobic outer membrane protein. Anti-Tromp1 antise rum was also used to identify native Tromp1 purified from whole T. pal lidum by Triton X-100 solubilization followed by nondenaturing isoelec tric focusing, Reconstitution of purified Tromp1 into planar lipid bil ayers showed porin activity based on the measured single channel condu ctances of 0.15 and 0.7 nS in 1 M KCl. These findings demonstrate that Tromp1 is a transmembrane outer membrane porin protein of T. pallidum .