Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete.
In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK+. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of treponemal physiology. At the same time, however, these findings also raise important issues regarding the use of OM preparations for identifying rare OM proteins of T. pallidum.
Shevchenko
D V
DV
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Akins
D R
DR
Robinson
E J
EJ
Li
M
M
Shevchenko
O V
OV
Radolf
J D
JD
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D005798:Genes, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D014210:Treponema pallidum / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D013879:Thioredoxins; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase; D019696:Peptidylprolyl Isomerase