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Journal of Bacteriology, July 1999, p. 4420-4423, Vol. 181, No. 14
Departments of
Microbiology1 and Internal
Medicine,2 The University of Texas
Southwestern Medical Center, Dallas, Texas 75235, and the
Division of Research Microbiology, Research Institute,
Hospital for Sick Children, Toronto, Ontario M5G 1X8,
Canada3
Received 30 November 1998/Accepted 3 May 1999
Although TroA (Tromp1) was initially reported to be a
Treponema pallidum outer membrane protein with porin-like
properties, subsequent studies have suggested that it actually is a
periplasmic substrate-binding protein involved in the transport of
metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our
prior contention that the native protein's amphiphilic behavior is due
to its uncleaved leader peptide. Whereas typical porins are trimers
with extensive The quest for outer membrane
proteins of Treponema pallidum as potential vaccine
candidates and virulence determinants is a formidable but exceedingly
important area of contemporary syphilis research (22). In
this regard, Blanco et al. (3, 4) recently reported on the
identification of a 31-kDa protein (designated T. pallidum
rare outer membrane protein 1, or Tromp1) in isolated T. pallidum outer membranes that formed ion-conducting channels in
planar lipid bilayers. However, others have noted that Tromp1 has
extensive sequence homology with a novel family of periplasmic substrate-binding proteins (designated cluster 9) involved in metal
(i.e., iron, zinc, or manganese) transport in other prokaryotes (1, 2, 10, 14, 15, 17, 19) and that the tromp1 gene is contiguous to and transcriptionally linked with homologs for
additional ATP-binding cassette (ABC) transporter components (11,
14). Moreover, Akins and coworkers (1) showed that Tromp1 contains an uncleaved signal sequence, is very hydrophilic downstream of the signal peptide, does not form aqueous channels in
liposomes, and is not surface exposed in T. pallidum.
Although these studies weigh heavily in favor of TroA
(transport-related operon A) as the appropriate nomenclature for this
protein (11), additional data are needed to resolve this
controversy. Here we present physicochemical evidence that TroA is a
zinc-containing protein with structural features inconsistent with
those of bacterial porins.
An uncleaved signal sequence confers amphiphilicity on TroA.
In a prior study (1), we used an in vitro-coupled
transcription-translation system to demonstrate that native TroA
contains an uncleaved N-terminal signal sequence. We also showed that
recombinant TroA lacking this signal sequence was hydrophilic, while
the native protein was amphiphilic (1). Based upon these
findings, we proposed that the signal sequence alone is responsible for
TroA's amphiphilic character (1). To garner further support
for this notion, we directly compared the Triton X-114 phase
partitioning behaviors of recombinant TroA constructs which differed
only by the presence or absence of the native polypeptide's
22-amino-acid signal sequence (14). The larger construct,
L-TroA, was PCR-amplified from T. pallidum DNA with the
forward and reverse primers
5'-CGCGGATCCTTGATACGTGAAAGAATATGTGCCTGC-3' and
5'-CGGAATTCTTACTAGCGAGCCAACGCAGCAACGATCG-3',
respectively, while the smaller construct, S-TroA, was
amplified with the forward primer
5'-CGCGGATCCTTCGGTAGCAAGGATGCCGCAGCGGA C-3'
and the reverse primer used for L-TroA. The PCR products
were cloned into the BamHI and EcoRI sites of the
expression vector pProEx1 (Gibco BRL, Gaithersburg, Md.); the
resulting polypeptides both contain a 28-amino-acid N-terminal
extension which includes the His6 tag used for affinity
purification. Interestingly, L-TroA was very poorly expressed in
Escherichia coli and required solubilization in 8 M urea for
affinity purification on the Ni-nitrilotriacetic acid agarose matrix,
whereas S-TroA was abundantly expressed and quantitatively recovered
from the E. coli cell supernatant (data not shown). As shown
in Fig. 1, L-TroA, like its native
counterpart, partitioned exclusively into the detergent-enriched phase
following solubilization in 2% Triton X-114; S-TroA, in contrast,
partitioned exclusively into the aqueous phase (Fig. 1). Identical
phase-partitioning results were obtained using L-TroA and S-TroA
constructs lacking the N-terminal extension (data not shown). It should
be noted that porins, unlike TroA, are amphiphilic even without their
leader peptides and completely partition into the Triton X-114
detergent-enriched phase (1). These results also support our
proposed topology, which places TroA within the periplasmic space where
it is anchored by its uncleaved leader peptide to the treponemal
cytoplasmic membrane (1).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Physicochemical Evidence that Treponema
pallidum TroA Is a Zinc-Containing Metalloprotein That Lacks
Porin-Like Structure
ABSTRACT
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Abstract
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References
-barrel structure, size exclusion chromatography and
circular dichroism spectroscopy revealed that TroA was a monomer and
predominantly alpha-helical. Neutron activation, atomic absorption
spectroscopy, and anomalous X-ray scattering all demonstrated that TroA
binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to
possess structural features consistent with those of bacterial porins.
TEXT
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FIG. 1.
An uncleaved signal sequence confers amphiphilicity on
TroA. Detergent-enriched (D) and aqueous (A) phases were collected
following Triton X-114 phase partitioning of T. pallidum,
and recombinant constructs possessed (L-TroA) or lacked (S-TroA) the
native polypeptide's 22-amino-acid leader peptide. Proteins were
visualized by immunoblot analysis by using a TroA-specific murine
monoclonal antibody. Note that both L-TroA and S-TroA contain
28-amino-acid N-terminal extensions derived from the pProEx1 expression
vector. Numbers at the left correspond to molecular mass markers.
TroA is a monomer with predominant alpha-helical secondary
structure.
Typical porins, such as E. coli OmpF, are
multistranded -barrels that form highly stable trimers (20,
25). It was of interest, therefore, to examine the oligomeric
state and secondary structure of TroA. Because porins attain their
final conformation and trimeric state following removal of their signal
peptides (24, 25), we reasoned that it was appropriate to
use S-TroA in the present studies. S-TroA (calculated molecular mass,
34,417 Da) resolved as a single peak with a deduced molecular mass of 34,457 Da by size exclusion chromatography (Superdex-S75 exclusion column) under nondenaturing conditions (20 mM Tris-HCl, pH 7.5, 20 mM
NaCl) (data not shown). Circular dichroism spectroscopy, performed on
an AVIV (Lakewood, N.J.) model 62DS spectrophotometer and analyzed by
using the self-consistent algorithm of Sreerama and Woody
(29), revealed that the protein (with or without the 28-amino-acid N-terminal extension created by the cloning vector) was
60% alpha-helix and only 3% beta-sheet, a result consistent with the
large amount of alpha-helical structure predicted by both the
Chou-Fasman algorithm (8) and the algorithm of Garnier et
al. (12). Also noteworthy was the observation that cleaved S-TroA was fully water soluble. While these results do not entirely preclude the possibility that native TroA is multimeric and
predominantly a -barrel, it seems unlikely that the native and
recombinant forms of the protein should adopt such different conformations.
TroA binds zinc in a 1:1 molar ratio.
As noted earlier, TroA
shares sequence homology with known periplasmic metal-binding proteins
(1, 2, 10, 14, 15, 17, 19). Unlike most of the other
bacteria containing TroA orthologs, genetic approaches cannot be used
to analyze metal uptake by T. pallidum. For this reason, we
applied to TroA physical methods which recently were used to examine
heavy-metal binding by Pzp1, the Haemophilus influenzae TroA
ortholog (17). Neutron activation of S-TroA (following
His-tag removal and extensive dialysis) revealed that zinc was present
at a molar ratio of 1:1 and at a level markedly above that found in the
control dialysate; other elements were present in only trace or
undetectable quantities. Because neutron activation has limited
sensitivity, particularly for iron, we also examined S-TroA by atomic
absorption spectroscopy. This technique also detected zinc in a 1:1
stoichiometric ratio, while iron was not detected (Table
1).
|
Anomalous scattering of S-TroA crystals.
As a final
confirmation of the metal-binding data, we analyzed the anomalous
scattering signal at the X-ray wavelength of the zinc K absorption edge
with S-TroA crystals. The crystals were grown from hanging drops of a
1:1 mixture of reservoir solution comprised of 30% polyethylene
glycol, 1 M LiCl, and 0.1 M HEPES-Cl (pH 7.0) and containing 30 to 40 mg of protein/ml. Hexagonal rods with a size of 0.1 by 0.1 by 0.6 mm
grew after 1 week of incubation (Fig. 2)
and diffracted to a resolution of 2.1 Å when measured by using a
Rigaku RU200 (Molecular Structure Corporation, The Woodlands, Tex.)
rotating copper anode X-ray generator and R-axis II detector. The
crystals were found to belong to a P61 space group
with a unit cell dimension of a = b = 125.2 Å, c = 74.5 Å, 
= = 90°, and
= 120°. Based on protein crystal volume considerations,
there are two copies of S-TroA in the crystallographic asymmetric unit.
That crystals with a high degree of order were readily obtained with
S-TroA is further proof that this construct is in a conformationally
native state.
|
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TroA and the quest for T. pallidum outer membrane proteins. The many difficulties inherent in the molecular characterization of T. pallidum outer membrane proteins (22) have spawned a variety of experimental approaches for accomplishing this elusive objective (5-7, 11, 13, 23, 30). Of these, isolation of T. pallidum outer membranes seemed particularly promising because it permitted direct identification of candidate outer membrane proteins. More recently, however, there has been increasing evidence that most of the uncharacterized polypeptides in outer membrane fractions are periplasmic or cytoplasmic membrane contaminants rather than authentic outer membrane proteins (26-28). This conclusion also appears to pertain to TroA despite its relative abundance in outer membrane preparations (6, 23).
Although bacterial porins have limited primary amino acid sequence homology, their three-dimensional structures are similar (9). Periplasmic substrate-binding proteins also have limited sequence homology but a high degree of similarity in their tertiary structures (21). Most importantly, the tertiary structures of porins and substrate-binding proteins are highly dissimilar. Porins consist of 16 anti-parallel strands in a-barrel
configuration, whereas substrate-binding proteins consist of two
distinct globular domains bisected by a cleft or groove for ligand
binding (21). In this regard, it is interesting to note that
the three-dimensional structure of PsaA, the pneumococcal TroA
homologue and a known virulence determinant, was recently shown to be
that of a substrate-binding protein with zinc as the probable metal
ligand (16). The results described here, therefore, lay the
groundwork for definitively resolving TroA structure and, in so doing,
the divergent viewpoints concerning its function and cellular location
in T. pallidum. An equally important implication of our work
concerns the fact that Tro-like ABC transporters in other bacterial
pathogens have been shown to influence transport-nonrelated
virulence-related processes (10, 15, 18). Thus, our findings
also set the stage for an investigation of the role of zinc, an
essential trace element, and zinc transporters in T. pallidum physiology and host-pathogen interactions during
syphilitic infection.
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ACKNOWLEDGMENTS |
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This research was supported in part by U.S. Public Health Service grants AI-26756 to J.D.R., AI-16692 to M.V.N., and DK-52089 to C.A.H. and by Robert A. Welch Foundation grants I-0940 to M.V.N. and J.D.R. and I-1318 to C.A.H.
We also are indebted to Martin Goldberg for technical assistance, to Ken Bourell for assistance with graphics and figure preparation, and to Eric Hansen for helpful suggestions.
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FOOTNOTES |
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* Corresponding author. Present address: Center for Microbial Pathogenesis, University of Connecticut Health Center, MC 3710, School of Medicine, 263 Farmington Ave., Farmington, CT 06030. Phone: (860) 679-8129. Fax: (860) 679-8130. E-mail: Jradolf{at}up.uchc.edu.
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REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Akins, D. R.,
E. Robinson,
D. V. Shevchenko,
C. Elkins,
D. L. Cox, and J. D. Radolf.
1997.
Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved leader peptide to the Treponema pallidum cytoplasmic membrane.
J. Bacteriol.
179:576-586 |
| 2. | Bartsevich, V. V., and H. B. Pakrasi. 1995. Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process. EMBO J. 14:1845-1853[Medline]. |
| 3. |
Blanco, D. R.,
C. I. Champion,
M. M. Exner,
H. Erdjument-bromage,
R. W. Hancock,
P. Tempst,
J. N. Miller, and M. A. Lovett.
1995.
Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).
J. Bacteriol.
177:3556-3562 |
| 4. |
Blanco, D. R.,
C. I. Champion,
M. M. Exner,
E. S. Shang,
J. T. Skare,
R. E. W. Hancock,
J. N. Miller, and M. A. Lovett.
1996.
Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.
J. Bacteriol.
178:6685-6692 |
| 5. | Blanco, D. R., M. Giladi, C. I. Champion, D. A. Haake, G. K. Chikami, J. N. Miller, and M. A. Lovett. 1991. Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector. Mol. Microbiol. 5:2405-2415[Medline]. |
| 6. |
Blanco, D. R.,
K. Reimann,
J. Skare,
C. I. Champion,
D. Foley,
M. M. Exner,
R. E. W. Hancock,
J. N. Miller, and J. N. Lovett.
1994.
Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.
J. Bacteriol.
176:6088-6099 |
| 7. |
Centurion-Lara, A.,
C. Castro,
L. Barrett,
C. Cameron,
M. Mostowfi,
W. C. Van Voorhis, and S. A. Lukehart.
1999.
Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.
J. Exp. Med.
189:647-656 |
| 8. | Chou, P. Y., and G. D. Fasman. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-148[Medline]. |
| 9. | Cowan, S. W., T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R. A. Pauptit, J. N. Jansonius, and J. P. Rosenbusch. 1992. Crystal structures explain functional properties of two E. coli porins. Nature 358:727-733[Medline]. |
| 10. | Dintilhac, A., G. Alloing, C. Granadel, and J.-P. Claverys. 1997. Competence and virulence of Streptococcus pneumoniae: Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases. Mol. Microbiol. 25:727-739[Medline]. |
| 11. |
Fraser, C. M.,
S. J. Norris,
G. M. Weinstock,
O. White,
G. C. Sutton,
R. Dodson,
M. Gwinn,
E. K. Hickey,
R. Clayton,
K. A. Ketchum,
E. Sodergren,
J. M. Hardham,
M. P. McLeod,
S. Salzberg,
J. Peterson,
H. Khalak,
D. Richardson,
J. K. Howell,
M. Chidambaram,
T. Utterback,
L. McDonald,
P. Artiach,
C. Bowman,
M. D. Cotton,
C. Fujii,
S. Garland,
B. Hatch,
K. Horst,
K. Roberts,
M. Sandusky,
J. Weidman,
H. O. Smith, and J. C. Venter.
1998.
Complete genome sequence of Treponema pallidum, the syphilis spirochete.
Science
281:375-388 |
| 12. | Garnier, J., D. J. Osguthorpe, and B. Robson. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120:97-120[Medline]. |
| 13. |
Hardham, J. M., and L. V. Stamm.
1994.
Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog.
Infect. Immun.
62:1015-1025 |
| 14. | Hardham, J. M., L. V. Stamm, S. F. Porcella, J. G. Frye, N. Y. Barnes, J. K. Howell, S. L. Mueller, J. D. Radolf, G. M. Weinstock, and S. J. Norris. 1997. Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transporter system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog. Gene 197:47-64[Medline]. |
| 15. |
Kolenbrander, P. E.,
R. N. Andersen,
R. A. Baker, and H. F. Jenkinson.
1997.
The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake.
J. Bacteriol.
180:290-295 |
| 16. | Lawrence, M. C., P. A. Pilling, V. C. Epa, A. M. Berry, A. D. Ogunniyi, and J. C. Paton. 1999. The crystal structure of pneumococcal surface antigen PsaA reveals a metal-binding site and a novel structure for a putative ABC-type protein. Structure 6:1553-1561. |
| 17. |
Lu, D.,
B. Boyd, and C. A. Lingwood.
1998.
Identification of the key protein for zinc uptake in Haemophilus influenzae.
J. Biol. Chem.
272:29033-29038 |
| 18. | Novak, R., J. S. Braun, E. Charpentier, and E. Tuomanen. 1998. Penicillin tolerance genes of Streptococcus pneumoniae: the ABC-type manganese permease complex Psa. Mol. Microbiol. 29:1285-1296[Medline]. |
| 19. | Patzer, S. I., and K. Hantke. 1998. The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli. Mol. Microbiol. 28:1199-1210[Medline]. |
| 20. | Phale, P. S., A. Philippsen, T. Kiefhaber, R. Koebnik, V. P. Phale, T. Schirmer, and J. P. Rosenbusch. 1998. Stability of trimeric OmpF porin: the contribution of the latching loop. Biochemistry 37:15663-15670[Medline]. |
| 21. | Quiocho, F., and P. S. Ledvina. 1996. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol. Microbiol. 20:17-25[Medline]. |
| 22. | Radolf, J. D. 1995. Treponema pallidum and the quest for outer membrane proteins. Mol. Microbiol. 16:1067-1073[Medline]. |
| 23. | Radolf, J. D., E. J. Robinson, K. W. Bourell, D. R. Akins, S. F. Porcella, L. M. Weigel, J. D. Jones, and M. V. Norgard. 1995. Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete. Infect. Immun. 63:4244-4252[Abstract]. |
| 24. |
Reid, J.,
H. Fung,
K. Gehring,
P. E. Klebba, and H. Nikaido.
1988.
Targeting of porin to the outer membrane of Escherichia coli. Rate of trimer assembly and identification of a dimer intermediate.
J. Biol. Chem.
263:7753-7759 |
| 25. |
Sen, K., and H. Nikaido.
1990.
In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli.
Proc. Natl. Acad. Sci. USA
87:743-747 |
| 26. |
Shevchenko, D. V.,
D. R. Akins,
E. Robinson,
M. Li,
T. G. Popova,
D. L. Cox, and J. D. Radolf.
1997.
Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete.
J. Bacteriol.
179:3188-3195 |
| 27. | Shevchenko, D. V., D. R. Akins, E. J. Robinson, M. Li, O. V. Shevchenko, and J. D. Radolf. 1997. Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete. Infect. Immun. 67:4179-4189. |
| 28. |
Shevchenko, D. V.,
T. J. Sellati,
D. L. Cox,
O. V. Shevchenko,
E. J. Robinson, and J. D. Radolf.
1999.
Membrane topology and cellular location of the Treponema pallidum glycerophosphodiester phosphodiesterase (GlpQ) ortholog.
Infect. Immun.
67:2266-2276 |
| 29. | Sreerama, N., and R. W. Woody. 1996. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 209:32-44. |
| 30. | Stebeck, C. E., J. M. Shaffer, T. W. Arroll, S. A. Lukehart, and W. C. Van Voorhis. 1997. Identification of the Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase homologue. FEMS Microbiol. Lett. 154:303-310[Medline]. |
Journal of Bacteriology, July 1999, p. 4420-4423, Vol. 181, No. 14
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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