Vol. 59, No. 10

INFECTION AND IMMUNITY, Oct. 1991, p. 3685-3693

0019-9567/91/103685-09$02.00/0 Copyright C) 1991, American Society for Microbiology

Treponema phagedenis Encodes and Expresses Homologs of the Treponema pallidum TmpA and TmpB Proteins DAVID B. YELTON,1* RONALD J. LIMBERGER,2 KRISTINA CURCI,1 FRANCIS MALINOSKY-RUMMELL, LINDA SLIVIENSKI,2 LEO M. SCHOULS,3 JAN D. A. VAN EMBDEN,3 AND NYLES W. CHARON' Department of Microbiology and Immunology, Health Sciences Center, West Virginia University, Morgantown, West Virginia 265061; Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, New York 1220J_05092; and Unit for Molecular Microbiology, National Institute of Public Health and Environmental Protection, 3720 BA Bilthoven, The Netherlands3 Received 17 April 1991/Accepted 24 July 1991

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase H and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive a-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.

One of the major immunoreactive proteins of T. pallidum is the lipoprotein TmpA (4, 22). TmpA was believed to be found only in the pathogen T. pallidum (4, 22). This protein migrates as a 44-kDa protein in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels (4, 22). In vivo it is produced as a 46-kDa precursor which is cleaved during maturation and addition of lipid (23). The termination codon for the gene encoding the TmpA protein overlaps with the initiation codon for the gene encoding the TmpB protein (4). The TmpB proprotein has a transmembrane leader sequence, but it is not modified by lipid addition during maturation. It does undergo cleavage from a 38-kDa primary transcript to a mature protein of 34 kDa (4, 22). Both TmpA and TmpB are expressed from a common promoter located upstream of the initiation codon for TmpA (4). Since T. pallidum can only be grown in vivo, it has not been possible to study these processing reactions in treponemes or to determine the role of these proteins in the biology of the

Proteins that are an integral part of the outer membrane (OM) of the spirochetes play an essential role in the growth of these organisms. They may be involved in transport, attachment to cells, and pathogenesis. Despite the presence of OM proteins, most pathogenic spirochetes are able to evade the immune response of the host. How spirochetes are able to circumvent the host immune response and still maintain the activities of OM proteins is an area of active research. Brandt and coworkers have shown that most of the OM proteins of Borrelia burgdorferi selectively extracted with Triton X-114 are reactive with immune sera from patients with Lyme disease (1). Furthermore, the majority of these proteins are lipoproteins (1). These include the OspA and OspB proteins, which are found abundantly in the OM (1). Chamberlain et al. and Swancutt et al. have shown that the majority of the proteins of Treponema pallidum subsp. pallidum (hereafter referred to as T. pallidum) that can be extracted with Triton X-114 not only are immunogenic in patients but also are lipoproteins (2, 3, 25). Others have identified and cloned the genes for several putative OM proteins of T. pallidum (4, 5, 22, 23, 26). Most of these are lipoproteins (23). Included in this group of putative OM lipoproteins are TmpA (4), TmpC (23, 26), and TpD (23). Another potential OM protein is TmpB (4, 22, 26). Although TmpB is not a lipoprotein, it contains a potential membranespanning leader sequence (4). The gene encoding TmpB is part of an operon that includes the tmpA gene (4, 22). *

organism. It has been difficult to obtain envelope proteins from T. pallidum for study, as this treponeme can only be grown in vivo. Hence, many investigators have turned to cloning techniques to isolate the genes encoding such proteins (2-5, 22, 23, 25, 26). In this paper, we report the cloning and sequencing of genes from Treponema phagedenis encoding homologs of the TmpA and TmpB proteins of T. pallidum. The genes were isolated from a Xgtll library of T. phagedenis Kazan 5. For comparison, the gene encoding the TmpB protein of T. pallidum was also sequenced. In contrast to

Corresponding author. 3685

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TABLE 1. E. coli K-12 strains, plasmids, and bacteriophage used in this study Strain, plasmid,

Relevant property

or bacteriophage

E. coli Y1088 Y1090

JM101 JA221

Host for Host for Host for Host for

Xgtll

Reference or source

6 6 15 31

Agtll M13 plasmids

Plasmids pRIT4633 pGEM3 pTPH39K1600 pTPH39K1000 pTPH39K800 pTPH39K600 pTPH39K400 pTPALB pTPALB2

Source of tmpB gene from T. pallidum Double-stranded DNA sequencing vector 1,600-bp EcoRI fragment from Xgtll.39K cloned into pGEM3 ExollI deletion of pTPH39K1600 ExolI deletion of pTPH39K1600 ExoIl deletion of pTPH39K1600 ExollI deletion of pTPH39K1600 HindIII-SstI fragment of pRIT4633 cloned into pGEM3 Derived from pTPALB by deletion of internal PstI fragment

4 18 This paper This paper This paper This paper This paper This paper This paper

Bacteriophage xgtll Xgtll.39K M13mpl8 mp18KS13 mpl8L9 mpl8L12

Cloning vector Immunoreactive clone Single-stranded DNA sequencing vector M13 subclone of the KpnI-SacI fragment from Xgtll.39K M13 subclone of the downstream Sau3A-EcoRI fragment from Xgt1l.39K M13 subclone of the upstream Sau3A-EcoRI fragment from Xgtll.39K

32 This 15 This This This

previous results (26), both tmpA and tmpB were found to be present and expressed in T. phagedenis Kazan 5 and Reiter. The presence of TmpA and TmpB homologs in a cultivable treponeme will allow the native proteins to be readily obtained for study and may allow an unambiguous determination of their locations within the cell to be made. Homologs of other T. pallidum proteins may also be present in T. phagedenis. If so, they also could be studied in this cultivable treponeme. MATERIALS AND METHODS Bacterial strains and plasmids. The origin of T. phagedenis Kazan 5 has been previously described (10). T. phagedenis Reiter was obtained from D. Thomas. T. pallidum was obtained from L. Schouls (4). The Escherichia coli strains and plasmids used in these studies are listed in Table 1. Media and materials. E. coli strains were grown in L broth or on L agar (1.5%) at 37°C (17). T. phagedenis strains were grown in PYG-RS broth (10). T. pallidum was grown and purified as previously described (26). Agtll clones were plated on tryptone agar plates (1.5%) with tryptone top agar (0.5%) and incubated at 43°C until plaques appeared (6, 12). M13 infections were done in YT medium at 37°C (15). Restriction endonucleases, T4 DNA ligase, and exonucleases were purchased from Bethesda Research Laboratories, Inc., New England BioLabs, Inc., and Boehringer Mannheim Biochemicals. Primers for DNA sequencing and for polymerase chain reactions (PCR) were produced on an Applied Biosystems model 380B DNA synthesizer in the West Virginia University Recombinant DNA Facility. DNA sequencing kits were purchased from Pharmacia and United States Biochemical. Isotopes were purchased from Amersham Corp. Cloning procedures. Chromosomal DNA was isolated from T. phagedenis Kazan 5 as previously described for Leptospira spp. (31). The DNA was sheared to about 20 kb in size by passage through a needle and then fragmented to

paper paper paper paper

a smaller size by digestion with DNase I in the presence of Mn2" as described previously (12). The DNA fragments were blunt ended by treatment with the Klenow fragment of DNA polymerase. Fragments ranging from 2 to 7 kb in size were selected by electrophoresis through agarose, eluted from the gel, phenol extracted, and precipitated with ethanol. The DNA fragments were methylated with EcoRI methylase. Phosphorylated EcoRI linkers (8-mers; New England BioLabs) were added by treatment with T4 DNA ligase. Excess linkers were removed by precipitation with ethanol. The DNA was digested with EcoRI restriction endonuclease, and the DNA fragments were ligated into the Agtll vector (6, 32). The recombinant molecules were packaged into phage particles with the Packagene system from Promega. The phages were amplified by passage through E. coli Y1088. The amplified phage lysate was used to infect E. coli Y1090, and the resulting plaques were blotted to nitrocellulose membranes impregnated with isopropyl-p-D-thiogalactopyranoside (6, 32). Immunoreactive clones were selected with antiserum directed towards the 39-kDa periplasmic flagellum protein of T. phagedenis (10). Before use, the antiserum was extensively absorbed against a boiled suspension of E. coli Y1090. DNA was isolated from one of the immunoreactive clones, and subclones were prepared in either plasmid pGEM3 or bacteriophage M13mpl8 for sequencing. The gene encoding TmpB of T. pallidum was obtained by subcloning from plasmid pRIT4633 (4). A 1.5-kb HindIII-SstI fragment containing the tmpB gene was subcloned into pGEM3 and used for sequencing. DNA sequencing and analysis. DNA sequencing was done by the dideoxy procedure of Sanger et al. (21). Both singlestranded and double-stranded DNA molecules were used for sequencing. Double-stranded DNA was purified through two cycles of equilibrium centrifugation in CsCl-ethidium bromide before use. Single-stranded DNA was extracted from purified M13 bacteriophage particles as described previously (15). Nested deletions (Erase-a-base kit; Promega) of cloned

VOL. 59, 1991

DNA molecules were prepared to aid in the sequencing procedures (11). [35S]dATP and T7 DNA polymerase (Pharmacia) were used for sequencing double-stranded DNA. Single-stranded DNA was sequenced with [35S]dATP and Sequenase (United States Biochemical). Sequence analyses were done with the DNASTAR program. Hybridization techniques. Southern blotting was performed as described previously (12, 24). Total DNA was extracted from the organisms (31) and digested with restriction endonucleases, and the fragments were separated by electrophoresis in 0.8% agarose with TBE buffer (12). At least 2 ,ug of DNA was used per lane. The DNA was blotted to a Zeta-Probe nylon membrane (Bio-Rad), dried, and prehybridized in 7% SDS-1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate). Hybridization was performed at 65°C in 1% SDS-5x SSC with a DNA probe labelled with 32p. The probe was a gel-purified fragment that was generated by PCR from the tmpA gene of T. phagedenis Kazan 5. The probe was labelled by the random primer method with an oligolabelling kit from Pharmacia (8). The blots were washed at high stringency and exposed to X-ray film (Kodak XAR5). Northern (RNA) blotting was performed as described previously (12). RNA was extracted from the spirochetes by standard techniques (27) and electrophoresed through 1.4% agarose gels containing formaldehyde. Three micrograms of RNA was used per lane. The RNA was blotted to a ZetaProbe nylon membrane and hybridized to a DNA probe labelled with 32p. The probe was prepared by random priming of an internal fragment of either the tmpA gene or the tmpB gene of T. phagedenis Kazan 5 produced by PCR

(8).

Western blotting. Western blotting was performed as described previously (4, 10, 26). The proteins were separated by SDS-PAGE in 10% polyacrylamide gels, blotted to polyvinylidene difluoride membranes (Millipore Corp.), and reacted with specific rabbit antisera. Antisera specific for either the TmpA or the TmpB protein of T. pallidum have been described previously (22). In addition to these previously described sera, we used an antiserum directed against a conserved portion of TmpA. The peptide representing amino acids 277 to 296 from T. pallidum was synthesized, coupled to tetanus toxoid, and used to immunize rabbits. The resulting antiserum was used in Western blots to identify the TmpA proteins from T. pallidum and T. phagedenis. Goat antirabbit serum coupled to horseradish peroxidase was used as the secondary antibody. Color was developed with 4-chloro-1-naphthol in the presence of H202. Nucleotide sequence accession numbers. The sequences reported here have been assigned GenBank accession numbers. The T. phagedenis tmpA gene has been assigned number M58475. The T. phagedenis tmpB gene has been numbered M58563. The T. pallidum tmpB gene has been assigned number M58562. RESULTS Isolation of clones encoding TmpA and TmpB. Eleven clones were selected from a Agtll clone bank of T. phagedenis Kazan 5 by use of rabbit antiserum prepared against the partially purified, 39-kDa, periplasmic flagellar protein from T. phagedenis (10). Restriction enzyme digestion of the DNA obtained from each of these clones showed them to be identical. One was picked for further analysis and DNA sequencing. While we expected sequence analysis to reveal the presence of at least part of a gene encoding the 39-kDa flagellum protein, we found instead genes encoding ho-

TmpA AND TmpB HOMOLOGS OF T. PHAGEDENIS

3687

mologs of the TmpA and TmpB proteins of T. pallidum. Subsequent Western blotting experiments revealed that antiserum to the 39-kDa, periplasmic flagellar protein was strongly reactive with TmpB from T. phagedenis (data not shown). It is likely that the partially purified, 39-kDa, flagellar protein used to raise the initial antiserum was contaminated with the TmpB homolog. Thus, this antiserum reacted with clones containing the gene encoding TmpB. Determination of nucleotide sequences. The nucleotide sequences were determined for both the tmpA and the tmpB genes of T. phagedenis Kazan 5. The nucleotide sequences of these genes and the deduced amino acid sequences of the proteins they encode are shown in Fig. 1. The tmpA gene is 1,032 nucleotides long and encodes a protein of 344 amino acids. The tmpB gene is 1,152 nucleotides long and encodes a protein of 384 amino acids. The initiation codons were chosen on the basis of the sizes of the open reading frames that they preceded and by alignment with the homologs found in T. pallidum. Each gene is preceded by a ShineDalgarno sequence. The gene organization of tmpA and tmpB in T. phagedenis is the same as that in T. pallidum, with the termination codon of tmpA overlapping the initiation codon of tmpB (4). The tmpB gene of T. phagedenis contains a direct repeat consisting of 88 (44 x 2) nucleotides located centrally within the gene (nucleotides 1587 to 1631 and nucleotides 1632 to 1676). A third consecutive repeat of this sequence begins at nucleotide 1677 but ends at nucleotide 1703, before it is completed (Fig. 1). To compare the tmpB gene of T. phagedenis with that of T. pallidum, we also sequenced the tmpB gene of T. pallidum. The nucleotide sequence of this gene and the amino acid sequence of the protein that it encodes are shown in Fig. 2. The gene is 975 nucleotides long and encodes a protein of 325 amino acids. The tmpB gene of T. pallidum also contains a direct repeat consisting of 186 (93 x 2) nucleotides in the central portion of the gene (nucleotides 490 to 582 and nucleotides 583 to 675). A third consecutive repeat of this sequence begins at nucleotide 676 but ends at nucleotide 697 before it is completed (Fig. 2). Southern blot analysis of treponemal DNAs. Since tmpA and tmpB were thought to be specific for the pathogen T. pallidum (4, 22), a Southern blot analysis was performed. A probe was created from cloned T. phagedenis Kazan 5 DNA by PCR with primers corresponding to nucleotides 248 to 269 and complementary to nucleotides 733 to 713 (Fig. 1). This fragment was used as a probe because it lies entirely within the coding region for TmpA, resulting in a specific probe for the tmpA gene, and because the primers needed to produce the probe by PCR gave rise to a single product. Total DNAs from T. phagedenis Kazan 5 and Reiter, T. pallidum, and E. coli were analyzed by hybridization with the probe (Fig. 3). The probe only hybridized to DNAs from the T. phagedenis strains. These results indicate that the cloned DNA was derived from T. phagedenis and that both T. phagedenis strains contain the genetic information needed to encode the TmpA and TmpB homologs. No reaction was seen with T. pallidum or E. coli. As these organisms are not closely related to T. phagedenis, no reaction was expected. Expression of tmpA and hmpB in T. phagedenis. Northern blot analysis was used to examine transcription of the tmpA and tmpB genes in T. phagedenis Kazan 5 and Reiter. A probe for the tmpA gene was produced by PCR amplification of T. phagedenis Kazan 5 chromosomal DNA by use of primers extending from nucleotides 248 to 269 and complementary to nucleotides 733 to 713 (Fig. 1). A probe for the tmpB gene was produced by PCR amplification of Kazan 5

3688

INFECT. IMMUN.

YELTON ET AL. 1 1

ACAATGGAGGTCAATGAGA ,TGAAATTAAAAAGTTTGGTTTTTAGCTTATCCGCCCTTTTC

14 60

15 61

L V L G F T G C K S K A Q A K A E Q E A CTTGTATTAGGATTTACCGGTTGTAAATCTAAAGCACAGGCAAAAGCGGAACAAGAAGCT

120

35 121

54 Q E R K A F M A E N A K I E K R L M Q A CAAGAACGAAAAGCATTCATGGCGGAAAATGCTAAAATTGAAAAAAGATTGATGCAAGCC 180

55 181

74 K N A A T E A E A N V Y Y P E K F A Q I AAAAATGCTGCAACTGAAGCGGAAGCAAATGTATATTATCCCGAAAAGTTTGCACAAATC 240

75 241

94 E D L E K Q S S E A K E Q D D L K K A N GAAGATTTGGAAAAACAATCATCGGAAGCAAAAGAACAGGATGATTTAAAAAAAGCGAAT 300

95 301

114 S L G S A A A D K Y E T L A N K M K I A AGCTTGGGATCTGCTGCCGCCGACAAATACGAGACGTTGGCGAATAAAATGAAGATAGCG 360

115 361

134 N Q R S K I E A N K L A K Y D E E S Y R AATCAACGCTCAAAAATTGAAGCAAATAAACTTGCAAAATATGACGAAGAAAGCTATCGA 420

135 421

154 L G E E A E K K I D G L Y K S D S V A A CTCGGGGAAGAGGCGGAGAAAAAGATTGACGGACTTTATAAAAGCGATTCCGTTGCCGCC 480

155 481

174 L Q T S N E S L N Y Y N K V I D A G Y K TTGCAGACATCAAATGAAAGCCTTATGTACTATAATAAGGTGATAGATGCGGGATATAAG 540

175 541

194 S L S Q D A K K T A D D A K A A L T A V TCTCTTTCGCAAGATGCAAAAAAAACAGCCGATGATGCAAAAGCGGCATTAACGGCGGTA 600

195 601

214 K V A A S L K P Q Q E E A D G I Y A K A AAGGTTGCCGCGAGTTTGAAACCCCAGCAAGAGGAAGCGGATGGAATATATGCTAAAGCT 660

215 661

234 E E A E N S A Q Y E Q S Y G G Y T S A A GAAGAAGCGGAGAATTCCGCTCAATATGAGCAATCATATGGAGGGTACACTTCTGCTGCT 720

235 721

254 Q A Y N D L T Q I I K A K R L E A Q K A CAAGCATATAACGATTTAACACAAATAATTAAGGCAAAACGATTGGAAGCTCAAAAGGCA 780

255 781

274 M Q A A K T K Q E L S A K L A N E A D K ATGCAGGCGGCAAAGACAAAACAAGAACTTTCCGCAAAGCTTGCAAATGAAGCGGATAAA 840

275 841

GAGAGCCCTCTACCTGAAAATGCCGAAGGTTTTTCAAAAGAACCGATAGAAGTTGAACCT 900

295 901

314 L P T D V L N A P Q D E K A E E T V P V CTCCCGACAGATGTGTTAAATGCACCTCAAGATGAAAAAGCTGAGGAAACAGTTCCCGTT 960

M K L

E

S

P

L

P

E

N

A

E

K

G

S

F

L

S

V

K

F

E

S

P

L

I

S

E

A

V

L

E

F

P

34

294

334 E E M N E N S S E E V N G N A E K I E S 315 961 GAGGAAATGAATGAAAATTCTTCGGAAGAAGTAAACGGGAATGCGGAAAAAATTGAATCG 1020

10 1 M K K I F L F L S L 344 335 T E E P I E G G V Q * 1021L ACTGAAGAGCCGATAGAG§§AG§TGTACAATGAAAAAGATTTTTTTATTCCTTTCACTC 1079

FIG. 1. Sequence of the tmpA and tmpB genes of T. phagedenis. The nucleotide sequences of the genes and regions immediately upstream and downstream are shown. The deduced amino acid sequences are also indicated. Potential Shine-Dalgarno sequences are underlined. The region encoding TmpA starts at nucleotide 19 and ends at nucleotide 1050. The gene for TmpB begins at nucleotide 1050 and ends at nucleotide 2201. Nucleotide repeats are underlined with arrows; the partial repeat is underlined with a broken arrow. Amino acid repeats shared with the TmpB protein of T. pallidum are indicated in boldface type. An amino acid repeat occurring 17 times in succession is outlined with boxes.

chromosomal DNA by use of primers extending from nucleotides 1071 to 1085 and complementary to nucleotides 1900 to 1883 (Fig. 1). The results are shown in Fig. 4. Both strains contained a 2.5-kb RNA molecule that hybridized to a probe specific for either the tmpA gene or the tmpB gene. Since this RNA molecule was large enough to encode both proteins and since the nucleotide sequences encoding these proteins overlapped, the results suggest that these genes are cotranscribed into a polycistronic mRNA molecule. Western blot analysis was used to determine whether translation of the tmpA-tmpB mRNA occurs in T. phagedenis (Fig. 5). Proteins reactive with specific antisera to the TmpA and TmpB proteins from T. pallidum were found in both Kazan 5 and Reiter. Their molecular weights differed

from those found for purified TmpA or TmpB from T. pallidum. TmpA from T. phagedenis migrates at 52 kDa; on the basis of its amino acid composition, one would expect it to migrate at about 38 kDa. TmpA from T. pallidum also migrates anomalously on SDS-PAGE gels. It migrates at about 44 kDa but, on the basis of its amino acid composition, should migrate at a position corresponding to approximately 38 kDa (4). This anomalous behavior may be related to the finding that TmpA from T. pallidum is a lipoprotein (23). Presumably, TmpA of T. phagedenis is also a lipoprotein (see below). While this immunoreactive band migrates anomalously on SDS-PAGE gels, several lines of evidence indicate that it is the TmpA homolog of T. phagedenis. It specifically

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VOL. 59, 1991

3689

30 11 V S V L G I T S L F A I S Y D D N E Y S 1080 GTATCCGTTTTAGGAATTACCTCTTTATTTGCAATTTCCTATGATGATAATGAGTATTCA1139 50 R K S R A Y T Q L A E K A Y D A G E Y D 1140 AGAAAAAGCCGCGCGTATACGCAATTAGCGGAAAAAGCATATGATGCCGGAGAGTATGAT1199

31

70 51 T A I E Y S Q L A E N F A Q E S A E Y I 1200 ACCGCAATAGAATATTCTCAACTTGCGGAGAATTTTGCGCAAGAATCCGCAGAGTATATC1259

90 K R MH A R N E A E D A M N K A R T R Y 1260 AAAAGAATGATGGCGAGAAATGAAGCGGAAGACGCGATGAATAAGGCAAGAACCCGCTAT1319

71

91 1320

A

W

A

KQ

Q

K

A

D

K

N

Y

P

T

E

Y

L

I

A

G

110

GCATGGGCAAAGCAGCAAAAAGCCGATAAAAACTATCCGACCGAATACCTTATTGCCGGA 1379

111

130 E A I K A G G I A F D N K N Y D V A V T 1380 GAAGCAATTAAAGCGGGCGGAATAGCTTTTGACAATAAAAACTATGATGTTGCGGTTACC1439

150 C A E K A L E S L K T V E P E D K V I A 131 1440 TGTGCGGAAAAAGCTTTGGAGTCACTGAAAACAGTAGAACCAGAAGATAAAGTTATAGCC1499

EJ|XI RZEW

ElZ

KC ZA

A A 170 IK A A A _l A A A A K 1500 AAAGCAGCTGCTGACAAAGCGGCAGCGGAAAAAGCCGCTAAAGAAAAGGCAGCAAGAGAA1559

151

171

K

S

A K

D_W K

A A

K

EIKI

A A K

EIIK

A

A

K

Di

190

1560 AAGTCAGCCAAAGACAAAGCCGCTAAGGAAAAGGCAGCCAAAGAAAAAGCGGCAAAAGAC1619 191 1620

211 1680

IK A A K EK A A KEK A A K DIK AAAK Rl 210 1679 AAAGC CCAAAQAA&&&GCGGCAAAAGACAAAGC A X Dl 230 A A K El K A A K EIl A A

CGGCAMGMaAGAAGGCGGACGCGAAATGGCGGCAAAGGAAAAAGCCGCTAAAGAT1739

K A A K E I8Z A a R 231 A A Z E A A A R Y A 250 1740 AAGGCAGCTAAAGAAGAAGCGGCACGAAAAGCTGCTGAAGAAGCAGCCGCAAGAAAAGCT1799

251 A Z E A A a R K a A Z E Z a A R I A A E 270 1800 GCTGAAGAAGCAGCCGCAAGAAAAGCCGCTGAAGAGGAAGCTGCAAGAATTGCCGCTGAA 1859 E Z a A R K A A Z EZ A a R X A A Z 271 E A 290 1860 GAGGAAGCCGCTCGTAAGGCTGCCGAAGAGGAAGCTGCACGAAAAGCCGCCGAAGAAGCT 1919

310 291 L Y N E K G E K V L P S E Y K V L T W K 1920 TTGTACAACGAAAAGGGCGAAAAAGTTCTTCCCTCGGAATACAAAGTGCTTACTTGGAAA 1979 L D R E C Y W N I A K N P A V Y N D P F 330 311 1980 TTGGATAGAGAGTGTTTCTGGAATATTGCAAAGAATCCTGCCGTATACAATGATCCCTT 2039

350 331 M W R K L Y E A N K D K I P E S N N P D 2040 ATGTGGAGAAAACTGTATGAAGCAAATAAGGACAAGATCCCTGAATCAAATAATCCCGAC 2099 370 W V E A E T I L V I P S I R G E R R E G 351 2100 TGGGTTGAGGCGGAAACCATCTTGGTAATTCCGAGCATCCGCGGTGAAAGACGTGAAGGT 2159 L Y D P D V K Y Q A L P K T 384 371 2216 2160 CTCTATGACCCCGATGTAAAATATCAGGCACTTCCCAAACGATAAGGAAAG

FIG. 1-Continued.

reacts with anti-TmpA serum prepared against cloned and purified TmpA, it specifically reacts with antiserum prepared against a conserved amino acid sequence from TmpA, and it also reacts with antibody obtained by absorption of antiTmpA serum with purified TmpA, elution of the absorbed antibody, and use of this eluted antibody in a Western blot. The nucleotide sequences of the tmpB genes predict that the TmpB protein from T. phagedenis should have a molecular mass of about 43 kDa and that the TmpB protein from T. pallidum should have a molecular mass of about 37 kDa. Unprocessed TmpB protein from T. pallidum has a mass in SDS gels of 38 kDa (4). The TmpB protein of T. phagedenis migrates at 51 kDa in SDS-PAGE gels. However, the TmpB homolog contains a central region of unusual structure that may prevent it from migrating at a rate proportional to its molecular mass (see below). That this immunoreactive band is the TmpB homolog is shown by the fact that it reacts with specific antiserum prepared against cloned and purified

TmpB and that it also reacts with antibody obtained by absorption of anti-TmpB serum with purified TmpB, elution of the absorbed antibody, and use of this eluted antibody in a Western blot. Alignment of amino acid sequences. The amino acid sequences of the two TmpA proteins were aligned with the algorithm in the DNASTAR program. Each of these proteins contains a leader sequence followed by a site for the addition of lipid (Fig. 6). The site in T. phagedenis is found at amino acids 19 through 23 and reads FTGC. This sequence differs somewhat from the consensus sequence of LXYC, in which X and Y are small, uncharged amino acids (29). TpD (34 kDa), another T. pallidum lipoprotein, uses phenylalanine at the start of the sequence (23, 25). Some lipoproteins contain serine as one of the uncharged amino acids (28, 29); however, this is the first instance of threonine being used as one of the uncharged amino acids. This sequence aligns with the sequence LGSC in T. pallidum at which lipid addition occurs

3690

YELTON ET AL.

INFECT. IMMUN. G

A

S

R

*

M K

T

R

L

V

S

V

L

i

AQAIMCCTCTCGATGAAGACACGTAATTTCTCGCTCGTATCCGCGTTGTACGTACTGCTG

16 60

17 61

G V P L F V S A A S Y D D N E F S R K S GGTGTTCCTCTGTTTGTGTCTGCCGCTTCCTACGACGACAATGAATTTTCTCGCAAGAGT

36 120

1

N

F

S

A

L

Y

L

56 R A Y S E L A E K T Y D A G E Y D V S A 37 121 CGTGCGTACTCGGAGCTTGCAGAGAAGACATACGACGCGGGAGAGTATGACGTCTCTGCA 180

E Y A R L A E D F A Q K S S V Y I K E T 57 76 181 GAGTACGCCCGGCTCGCTGAGGATTTTGCGCAAAAATCCTCGGTCTACATCAAGGAAACT 240

M A R T T A E D A M N A A A T R H G W A 77 241 ATGGCGCGCACCACTGCCGAGGACGCTATGAACGCTGCGGCCACCCGCCACGGCTGGGCG

96 300

97 D R P Y P T E Y L L A S E A I K N E R 301 AAAAATGAGCGCATCGATCGGCCCTATCCGACCGAGTATTTGCTCGCTAGCGAGGCTATC

116 360

117 K T G G L S F D S K Q Y D V A L T W A R 136 361 AAGACCGGAGGGCTCGCTTTTGACAGCAAGCAGTACGACGTAGCGCTCACGTGGGCGCGT 420

137 K A L D A L K N V K P E S Q L L A K A A 421 AAGGCGTTGGACGCACTCAAAAACGTAAAGCCTGAAAGTCAGTTGCTTGCAAAGGCCGCG

156 480

157 K E IB a A R X a a B A R K L E E Q R I A 481 AAGGAGGAGGCTGCGCGCAAGGCCGCCGAGGCACGAAAACTCGAAGAACAAAGAATTGCA

176 540

196 177 A Q K A Q E E R K R A E E E A A R X A A 541 GCCCAGAAAGCGCAGGAAGAACGTAAGCGTGCGGAGGAGGAAGCTGCGCGCAAGGCCGCC 600 2 A R K L E E Q R I A A Q K A Q ,E E RK 197 601 GAGGCACGAAAACTCGAAGAACAAAGAATTGCAGCCCAGAAAGCGCAGGAAGAACGTAAG

217

661

R

A

E

E

,CGTGCGGAGGAgo

A A AR

X

216 660

ZUA A R I A Z E 236 A,AGCAGCGCGAAAGGCGGAGGAA 720

AA

=G >GCGCCCAA,GGCG

256 L E K G R V L P A Q Y K V T T W S I D R 237 721 CTCGAGAAGGGTCGTGTGCTACCTGCGCAATACAAGGTGACTACGTGGTCCATTGACCGG 780

257 781

E

C

F

W

N

I

A

K N

P

A

V

Y

G

N

P

F

L

W

K

276 840

296 K L Y E A N K D K I P Q S K N P N W V E 277 841 AAGTTGTATGAGGCGAACAAGGACAAAATTCCTCAGTCCAAAAACCCCAATTGGGTAGAG 900

P E T V L V I P S L K G E E R E G L Y E 297 901 CCTGAGACAGTCCTGGTCATCCCCAGTCTCAAGGGAGAGGAGCGCGAGGGTCTGTATGAG

316 960

317

325

P

N

V

K

Y

R

P

L

P

*

961 CCCAACGTGAAATACCGTCCTCTGCCGTAACGGATAGACAAGAGGTATACGCTTTTTCCC 1020 1021 CTTTTCCACAAGGGTGCAAGG

1041

FIG. 2. Sequence of the tmpB gene of T. pallidum. The nucleotide sequence of the gene and the regions immediately upstream and downstream is shown. The deduced amino acid sequence is also indicated. A potential Shine-Dalgarno sequence is underlined. The region from nucleotides 1 through 86 is identical to that published previously by Hansen et al. (4). Nucleotide repeats are underlined with arrows; the partial repeat is underlined with a broken arrow. Amino acid repeats shared with the TmpB protein of T. phagedenis are indicated in boldface type.

(23). Overall, these proteins show 42% identical amino acids and 25% similar amino acids over their entire lengths. A very highly conserved amino acid sequence (19 of 20 identical amino acid residues, with a substitution of aspartate for ghutamate at the other position) is found from amino acid 277 through 296 in each of the TmpA proteins (Fig. 6). The TmpB proteins were also aligned (Fig. 7). Alignment was more difficult for these proteins, as they are dramatically different in size. Optimal results were obtained by aligning the first 150 amino accids with each other, aligning the carboxy-terminal 85 residues with each other, and aligning the central regions of the proteins with each other. The carboxy-terminal ends of the proteins show a high degree of sequence conservation. Of the 85 amino acids at the carboxy-terminal ends of these proteins, 75% are identical and 20% are related substitutions. The amino-terminal ends

show a lower degree of sequence conservation. Of the first 150 amino acids, 61% are identical and l9o are related substitutions. Each of the proteins has at its amino-terminal end a leader sequence which may be cleaved following transport of the protein to the periplasmic space (4). The differences in the proteins are found in their central regions. These regions contain a series of repeated amino acid sequences rich in alanine, lysine, and glutamate. A series of identical repeats (EAAKKAAE) are found in both organisms (Fig. 1, 2, and 7). While a series of conserved repeats is seen in the TmpB proteins from both organisms, other amino acid repeats are seen in the central regions of the individual proteins. In T. pallidum, a long amino acid repeat is seen. It extends from amino acids 158 to 188 and is repeated from amino acids 189 to 219 (Fig. 2). Dot matrix analysis revealed additional

VOL. 59, 1991

TmpA AND TmpB HOMOLOGS OF T. PHAGEDENIS

A BC D 21.3 9.4 6.6 4.4 2.3

45

-

31

-

21.5-

B 97.4 66.2 -

FIG. 3. Southern blot analysis of treponemal DNAs. Chromosomal DNAs were digested with restriction endonuclease Hincl, electrophoresed through agarose, and blotted to nitrocellulose membranes. The blots were probed with radiolabelled DNA under conditions of high stringency. The probe was prepared by PCR with primers located within the tmpA gene of T. phagedenis Kazan 5 DNA; the probe represents nucleotides 248 through 733 (Fig. 1). Lanes: A, T. phagedenis Reiter; B, T. phagedenis Kazan 5; C, T. pallidum; D, E. coli. Numbers are in kilobases. repeat motifs within this protein between amino acid resi-

dues 150 and 225. In the case of TmpB from T. phagedenis, a second structure, having the consensus sequence KAAKE/D, is repeated 17 times, beginning with amino acid number 151 and ending with amino acid number 235 (Fig. 1). This additional repeat motif accounts for the extra mass of the TmpB protein from T. phagedenis compared with the TmpB protein from T. pallidum. A series of complex repeat motifs were found by dot matrix analysis between amino acids 150 and 240; a second series of complex repeat motifs were found between amino acids 240 and 290.

DISCUSSION

Agtll clone bank of T. phagedenis

we

have

isolated recombinants which express homologs of the TmpA

A

B

C

D

9.49

7.46 4.40

4

-

0.6

a

3

97.4_ 66.2_

2.0

From

2

A

3691

-

2.37 1.35-

0.24-

FIG. 4. Northern blot analysis of RNA. The probes were labelled with [32P]dCTP and hybridized to the blots at high stringency. Following a high-stringency wash, the blots were exposed to X-ray film overnight. Lanes: A and C, RNA from T. phagedenis Kazan 5; B and D, RNA from T. phagedenis Reiter. Lanes A and B were probed with the tmpA probe (nucleotides 248 through 733; Fig. 1); lanes C and D were probed with the tmpB probe (nucleotides 1071 through 1900; Fig. 1). Each lane contains 3 ,ug of RNA. Numbers are in kilobases.

45

-

31

-

2

3

4

21.5_FIG. 5. Western blot analysis of proteins from T. phagedenis and T. pallidum. Blot A was reacted with rabbit antiserum prepared against a conserved peptide fragment of the TmpA protein from T. pallidum. Lanes: 1, T. pallidum whole-cell extract; 2, purified TmpA protein from T. pallidum; 3, T. phagedenis Kazan 5 whole-cell extract; 4, T. phagedenis Reiter whole-cell extract. Blot B was reacted with rabbit anti-TmpB serum prepared against purified TmpB protein from T. pallidum. Lanes 1, 3, and 4 are as above; lane 2 contains purified TmpB protein from T. pallidum. Numbers are in kilodaltons.

and TmpB proteins of T. pallidum. Several laboratories have assessed the possibility of using cloned genes expressing T. pallidum proteins as serodiagnostic reagents for diagnosing syphilis (11, 19, 22). The results that we report here suggest that this approach must be used with caution. Our finding that nonpathogenic spirochetes possess homologs of at least two envelope proteins of T. pallidum raises questions about the source of the immunogens which induce the formation of antibodies directed against T. pallidum proteins in the human population. Riviere et al. reported finding antibodies in serum from patients with acute necrotizing ulcerative gingivitis that reacted with the 47-, 14-, and 12-kDa proteins of T. pallidum (20). Previously, these proteins were identified as being pathogen specific (2, 3). TmpA and TmpB were also thought to be pathogen specific (4, 22). Antiserum prepared against a whole-cell preparation of T. phagedenis was shown to cross-react with several proteins of T. pallidum, including TmpA and TmpB (26). However, this cross-reactivity was very weak and was thought to be nonspecific. In this study, specific antisera directed against cloned and purified TmpA and TmpB proteins were available. Each of these antisera specifically reacted with the cloned protein which induced its formation and with the homologs from T. phagedenis, as shown in Fig. 5. In the case of the TmpA protein from T. pallidum, previous serological surveys indicated that very few nonsyphilitic individuals contain antibodies which react with this protein (7). Thus, the amino acid homology that we found among the TmpA proteins from various treponemes

3692

YELTON ET AL. 1

*

INFECT. IMMUN. *

*

*

50

MNAHTLVYSGVALACAAMIASGAKEEAEKKAEQRALLVESAHADR

T. pal.

M: .:LV:S AL

.

:.:C S A:..AE:.A.E::A::.E:A. ::R

MKLKSLVFSLSALFLVLGTKSKAQAKAEQEAQERKAFAENAKIEKR

T. phage.

1

*

*

*

*

*

50

*

*

*

100

*

LIMEARIGAQESGADTQHPELFSQIQDVERQSTDAKIEGDLKKAAGVASEAADKYEILRNR LM:A: :A E:.A:. .PE F:QI:D:E:QS::AK ::DLKKA.:::S.AADKYE.L N: LMQAKNAATEAEANVYYPEKFAQIEDLEKQSSEAKEQDDLKKANSLGSAAADKYETLANK *

*

*

*

100

*

*

*

*

150

*

*

VEVADLQSKIQTHQLAQYDGDSANAAEESWALELYETDSAQCLQSTVEALESYRKVAH :.:A: :SKI::::LA:YD. :S . :EE: KK

.LY.:DS.. LQ:: E:L Y.KV:

MKIANQRSKIEANKLAKYDEESYRLGEEAEKXIDGLYKSDSVAALQTSNESLMYYNKVID *

*

*

*

*

200

150

*

*

EGFGRLLPDMXARAGAAKTDVGGLKVAVELRPQLEEADSQYQEAREAEEVNARAKAFSGY

.G: .L .D K A:.AK:.:.::KVA..L:PQ EEAD: Y..A EAE:... .::::GY AGYKSLSQDAKKTADDAKAALTAVKVAASLKPQQEEADGIYAKAEEAENSAQYEQSYGGY * * * * * 200 *

*

250

*

*

*

HRALEIYTELGKVVRLKKTEAEKALQSAKTKQKASSDLARSADKSAPLPENAQGFSKEPI .A: Y.:L.:::: K: EA:KA:Q:AKTKQ. S:.LA..ADK.:PLPENA:GFSKEPI TSAAQAYNDLTQIIKAKRLEAQKAMQAAKTKQELSAKLANEADKESPLPENAEGFSKEPI *

*

250

300

*

*

*

*

*

does not seem to affect the specificity of a serological test (enzyme-linked immunosorbent assay) in which the TmpA protein of T. pallidum is used as the test antigen (7). However, a population of nonsyphilitic patients with acute necrotizing ulcerative gingivitis has not been specifically examined for the presence of antibodies to the TmpA protein of T. pallidum. The gene encoding TmpA from T. phagedenis Kazan 5 consists of 1,032 nucleotides and encodes a protein of 344 amino acids. It contains the tetrapeptide sequence Phe-ThrGly-Cys immediately following a hydrophobic leader sequence. The former sequence allows proteins to be modified by the addition of diacylglycerol and then cleaved by signal peptidase 11 (29). Such lipid modification has been shown to occur with the TmpA protein of T. pallidum when the gene encoding it is introduced into E. coli (23). The termination codon of the gene encoding TmpA overlaps the initiation codon of the gene encoding TmpB. Similar gene overlaps have been found elsewhere. It has been suggested that such overlaps ensure that the two gene products are produced in equimolar amounts (30). Such overlaps are often found when two proteins form subunits of a functional enzyme (30). Whether a similar situation exists with proteins in treponemes requires further study. The T. phagedenis gene encoding TmpB is 1,152 nucleotides long and encodes a protein of 384 amino acids. The gene for TmpB from T. pallidum is 975 nucleotides long and encodes a protein of 325 amino acids. The amino acid sequence of this protein is very similar to that of the TmpB protein of T. phagedenis. Both of these proteins have an extensive a-helical region composed of repeated amino acid sequences, and both contain a leader sequence which directs the protein to the membrane of the cell.

50

*

*

*

*

*

*

100

*

*

EYDVSAEYARLAEDFAQKSSVYIKETMARTTAEDAMNAAATRHGWAKNERIDRPYPTEYL EYD.: EY::LAE:FAQ.S: YIK MAR..AEDAMN A TR.:WAK::: D: YPTEYL EYDTAIEYSQLAENFAQESAEYIKRUOARNEAEDAMNKARTRYAWAKQQKADKNYPTEYL *

50

*

*

*

*

*

100

*

150

LASEAIKTGGLAFDSKQYDVALTWARKALDALKNVKPESQLLA---------------:A:EAIK GG:AFD:K:YDVA:T A KAL::LK.V.PE.:::A IAGEAIKAGGIAFDNKNYDVAVTCAEKALESLKTVEPEDKVIAKAAADKAAAEKAAKEKA *

*

*

150

*

*

------------------------------------------------------------

AREKSAKDKAAKEKAAKEKAAKDKAAKEKAAKEKAAKDKAAKEKAAKEKAARENAAKEK *

*

*

*

*

200

*

*

*

*

200

----KAAKEEAARKAAEARKLEEQRIAAQKAQEERKRAEEEAARKAAE--ARKLEEQRI KAAKEEAARKAAE E AARKAAE ARK AAKDKAAKEEAARKAAE----------------------EAAARKAAEEAARK-----* * 250 *

*

*

250

AAQKAQEERKRAEEEAARKAAEE-AARKA-EE-L--EKGR-VLPAQYKVTTWSIDRECF AA:.... AEEEAARKAAEE AARKA EE L EKG VLP::Y KVTW.:DRECF AAEEEAARIA-AEEEAARKAAEEEAARKAAEEALYNEKGEKVLPSEYKVLTWKLDRECF *

*

FIG. 6. Alignment of the amino acid sequences of the TmpA proteins. The upper line is the sequence from T. pallidum, the lower line is the sequence from T. phagedenis, and the middle line shows the conserved sequence. When necessary, gaps were introduced into the amino acid sequences to enhance the alignment. Conserved amino acids are indicated by letters; related substitutions are indicated by colon. The potential sites for lipid addition and processing by signal peptidase II are underlined.

*

*

*

EVEPLPTDVLNAPQDEKAEETVPVEEMNENSSEEVNGNAEKIESTEEPIEGGVQ *

*

1

*

EVEPLPNDRLNTTQADES-APIPISDTSSPSRVQSRGVEDGGRSPKSSINEEGASR EVEPLP.D LN:.Q.:.: ..:P:.: :. S. : .G .: S...:::.. 300

1

NKTRNFSLVSALYVLLGV-PLFVSAASYDDNEFSRKSRAYSELAEKTYDAG XK : F L :L :LG: :LF A SYDDNE:SRKSRAY::LAEK:YDAG T. phage. NK-KIF-LFLSLVSVLGITSLF--AISYDDNEYSRKSRAYTQLAEKAYDAG

T. pal.

*

*

*

*

*

*

*

300

* *

300

WNIAKNPAVYGNPFLWKKLYEANKDKIPQSKNPNWVEPETVLVIPSLKGEEREGLYEPN WNIAKNPAVY.:PF:W:KLYEANKDKIP:S:NP:WVE:ET:LVIPS: :GE REGLY:P: WNIAKNPAVYNDPFNWRKLYEANKDKIPESNNPDWVEAETILVIPSIRGERREGLYDPD *

*

*

350

*

*

VKYRPLP VKY:: LP

VKYQALPKR

FIG. 7. Alignment of the amino acid sequences of the TmpB proteins. The upper line is the sequence from T. pallidum, the lower line is the sequence from T. phagedenis, and the middle line shows the conserved sequence. When necessary, gaps (-) were introduced into the amino acid sequences to enhance the alignment. Conserved amino acids are indicated by letters; related substitutions are indicated by colons.

The repeated units containing alanine, lysine, and glutamic or aspartic acid in both of the TmpB proteins are unusual. These repeats are not artifacts, since PCR amplification of this region of the chromosome resulted in a DNA fragment of the expected length. A similar amino acid sequence has been reported in the TolA protein of E. coli (9). TolA is an integral membrane protein that is involved in the translocation of colicins and bacteriophage DNA across the membrane (9). The presence of alanine-rich regions within a protein is known to help stabilize a-helical regions of proteins (14). The presence of lysine, arginine, aspartate, and glutamate residues within this region of repeated structure would also help stabilize the a-helical configuration by the formation of salt bridges (13). Chou-Fasman analysis of the TmpB proteins predicts that almost the entire length of these proteins will be in the a-helical configuration (3a). The KAAKE/D repeats seen in TmpB from T. phagedenis would allow the basic residues to stack above and below the acidic residues in an a-helix. This configuration not only would help stabilize the helix but also would confer a net positive charge to the outside of the helix. The role of such a peculiar helical protein in the biology of treponemes will require further study. By analogy with TolA, such a protein might serve as a porin or transport protein for large molecules. The Reiter and Kazan 5 strains of T. phagedenis are

VOL. 59, 1991

closely related. They both have a GC content of 38 to 39%, and their DNAs reassociate to greater than 93% (16). Neither of these strains is closely related to T. pallidum, which has a GC content of about 53%. Reassociation of T. phagedenis DNA with T. pallidum DNA occurs to only 5% (16). Despite the genetic divergence of these organisms, a very high degree of amino acid sequence conservation is seen in their TmpA and TmpB homologs, suggesting that these proteins play important roles in the biology of the treponemes. A determination of their exact role is needed. ACKNOWLEDGMENTS This work was partially supported by USPHS grant DE04645 to N.W.C. and by USPHS grant S07 RR 05433-29. We thank Susan Bartz for technical assistance. REFERENCES 1. Brandt, M. E., B. S. Riley, J. D. Radolf, and M. V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect. Immun. 58:983-991. 2. Chamberlain, N. R., M. E. Brandt, A. L. Erwin, J. D. Radolf, and M. V. Norgard. 1989. Major integral membrane protein immunogens of Treponema pallidum are proteolipids. Infect. Immun. 58:2872-2877. 3. Chamberlain, N. R., L. DeOgny, C. Slaughter, J. D. Radolf, and M. V. Norgard. 1989. Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity. Infect. Immun. 57:2878-2885. 3a.Chou, P. Y., and G. D. Fasman. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. 47:45-148. 4. Hansen, E. B., P. E. Pedersen, L. M. Schouls, E. Severin, and J. D. A. van Embden. 1985. Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli. J. Bacteriol. 162:1227-1237. 5. Hindersson, P., A. Cockayne, L. M. Schouls, and J. D. A. van Embden. 1986. Immunochemical characterization and purification of Treponema pallidum antigen TpD expressed by Escherichia coli K12. Sex. Transm. Dis. 13:237-244. 6. Huynh, T. V., R. A. Young, and R. W. Davis. 1984. Construction and screening cDNA libraries in AgtlO and Agtll, p. 49-78. In D. M. Glover (ed.), DNA cloning: a practical approach, vol. 1. IRL Press, Oxford. 7. I;sselmuiden, 0. E., L. M. Schouls, E. Stolz, G. N. M. Aelbers, C. M. Agterberg, J. Top, and J. D. A. van Embden. 1989. Sensitivity and specificity of an enzyme-linked immunosorbent assay using recombinant DNA-derived Treponema pallidum protein TmpA for serodiagnosis of syphilis and the potential use of TmpA for assessing the effect of antibiotic therapy. J. Clin. Microbiol. 27:152-157. 8. Innis, M. A., and D. H. Gelfand. 1990. Optimization of PCRs, p. 3-12. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., New York. 9. Levengood, S. K., and R. E. Webster. 1989. Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in

Escherichia coli. J. Bacteriol. 171:6600-6609. 10. Limberger, R. J., and N. W. Charon. 1986. Treponema phagedenis has at least two proteins residing together on its periplasmic flagella. J. Bacteriol. 166:105-112. 11. Lukehart, S. A., S. A. Baker-Zander, and E. R. Gubish, Jr. 1982. Identification of Treponema pallidum antigens: compari-

TmpA AND TmpB HOMOLOGS OF T. PHAGEDENIS

3693

son with a nonpathogenic treponeme. J. Immunol. 129:833-838. 12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 13. Marqusee, S., and R. L. Baldwin. 1987. Helix stabilization by Glu- Lys' salt bridges in short peptides of de novo design. Proc. Natl. Acad. Sci. USA 84:8898-8902. 14. Marqusee, S., V. H. Robbins, and R. L. Baldwin. 1989. Unusually stable helix formation in short alanine based peptides. Proc. Natl. Acad. Sci. USA 86:5286-5290. 15. Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol. 101:20-78. 16. Miao, R., and A. H. Fieldsteel. 1978. Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes. J. Bacteriol. 133:101-107. 17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Promega Corp. 1990. Biological research products: protocols and applications. Promega Corp., Madison, Wis. 19. Radolf, J. D., E. B. Lernhardt, T. E. Fehninger, and M. A. Lovett. 1986. Serodiagnosis of syphilis by enzyme linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D. J. Infect. Dis. 153:1023-1027. 20. Riviere, G., M. Wagoner, S. Baker-Zander, and S. Lukehart. 1990. Abstr. Annu. Meet. Am. Soc. Microbiol. 1990, abstr. D-64, p. 91. 21. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 22. Schouls, L. M., 0. E. Usselmuiden, J. Weel, and J. D. A. van Embden. 1989. Overproduction and purification of Treponema pallidum recombinant DNA-derived proteins TmpA and TmpB and their potential use in serodiagnosis of syphilis. Infect. Immun. 57:2612-2623. 23. Schouls, L. M., R. Mout, J. Dekker, and J. D. A. van Embden. 1989. Characterization of lipid-modified immunogenic proteins of Treponema pallidum expressed in Escherichia coli. Microb. Pathog. 7:175-188. 24. Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-506. 25. Swancutt, M. A., J. D. Radolf, and M. V. Norgard. 1990. The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein. Infect. Immun. 58:384-392. 26. van Embden, J. D., H. J. van der Donk, R. V. van EiJk, H. G. van der Heide, J. A. de Jong, M. F. van Olderen, A. D. Osterhaus, and L. M. Schouls. 1983. Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12. Infect. Immun. 42:187-196. 27. Von Gabain, A., J. G. Belasco, J. L. Scholtell, A. C. Y. Chang, and S. N. Cohen. 1983. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc. Natl. Acad. Sci. USA 80:653-657. 28. Watson, R. J., P. C. K. Lau, T. Vemet, and L. P. Visentin. 1984. Characterization and nucleotide sequence of a colicin-release gene in the hic region of plasmid ColE3-CA38. Gene 29:175-184. 29. Wu, H. C., and M. Tokunaga. 1986. Biogenesis of lipoproteins in bacteria. Curr. Top. Microbiol. Immunol. 125:127-157. 30. Yanofsky, C. 1984. Comparison of the regulatory and structural genes of tryptophan metabolism. Mol. Biol. Evol. 1:143-161. 31. Yelton, D. B., and N. W. Charon. 1984. Cloning of a gene

required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli. Gene 28:147-152. 32. Young, R. A., and R. W. Davis. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80:11941198.