Homology of a Plasmid from the Spirochete Treponema denticola with the Single-Stranded DNA Plasmids


The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate. 
The order Spirochaetales comprises two families of mor- phologically similar but otherwise diverse bacteria (9, 16); these include species that are pathogenic for both humans and animals. Despite their medical significance, genetic manipulation of these organisms is still at a preliminary level, in part because of the lack of any known system of genetic transfer. The identification of a small, circular 2.6-kb plasmid, pTD1, in the cultivable oral spirochete Treponema denticola raised the possibility of a plasmid-based genetic transfer system within this group of bacteria (8). As the first step in the development of pTD1 as a shuttle vector, we determined its nucleotide sequence to distinguish between essential regions required for replication and nonessential areas that would be suitable as cloning sites. This is the first reported sequence of an extrachromosomal element from within this genus. 
Identification of proteins encoded by pTDI. Gene expres- sion in pTD1 was demonstrated in a cell-free prokaryotic DNA-directed in vitro transcription-translation assay, based on a 30S ribosomal Escherichia coli extract (Amersham, France SA). The gene products were identified by autoradi- ography of incorporated L-[35S]methionine (37 TBq/mmol; Amersham, France SA), after conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 10% discon- tinuous system (11), and this revealed the presence of four major protein bands. ). These size estimations suggest total coding regions in the plasmid of approximately 3,300 bp. Since the plasmid is only 2,647 bp long, the amino acid sequence of each of the four protein products cannot be unique. This could be a consequence, for example, of the initiation of translation at alternate sites within a coding region. 
pTD1 structure and DNA sequence analysis. Plasmid pTD1 was obtained from T. denticola ATCC 33520 which was maintained anaerobically as previously described (8). ), was determined for both strands.  indicates the restriction endonuclease sites utilized to subclone fragments of pTD1 into pTZ18R (Pharmacia) for sequencing by the dideoxy method of Sanger et al. (19). The methods used for ligation, transformation, preparation of single-stranded DNA (ssDNA), running of agarose gels, and restriction endonuclease reactions were as described by Sambrook et * Corresponding author. 

al. (18). The relative ease with which clones could be made, and also their stability, varied considerably; ssDNA could not be obtained from clones carrying some regions of the plasmid. These latter regions were sequenced from double- stranded templates obtained by transformation of the recom- binant plasmids into E. coli XL-1 Blue (endAl hsdRI7 supE44 thi-1 A-recAl gyrA96 relAI[F'proAB lacIZlM15 TnlO]) and Qiagen preparation of the DNA (Hybaid Ltd.). 
The nucleotide sequence was searched for regions con- taining inverted repeats of 10 bp or more, and three such regions were found. Between bp 152 and 344 are one inverted repeat of 10 bp and two 10-bp direct and two 10-bp indirect repeats with greater than 80% homology (Fig. 2). A further three inverted repeats, the longest of which spans 20 bp, occur at the end of the sequence. Such regions have the potential to form hairpin loops and secondary structures in RNA transcripts and may also be important for regulation of plasmid replication (3). 
Analysis of the sequence revealed the presence of three open reading frames (ORFs) of more than 50 codons. The largest ORF, A, extends from bp 167 to 1220 and contains 336 codons (Fig. 2). The first ATG codon in ORF A is at bp 212 to 215. A protein expressed from ORF A, initiated by this codon, would have a molecular weight of 38,999; however, this does not correspond to the data from the in vitro assay, according to which the largest protein product has an apparent mobility of 32 kDa (Fig. 1). The sequence of the 3' end of the 16S rRNA of T. denticola is 5'-CCGAAG GUACGUUU-3' (15). The sequence 5'-ACCT-3' is thus a possible ribosome-binding site (20), 7 bp from the ATG at bp 212 to 215 (Fig. 2). A sequence reading 5'-GAAG-3', which bears a resemblance to the consensus sequences described for E. coli ribosome-binding sites, occurs at bp 371 to 374, 17 bp from the third ATG codon at bp 392 to 394. If this served as a site for translation initiation in E. coli, it would account for the accumulation of a protein smaller (32 kDa) than that predicted (39 kDa) in the in vitro transcription-translation assay. 
ORF A homology with replicative proteins and conservation of the active site. Comparison of ORF A both as a nucleotide sequence and as a peptide sequence with the EMBL (release 26), GenBank (release 66), and NBRF (release 27) data banks showed similarity between this ORF and the se- quences for the replicative (Rep) proteins from Bacillus subtilis plasmid pBAA1 (4), pFTB14 from B. amyloliquefaciens (14), pLAB1000 from Lactobacillus hilgardii (10), pTB913 from Bacillus sp., and pMV158 from Streptococcus of the reaction product loaded in lanes 1 to 3, respectively. Protein molecular sizes are given in kilodaltons. The arrows mark the positions of the proteins thought to be the products of ORFs A (32 kDa) and B (28 kDa). agalactiae (21). If two peptide sequences were aligned by class of amino acid (e.g., hydrophilic) rather than by strict conservation of the amino acid, the level of homology between the protein encoded by ORF A of pTD1 and the Rep protein of pBAA1 could be increased to 45%, and that with the other three Rep proteins could be increased to approxi- mately 40% (Fig. 3A). 
An interesting feature of this homology search was that these replicative proteins are all products of plasmids from the group known as ssDNA plasmids, which are highly interrelated and widespread in gram-positive organisms (6). The Rep proteins of those plasmids which share the pBAA1- type consensus sequence for the origin of replication have a conserved amino acid motif which is also present at the active site of the bacteriophage 4~X174 Rep protein (6). Figure 3B shows that this motif is also highly conserved within ORF A of pTD1, at amino acids 217 to 225. 
ORF B, which is encoded by the strand opposite to that which encodes ORF A, commences at bp 2297 and termi- nates at the TAA at bp 1590. The first ATG codon is at bp 2297 to 2295. A protein initiated at this codon would have a predicted molecular weight of 27,000 and could correspond to the 28-kDa protein band observed in the in vitro transcrip- tion-translation assay (Fig. 1). A hypothetical T. denticola ribosome-binding site, 5'-GACT-3', is within 3 bp of this ATG (Fig. 2), immediately adjacent to an E. coli ribosome- binding site. Computer searches revealed weak homology between ORF B and the plasmid-encoded recombinase proteins in pG12 from B. thuringiensis (13) and pLB4 from L. plantarum (2), the "hypothetical protein" from Staphy- lococcus aureus plasmid pC403 (7), and regions of plasmids pTB913 and pMV158 (21). 
The third ORF, C, runs from bp 2567 to 2316. Although this ORF is large enough to code for a peptide of 84 amino acids, in the absence of a product of the predicted molecular weight from the in vitro transcription-translation assay it was concluded to be a noncoding region. 
Features of ssDNA plasmids. ssDNA plasmids are alike in that they all replicate via an ssDNA intermediate, by rollingcircle replication. Three plasmid-encoded elements are essential for rolling-circle replication: the plus origin, a repli- cation protein (Rep), and a minus origin. The sequence homology between ORF A and known Rep proteins suggested that it too could function as a Rep protein and that pTD1 is an ssDNA plasmid. Further examination of the sequence revealed that there are also regions in pTD1 which could function as the plus and minus origins. In ssDNA plasmids, the minus origin serves as an efficient initiation site, recognized by host factors for conversion of circular plus-strand ssDNA to double-stranded DNA, and marks the completion of one productive cycle of replication. One major feature of the minus origin is the presence of palindromic sequences forming secondary structures. One such region of the pTD1 sequence is located between bp 2306 and 2467 (Fig. 2). 
Plus origin of replication. Consensus sequences for the plus origin of replication in ssDNA plasmids fall into three groups. There are two regions within the sequence of pTD1 that have a degree of homology with known plus origin sequences from plasmids which have bacteriophage +X174type Rep proteins (6). One is within ORF A, from bp 915 to 923, and has the sequence 5'-CTCTTGATA-3'. For compar- ison, the sequence of the origin of plasmid pBAA1 is 5'-CT-ATCTTGATA-3' and that of bacteriophage +X174 is 5'-CCCGCAAC ¶TGATA-3'. The other is upstream of ORF A, between bp 2338 and 2351, and has the sequence 5'- lTr1CTTCTTAGATA-3'. This latter region shows better homology over a longer-length sequence and is rich in palindromic motives (Fig. 2) which would allow formation of hairpin loops and secondary structures, which are common features of this type of origin. The plus origin is typically situated either upstream from or within the Rep protein (6), so each of the above-described regions satisfies this crite- rion. 
Conclusion. According to the criteria specified by Gruss and Ehrlich (6), plasmid pTD1 has sufficient features in common with ssDNA plasmids to be classified as a novel member of this group. Identification of pTD1 as an ssDNA plasmid has been based on homologies found between both nucleotide and peptide sequences; however, the high inci- dence of unstable clones and deletions may be another indication of its ssDNA nature (6). Replication of pTD1 through an ssDNA intermediate could be confirmed, using B. subtilis as the host, by the observation in vivo of pTD1 ssDNA. Alternatively, hybrid derivatives of pTD1 containing inserts of foreign (e.g., from E. coli) DNA should yield readily observable high-molecular-weight tandem multimers (5). The presence of a plasmid with ssDNA features suggests that expression of spirochete genes, previously reported to be poor in E. coli (1,12,17,22), may be higher in a gram-positive host and that use of shuttle vectors designed 
G D N G K K. V K R .L K K T S. G K. K R M .N G Y L S K T AAAAATGTaTGACAATTATCGGGGATAATGAAAAAAAAGTCAAGCGATTAAAAAAAATAGATGAAAAAAAGAGAATGAACGAATACCTTTCTAAAAC 201 1 10 20 30 Y R K LGE G K K A. K K L C A N H F. V F A .E G K I I E A. M Y C
CTACAGAAAGCTAG,G'GXTTAAGAAAGAcTAAAGAT, TAAAAbXTTTGTGCTAATCATTTTGTTTTTGCCGAAGGTAAAATAATCGAGGCTATGTATTGC 
M C E .F R R S F E G. T F K 1A L E I T V N. K K T .G E Y H P H Y. H I L .
TATGTGTGAATTTAGACGTTCTTTTGAAGGGACTTTTAA GCTTTAGAAATCACTGTAAACAAAAAAACAGGTGAGTATCATCCGCACTATCATATCTTG 601 140 HindIII 150 160 TAAGGAATAGCTCGCGGAAAAAATCGAGAAAAAAGACTTGAAAACTATTACGCAAACTTTTAAAGTTTTCACGAAGAACA'AATCGTAAATTAACAAAAAG 
A A V K K G Y. F R K .S N P D Y I S. Q E N .L I K L W Q K. V C K .L D Y E GCCGCTGTTAAAAAAGGTTATTTTAGAAAATCAAATCCTGATTATATATCTCAAGAAAATCTTATAAAATTATGGCAAAAGGTTTGTAAATTGGACTATG 701 170 180 190 P N V D I R .R V K N S T Y. K A V .A E V A K Y S_ V K A ,T _ Y I. K D E. AGCCGAATGTCGATATAAGACGTGTTAAGAACTCAACATATAAGGCTGTTGCTGAAGTTGCAAAATACAGCGTTAAAGCGACCGATTACATCAAGGACGA 801 200 210 220 230 E I V .K T L D K V L. F R R .R L L A Y G G. I F Q .V V R K K L K. L R R . GGAAATAGTCAAAACTCiTTGAT,AAAGTGCTGTTTAGACGCCGTTTATTGGCTTATGGAGGCATTTTTCAGGTAGTCCGTAAAAAATTAAAGCTTCGAAGA 901 240 250 260 R A D R R H D. S F N .A S Q I L A N. P N I .V K E I Y K W. N F G .T K T Y AGAGCCGATCGAAGGCATGACTCTTTTAATGCTTCTCAAATATTGGCAAATCCGAATATCGTGAAAGAGATTTACAAGTGGAATTTCGGAACTAAGACTT 1001 270 280 290 E L V A K T .K L N L N K K. R L S .P V G L I P T. Q S S .L K N P1 G A T.
F E K D L A K K L E K K 0 V K S L A N S F K L L A E 0 K A N L Q K E 1601 230 220 210 200 AAACAG %CGGTTAAGAACGGGAAAAAATCGATTTATCAGGGACCTTAATTCCACAAATCGGAATCATTAGCGACGGCCAGCAGGAGATAGGTAGAATTTA K D A. L E 0 .G K K A L Y D. R S N .L H K A K T I. A A P .R G R D M K F. 1701 190 180 HindIII 170 AAAACAGGCTGTAAAAACATTTTTAGCAGAACTTCGAGATAGTAAAATACAAAACTCGGCGGCTACATCATCAAACGTAATTCGA,AAAAAAAGAAATTCGC K 0 G .V N K Y F D D. 0 L E .I M K H K S G. G I Y .Y N A N L 1K K. E K L. 1801 160 150 140 CGTATTTTTAGTATACGTTTACACC CCAACGAAGTAGGGACACTTACGT CCGTGAATCTTCTTAAAGTGCCGGAGGTTCGACCAAAAGTCGGGTT CGATT P M F I M H L. H P T .A E D R H I C. A S L .L I E R G G L. R N E .A W A L 1901 130 120 110 100 TCTGACCAACTTCATCAAAAAAACGAGAACTTTCCAGTAAAAACTTTCTTAAAGTCCTCTGCGCCAATACTCTTAAAGATGTCGCACTTGACGTAAAAAA S Q N. F Y N .K Q E 0 F T M. K Q L .I E P S A T I. L I E .V A H V A N K. 2001 90 80 70 GCGTGAAAAAGTCCTTCGACAAAAAACATTTAAAGGTTTCGGAGTAGTATGCTCAATTTTATGACTAAATCTAAACTAAAAAGTCCTAtCCGTAAGAGTC R V K .E P L 0 K K Y. I E L .A E D Y S N F. Y Q N .L N S K E P N. A N E 2101 60 50 40 CAACTA1tATAAGAACTCAATACGAAGTTAGCGGAAGCCTGTCGATAACGGCTGAACTAAAAAACTTTTGCGTCCTATGTTTTCAATAACGAACGGTATTr P 0 N I R S N. H K L .A R R .V A I A. S K I .K 0 F R L I C. F N N .S A M 2201 30 . . . ,20 . . 10 . 
GTCATTCTACCGTATCaAAGAAT)AAATATCAATGCT-oCTTTCTTCTTAGATACGGACTTACTCCGGTTTTTAACAAAACCAGTAAGCCAAAGGGGCATACT- are marked with vertical arrows, and the primers constructed to complete the sequence are marked with asterisks. The primer sequences read as follows: I, 5'-TGGCATTCTCAGGTTGGTTGATT-3' (bp 2189 to 2207); II, 5'-GGCATAGTAGCAAAGGA-3' (bp 2472 to 2488); III, 5'-TAGTCGAAATTCAGCT-3'(bp 2569 to 2584); IV, 5'-ACCTAGCTTTCTGTAGG-3' (bp 300 to 316); V, 5'-TTGCTATGAATAAAGGC-3' (bp 70 to 86). The bases at the start and finish of each of the three ORFs are underlined, as are the ATG start codons and the conserved amino acid region. Potential ribosome-binding sites are indicated by brackets, and the two possible locations of the plus origin of replication are indicated by half brackets. The positions of repeats are indicated by horizontal arrows. The translations of the ORFs are in the single-letter amino acid code (the first base of each codon) at the top for ORF A and the bottom for ORF B. The numbering at the amino acid level is given below every 10th letter. ---------------------------- Comparison is with the active sites of replicative proteins from bacteriophage (X174 and other ssDNA plasmids that share the pBAA1-type origin of replication (6). Conserved amino acids are in boldface. 
for gram-positive organisms could enable genetic transfor- mation within spirochetes. 
Nucleotide sequence accession number. The sequence shown in Fig. 2 has been submitted to GenBank and assigned accession no. M87856. 

FIG. 1 .

 1 FIG.1. In vitro transcription-translation products of pTD1 in a sodium dodecyl sulfate-10% polyacrylamide gel, with 6, 4, and 2 PI1 


I

  Downloaded from https://journals.asm.org/journal/jb on 01 October 2023 by 149.165.159.222. 




  L G-P V. C S W .R R A D R T F. H N V .F Y I I S Q P. Ej F K .D L . T L T .V K N C S A 1D. E L P .A T L E M M T. K G W .R R L A M T A. 


FIG. 2 .

 2 FIG.2. Complete nucleotide sequence of pTD1 linearized through the unique SpeI site at bp 1. Restriction enzymes utilized for cloning 


FIG. 3 .

 3 FIG. 3. (A) Alignment of the deduced amino acid sequence of the ORF A gene product and the Rep proteins from four different ssDNA plasmids. The amino acid sequences, in the single-letter code, were aligned to find homology by class of amino acid. Numbers indicate amino acids omitted to maximize homology; dashes show where insertions are required. Triangles indicate amino acids common to all five sequences, and asterisks mark conservative substitutions of amino acids. The conserved amino acid motif of the pBAA1 Rep protein is underlined. (B) Conserved amino acid motif of the putative Rep protein from ORF A of pTD1. Comparison is with the active sites of 




  Ala Lys Tyr Ser Val Lys Ala Thr Asp T. denticola Ala Lys Tyr Val Asn Lys Lys Ser Asp E. coli Ser Lys Tyr Pro Val Lys Asp Thr Asp B. amyloliquifacices Ala Lys Tyr Gln Val Lys 6gap Asn Asp L. hilgardii Ala Lys Tyr Pro Val Lys Asp Thr Asp S. aureus Ala Lys Tyr Pro Val Lys Asp Thr Asp Bacillus sp. S. agalactiae Ser Lys Tyr Pro Val Lys Asp Thr Asp B. subtilis 


		FSI IAMWNWERKNYFIKE
	PBAA1	GYGGILKEIHKELNLGDAEDGDLVKIEEEDDEVANGAFEVMAYWHPGIKNYI IK
		B pTDI
		*X174
		pFTB 14
		pLABIOOO
		pBD64
		pTB913
		PMVI58 pBAAI I

Downloaded from https://journals.asm.org/journal/jb on 01 October 2023 by 149.165.159.222. 

We thank Barrie Davidson for critical review of the text and many helpful suggestions with the sequence analysis and Iain Pemberton for oligonucleotide III. We are also grateful to Charles Penn and Roy Russell for continued interest in and encouragement of this project. 
This work was supported by CNRS (UA1129) and by an ADIP fellowship (Institut Pasteur) awarded to Jane MacDougall.
Jane Macdougall, AND ISABELLE SAINT GIRONS Danielle Margarita
Homology of a Plasmid from the Spirochete Treponema denticola with the Single-Stranded DNA Plasmids