Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids

Abstract

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 morphologically 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 expression 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 autoradiography of incorporated L-[35S]methionine (37 TBq/mmol; Amersham, France SA), after conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 10% discontinuous system (11), and this revealed the presence of four major protein bands. In comparison to molecular size markers, these corresponded to 32, 28, 25, and 21 kDa (Fig. 1). 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). The entire nucleotide sequence, comprising 2,647 bp (Fig. 2), was determined for both strands. Figure 2 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 consider...

Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola

“…The ribosome binding site proposed for the msp gene is highly complementary to published sequences of the 3Ј ends of the 16S RNA of E. coli and several spirochetes, with the exception of T. denticola ATCC 33520 (42,49). However, the 16S RNA sequence reported for T. denticola ATCC 33520 is 60 to 70 bp shorter than those reported for other spirochetes, suggesting that this sequence may be incomplete.…”

High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori

“…2). In addition to exhibiting these homologies, the H. pylori vapD nucleotide sequence was 70.9% identical to a portion of plasmid pTD1 from Treponema denticola (28); however, the reported T. denticola sequence did not encode an ORF. (18), N. gonorrhoeae plasmid ORF5 (25), and H. influenzae vapD (15).…”

The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes

“…Some sequences of cp8.3 were similar to those of plasmids with a rolling-circletype replication (15,28). A circular plasmid of the spirochete Treponema denticola appears to use this method of replication (24).…”