Isolation and characterization of a plasmid from Treponema denticola

Ivic, A

doi:10.1016/0378-1097(91)90156-5

Cited by 16 publications

(16 citation statements)

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“…The existence of DNAfilled membrane vesicles on the surfaces of Borrelia burgdorferi cells has been reported, although their possible role in gene transfer is unknown (7). In addition, several extrachromosomal elements have been identified in B. burgdorferi, and an extrachromosomal plasmid of 2.6 kb has been isolated from Treponema denticola and characterized (10,25). In cultures of various spirochetes, bacteriophages have been observed free, attached to cells, or within cells as noted previously (9).…”

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confidence: 69%

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae

et al. 1997

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Serpulina hyodysenteriae B204 cells treated with mitomycin (20 g of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsCl density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm 3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (⌬flaA1 593-762::cat) were added to growing cells of strain A216 (⌬nox 438-760::kan), transductants (Cm r Km r ) were obtained at a frequency of 1.5 ؋ 10 ؊6 per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.Natural gene transfer mechanisms (conjugative plasmids and transducing bacteriophages) have not been demonstrated for spirochetes. Indirect evidence of lateral gene transfer has been obtained for Borrelia spp. (16). The existence of DNAfilled membrane vesicles on the surfaces of Borrelia burgdorferi cells has been reported, although their possible role in gene transfer is unknown (7). In addition, several extrachromosomal elements have been identified in B. burgdorferi, and an extrachromosomal plasmid of 2.6 kb has been isolated from Treponema denticola and characterized (10,25). In cultures of various spirochetes, bacteriophages have been observed free, attached to cells, or within cells as noted previously (9). Three lytic phages of Leptospira biflexa have been isolated and characterized (24). However, to our knowledge, there is no evidence that these three phages play a role in gene transfer.The spirochete Serpulina hyodysenteriae causes swine dysentery, an enteric disease producing a severe mucohemorrhagic diarrhea in infected swine (8). Potential genetic transfer elements for S. hyodysenteriae have been identified. Ritchie and colleagues observed bacteriophages with the same morphology in 18 different cultures of S. hyodysente...

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Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae

et al. 1997

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“…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.…”

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confidence: 99%

“…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.…”

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

1992

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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...

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“…The pTS1 plasmid of T. denticola contains ≈37.5 % G+C pairs (Ivic et al 1990) while, in the case of the pTD1 plasmid, in T. denticola it is 34.2 % G+C pairs (Chauhan et al 2004) only. The relatively high G+C content of the pKT plasmid is thus much more similar to the value of 53 % of the causative agent of syphilis, T. pallidum, than to other rumen treponemes (37-46 %; Canale-Parola et al 1977).…”

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confidence: 95%

Genome organization of Treponema zioleckii

et al. 2008

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Genome analysis of Treponema zioleckii proved that, in this bacterium, besides chromosomal DNA, a relatively small extrachromosomal DNA element is present. This element was shown to be a double-stranded circular plasmid DNA of approximately 7 kbp; it was designated as pKT. The plasmid was characterized by molecular and bioinformatic analysis. No pKT homologous DNA sequences were detected in other rumen Treponema strains. The overall G+C content of the pKT plasmid is approximately 56 %, which is higher than in other Treponema plasmids or genomes. The Rep module of the pKT plasmid consisting of the rep gene and the region of repeats was identified within a 1.6-kbp fragment. The putative rep gene encodes the replication protein belonging to the pfam04796 RepA_C family of proteins with the highest similarity (25 % within 249 amino acids) to the RepA protein from the green sulfur bacterium Prosthecochloris aestuarii.

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Isolation and characterization of a plasmid from Treponema denticola

Cited by 16 publications

References 4 publications

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae

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

Genome organization of Treponema zioleckii

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