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Applied and Environmental Microbiology, October 2008, p. 6461-6464, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.01424-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of a Transposon Mutagenesis System in the Oral Spirochete Treponema denticola{triangledown}

Yu Yang,1 Philip E. Stewart,2 Xiaoguang Shi,1 and Chunhao Li1*

Department of Oral Biology, State University of New York at Buffalo, Buffalo, New York 14214,1 Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 598402

Received 25 June 2008/ Accepted 11 August 2008


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ABSTRACT
 
Here, we report successful transposon mutagenesis in the oral spirochete Treponema denticola. A modified Himar1 transposon, including a new antibiotic selection cassette for T. denticola, generated mutations affecting cell division, transport, and chemotaxis, among other processes. This random mutagenesis system should facilitate research on the biology and pathogenesis of this spirochete, which is associated with human periodontal diseases.


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INTRODUCTION
 
The anaerobic spirochete Treponema denticola lives among the human oral microflora (3, 26). Clinical and basic studies have shown that this spirochete is highly associated with human periodontal diseases (6, 24). T. denticola produces many virulence factors that are involved in adherence and invasion and in cytotoxicity to a variety of different host cells, e.g., human gingival fibroblasts and immune cells (6, 7, 24). The recent decoding of the genome of T. denticola identified potential virulence genes encoding internalin-like proteins, bacteriocin, ABC transporters, and dipeptide YD repeat proteins (22, 25). Understanding the roles of these genes in the biology and pathogenesis of T. denticola has been hampered by a paucity of genetic tools and the fastidious growth requirements of the organism (3, 4).

Transposon mutagenesis is a powerful tool to study gene function and permits the generation of large numbers of independent mutations throughout the entire genome of an organism. The creation of mutant libraries, followed by in vitro and in vivo analyses, has been applied to the study of the biology and pathogenesis of a variety of microorganisms, and many new virulence factors have been identified using this approach (12, 13, 17, 18, 28). Recently, the Himar1 mariner transposon (11) has been used successfully for mutagenesis in the spirochete Borrelia burgdorferi and two Leptospira species (2, 16, 19, 27). The successful adaptation of transposon mutagenesis for these spirochetes implied that the Himar1 mariner transposon might be successfully adapted for use in T. denticola. Here, we describe how the Himar1 transposon was modified and used successfully in T. denticola.


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Development of a functional transposon mutagenesis system in T. denticola.
 
In B. burgdorferi, the transposon vector pMarGent was used previously to create saturated, random libraries (19, 27). However, this vector contains a gentamicin resistance marker, aacC1, which thus far had not been utilized in T. denticola. In order to apply aacC1 to mutagenesis of T. denticola, we first evaluated the sensitivity of T. denticola to gentamicin by measuring the MIC of gentamicin as described before (23). The lowest tested MIC of gentamicin for T. denticola is approximately 20 µg per ml. Initial attempts to introduce pMarGent into T. denticola failed. Within this vector, the expression of both Himar1 and aacC1 is driven by flgBp, which is a motility-specific promoter in B. burgdorferi (27). One potential reason for the failure may be the dysfunction of flgBp in T. denticola.

To overcome this potential problem, we modified pMarGent by replacing flgBp with tap1p, which is a well-characterized promoter in T. denticola (15). To do so, the fragment containing tap1p was amplified by PCR with two primers (Tap1F, 5'-AGCTGGGCCCGCGGCCGCTATTATACTTCTCCTTAAAACAGC-3', where underlining corresponds to an NotI site, and Tap1R, 5'-AGCTCCATGGATGAACCTCCATAAAAACTTTTTGC-3', where underlining corresponds to an NcoI site) and respectively fused to Himar1 and aacC1 via multiple-step cloning. The resulting vector (pTapMarGent), which contained Himar1 and the inverted repeats flanking aacC1 (Fig. 1), was used to deliver Himar1 into T. denticola strain ATCC 35405 (the wild type). This strain was provided by H. Kuramitsu and has been used in previous studies (14). The genome of this strain was recently sequenced (25). Transformation was performed by electroporation as described before (14, 15). Briefly, a 50-µl sample of T. denticola competent cells (~1010 spirochetes/ml) was subjected to electroporation in the presence of 2 µg (in the first attempt) or 10 µg (in the second attempt) of plasmid DNA. The resultant transformants were transferred into 4 ml of tryptone-yeast extract-gelatin-volatile fatty acids-serum (TYGVS) medium. After a 12-h incubation at 37°C in an anaerobic chamber (14, 15), cells were collected by centrifugation, resuspended in 100 µl of fresh TYGVS medium, and plated onto soft agar containing 40 µg of gentamicin/ml. After a 7- to 10-day incubation at 37°C in an anaerobic chamber, a total of 109 gentamicin-resistant colonies (30 colonies from the first attempt and 79 from the second) were obtained. The average transformation efficiency (approximately 10 transformants/µg of input DNA) was much lower than that for B. burgdorferi (27). Thirty colonies were selected at random and transferred into 5 ml of TYGVS medium for further analyses as described below. The liquid culture derived from an individual colony was referred to as a clone.


Figure 1
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  		FIG. 1. Construction of the Himar1 delivery vector pTapMarGent for T. denticola. This vector was constructed by modifying the vector pMarGent, which was used previously for the delivery of Himar1 in B. burgdorferi (27). The flgB promoter of B. burgdorferi on pMarGent was replaced with the tap1 promoter of T. denticola. IR, inverted repeats. P1 and P2 are primers for sequencing the transposon insertion site after recovery from E. coli. (Adapted from reference 27.)

All 30 clones were positive for aacC1 by PCR with primers GenF (5'-CATATGTTACGCAGCAGCAACGATGT-3') and GenR (5'-GCTAGCCGATCTCGGCTTGAACG-3'), but the parental T. denticola strain was not. This result indicates that aacC1 was integrated into the genomes of these clones. Genomic DNA from 11 of these PCR-positive clones was extracted, digested with HindIII, separated, blotted, and hybridized with an aacC1-specific probe (27). As shown in Fig. 2, a single DNA fragment hybridized to the probe, further confirming that aacC1 was integrated into the chromosomes and also showing that the genetic transposition mediated by Himar1 occurred only once per cell in these clones. These results demonstrate that pTapMarGent was able to mediate transposition events in the T. denticola genome. The results also indicate that the gentamicin resistance marker tap1p::aacC1 functions in T. denticola and may be used for targeted mutagenesis mediated by allelic exchange recombination.


Figure 2
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  		FIG. 2. Southern blot analysis of genomic DNA from transposon mutants of T. denticola. The purified genomic DNA was digested with HindIII, separated on 0.7% agarose gel, and transferred onto a nylon membrane. Blots were hybridized with a probe specific to the biotin-labeled aacC1 gene. M, DNA marker generated from a 1-kb DNA ladder; numbers indicate sizes in kilobases.


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Identification of transposon mutants.
 
Vectors pMarGent and pTapMarGent were designed to easily recover the transposon and the DNA flanking the insertion site from Escherichia coli, as described previously (27). Using this procedure, we successfully identified Himar1 insertion sites in 23 clones. Briefly, genomic DNA was isolated, treated with HindIII (this enzyme cuts the T. denticola chromosome into 1,709 fragments with average sizes of 1.6 kb but does not cut the transposon), and religated, and E. coli cells were transformed with the DNA products. The transposon, along with flanking DNA, was recovered from E. coli and sequenced as described previously (27). Sequences were identified using the BLAST nucleotide algorithm from the National Center for Biotechnology Information (1). Sequence analysis demonstrated that all insertions occurred after a TA dinucleotide, indicating that all insertions arose by transposition (11). The insertion sites in 23 clones, which appeared to be randomly distributed throughout the T. denticola chromosome, were successfully identified. The insertion site in clone 1 was not definitively identified. The majority of insertions (20 of 23) were mapped to putative open reading frames on the T. denticola chromosome, and three were mapped to intergenic regions (Table 1).


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  		TABLE 1. Transposon insertion sites in T. denticola mutants


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Characterization of mutants with altered morphologies or growth rates.
 
Among these 23 mutants, we found that clone 13 was defective in cell division and that the mutant grew in long and unseparated chains (Fig. 3). The targeted gene (TDE1714) encodes a putative N-acetylmuramyl-L-alanine amidase (MurNac-LAA). In E. coli, MurNac-LAAs are periplasmic enzymes that remove murein cross-links by cleaving the peptide moiety from N-acetylmuramic acid during cell division, and the E. coli mutants showed phenotypes similar to that of clone 13, indicating that the TDE1714 protein is a homolog of MurNac-LAAs that contributes to T. denticola cell division (9, 10).


Figure 3
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  		FIG. 3. Alteration of cell morphology of clone 13. Images were taken by using phase-contrast microscopy. At least 20 fields for each strain were examined. WT, wild type.

The growth rates of clones 16 and 18 were significantly decreased compared to that of the wild type (Fig. 4). The insertion site in clone 16 occurred in TDE0844, encoding a pyruvate phosphate dikinase involved in the synthesis of ATP and pyruvate (29). The insertion site in clone 18 disrupted a 16S rRNA gene (TDE16SB). There are two 16S rRNA genes (TDE16SA and TDE16SB) in T. denticola (25). The inactivation of TDE16SB may have partially influenced the assembly of the ribosome and protein synthesis in this mutant (8, 21). The inactivation of these two genes may influence either metabolism or protein synthesis in T. denticola, leading to lower growth rates. Except for clones 13, 16, and 18, the mutants did not have obvious alterations in cell shape or growth rate.


Figure 4
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  		FIG. 4. Comparison of growth rates of the wild type (WT) and clones 16 and 18. Aliquots of 1 µl of mid-logarithmic-phase cultures of the wild type and the mutants were each inoculated into 1 ml of fresh TYGVS medium, and the cells in the samples were enumerated every 24 h for 120 h by using Petroff-Hausser counting chambers. The data are the means of results for triplicate independent cultures. The standard deviations were less than 2% of the means.


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Conclusion.
 
We have clearly demonstrated that the modified Himar1 transposon generates random mutagenesis in T. denticola. Although the transformation efficiency is relatively low, this approach provides a starting point for generating random, identifiable insertion mutations in T. denticola. This ability should facilitate studies on the biology and pathogenesis of T. denticola. In addition, there have been more than 60 oral Treponema species identified among human oral microflora (5, 20). The results described in this report imply that a similar study can be applied to other oral spirochetes.


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ACKNOWLEDGMENTS
 
We thank R. Limberger, L. Gebhardt, and H. Kuramitsu for providing T. denticola strains. We also thank N. Charon and P. Rosa for helpful discussions, suggestions, and comments on the manuscript.

This research was supported by U.S. Public Health Service grants AR050656 and DE018829 and American Heart Association grant SDG 0735236N.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Oral Biology, SUNY at Buffalo, 3435 Main St., Buffalo, NY 14214-3092. Phone: (716) 829-6014. Fax: (716) 829-3942. E-mail: cli9{at}buffalo.edu Back

{triangledown} Published ahead of print on 22 August 2008. Back


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Applied and Environmental Microbiology, October 2008, p. 6461-6464, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.01424-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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