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Journal of Bacteriology, February 2000, p. 1176-1180, Vol. 182, No. 4
Divisions of Associated Clinical
Sciences1 and Oral Biology and
Medicine,2 UCLA School of Dentistry, Los
Angeles, California
Received 15 July 1999/Accepted 19 November 1999
Three native plasmids of Fusobacterium nucleatum were
characterized, including DNA sequence analysis of one plasmid, pFN1. A
shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was
constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation
efficiencies suggested the presence of a restriction-modification
system in F. nucleatum.
Fusobacterium nucleatum
is a gram-negative anaerobe of interest due to its central role in the
ecology of dental plaque, a complex microbial biofilm that forms on
teeth (6, 17), and its association with human infections
(5, 12, 21, 22). A significant hindrance to the study of
F. nucleatum is the lack of genetic and molecular systems
for the construction of trait-specific isogenic mutants which are
essential for delineation of gene function in the native cell
background. A homologous family of native cryptic plasmids has been
reported to occur in 18% of the F. nucleatum strains
examined (20). This study was initiated to investigate the
utility of native plasmids in the development of gene transfer systems
for F. nucleatum. Data are presented on the characterization of three native F. nucleatum plasmids isolated in our
laboratory, including determination and analysis of the complete DNA
sequence of one plasmid, pFN1. Also described are the use of pFN1 in
the construction of an intergeneric shuttle plasmid in
Escherichia coli, transformation of F. nucleatum
with the shuttle plasmid, and analysis of its stability in the F. nucleatum host cell background. To our knowledge, this is the
first report of transformation of F. nucleatum by electroporation.
Isolation and characterization of F. nucleatum
plasmids.
Three native plasmids (pFN1, pFN2, and pFN3) (Table
1; Fig. 1A)
were isolated from strains of F. nucleatum by routine
techniques (Wizard Plus Minipreps [Promega, Madison, Wis.]; Midi
Preps [Qiagen, Inc., Valencia, Calif.]) and visualized on
ethidium-stained 0.8% agarose gels. Restriction endonuclease mapping
(16) demonstrated that the plasmids varied in size and in
the occurrence of several restriction endonuclease sites (Fig. 1A),
suggesting that the plasmids were unrelated. However, Southern
hybridization studies (15) indicated that pFN1 and pFN2
share homology with each other but not with pFN3 (Fig. 1B and C).
Nitrocellulose blots of plasmid and chromosomal DNA preparations from
the plasmid-containing host strains were probed with pFN1 and pFN3 DNA.
The pFN1 probe hybridized to pFN1 and pFN2 DNA but not pFN3 DNA (Fig.
1B), whereas the pFN3 probe hybridized only to pFN3 DNA (Fig. 1C). No
hybridization to chromosomal DNA from any of the host strains was
evident (data not shown). The strain harboring pFN3, ATCC 10953, was
previously reported to lack plasmid DNA (20). Due to this
discrepancy, we obtained a new culture from the American Type Culture
Collection (Rockville, Md.) and confirmed the presence of pFN3 in this
strain. These data reveal the existence of two nonhomologous groups of plasmids indigenous to F. nucleatum, the first represented
by pFN1 and pFN2 and the second represented by pFN3.
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Native Plasmids of Fusobacterium
nucleatum: Characterization and Use in Development of
Genetic Systems
ABSTRACT
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TABLE 1.
Relevant characteristics of the bacterial strains and
plasmids used in this study
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FIG. 1.
Restriction maps and Southern blots of the F. nucleatum plasmids. (A) Partial restriction map of F. nucleatum plasmids pFN1, pFN2 and pFN3 and shuttle plasmid pHS17.
Selected restriction endonuclease sites in the native plasmids are
presented. Restriction endonuclease sites indicated for pHS17 relate to
the plasmid construction. The pFN1 portion of pHS17 is indicated by the
thick solid bar, with the position of the repA homologue
(ORF5) and putative ori indicated. (B and C) Plasmids from
F. nucleatum strains 12230 (pFN1, lanes 1), 10113 (pFN2,
lanes 2), and ATCC 10953 (pFN3, lanes 3) were probed with pFN1 DNA with
EcoRI digests (B) or pFN3 DNA with EcoRV digests
(C). The HincII-digested pFN1 and
AseI-digested pFN3 probes were 32P labeled
(specific activity of 25 × 108 and 4.5 × 108 dpm/µg of DNA, respectively). The positions of
molecular size markers are indicated on the left, and the linear forms
of pFN1, pFN2, and pFN3 are indicated on the right.
Determination and analysis of pFN1 DNA sequence.
Due to its
small size and superior plasmid yields, pFN1 was chosen for further
analysis. The DNA sequence was determined for both strands. Analysis of
the compiled sequence revealed a circular structure of 5,887 bp with
23% G+C content and seven putative open reading frames (ORFs) (defined
as 
150 bp [Fig. 2A]). Similarity searches were performed using the National Center for Biotechnology Information BLAST server (1, 2). The sequence of pFN1
was highly homologous to the sequence of a 6,281-bp F. nucleatum plasmid (pAD52; GenBank accession no. AF022647). No
similarity was found to any gene encoding antibiotic resistance or
other selectable phenotypic marker. Antibiotic susceptibility testing
indicated that the pFN1 host strain F. nucleatum 12230 was
susceptible to penicillin G, tetracycline, chloramphenicol,
clindamycin, cefoxitin, ampicillin-sulbactam, imipenem, metronidazole,
and streptomycin and resistant to erythromycin at a concentration of 25 µg/ml, as is common in other strains of F. nucleatum
(7). These data suggested that pFN1 is a cryptic plasmid
with respect to antibiotic resistance, comparable to previous findings
with this group of plasmids (20).
|
) termed iterons, and several putative
DnaA-binding sites (
).
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Transformation of F. nucleatum with shuttle plasmid
pHS17.
The shuttle plasmid pHS17 (Fig. 1) was constructed
sequentially in E. coli DH5
cells (Life Technologies,
Gaithersburg, Md.) as follows: AvrII-digested pFN1 was
cloned into the XbaI site of pBluescript; an
ermF-ermAM cassette (8) was added by cloning into
KpnI-PstI sites; and the pBluescript ampicillin
resistance determinant was deleted by digestion with flanking
BspHI sites. The resulting construct included both E. coli and F. nucleatum origins of replication from
pBluescript and pFN1, respectively. The junctions of DNA fragments
joined by cloning were confirmed by DNA sequencing, and the phenotypic
properties of the construct were confirmed on selective medium.
; field strength of 24 or 25 kV/cm; capacitance of 25 or 50 µF), the concentration of
MgCl2 in the Columbia broth (0.5, 1.0, and 2.0 mM), and the clindamycin concentration used in the selective medium (0.2 or 0.4 µg/ml).
Initial attempts to transform F. nucleatum ATCC 10953 with
pHS17 were successful under previously defined conditions (9, 24,
27). Preliminary results indicated optimal recovery of transformants with a field strength of 25 kV/cm, a capacitance of 25 µF, and resistance ranging from 200 to 400 . Analysis of the
transformants revealed the presence of pHS17 and the ATCC 10953 native
plasmid pFN3. The two plasmids were easily distinguished, based on
their sizes and restriction endonuclease digestion patterns (Fig. 3).
Electroporation controls included nonelectroporated cells with or
without the addition of DNA as well as electroporated cells without DNA
added, and all yielded negative results. Electroporation with pHS19
also yielded negative results, suggesting that pFN1 is essential for
replication in F. nucleatum. The single parameter found to
have a major effect on transformation efficiency was the pHS17 DNA
source. Transformation efficiency using 1 µg of plasmid DNA ranged
from 1.6 × 102 to 2 × 102
transformants per µg of DNA from the homologous F. nucleatum host, as compared to no transformants with DNA from the
heterologous E. coli host (Table
3). Transformation efficiency was optimal at a resistance setting of 200 with the homologous host DNA, although pronounced differences were not evident over the range examined. Transformation with 5 µg of E. coli pHS17 DNA
was demonstrated; however, the efficiency was still less than that
observed with 1 µg of homologous DNA. The 100-fold or greater
increase in transformation efficiency with homologous DNA suggests the
presence of a functional restriction-modification system in
F. nucleatum ATCC 10953. Restriction-modification systems have been found in F. nucleatum (18, 19).
Growth phase also influenced the transformation efficiency, but to a
lesser extent than the DNA source. Increased transformation
efficiencies were routinely obtained with early-log-phase cells. For
example, in one experiment using early-log-phase, mid-log-phase, and
stationary-phase recipient cells and an outgrowth period of 5 h
(approximately two generations), the transformation efficiencies were
7.2 × 103, 4.8 × 103, and 5.0 × 103, respectively. No significant differences were
observed with variations in the concentration of MgCl2 in
the outgrowth broth or with 0.2 versus 0.4 µg of clindamycin per ml
in the selective agar medium.
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Stability of shuttle plasmid in F. nucleatum transformants. The structural stability of pHS17 in representative transformants was evaluated by restriction endonuclease mapping, PCR amplification of pHS17-specific DNA regions, and Southern analysis of the transformant DNA with pFN1 and pHS17 DNA probes (data not shown). In all of the analyses done, no evidence of DNA rearrangement or deletion was detected. The segregational stability of pHS17 was examined in the transformant strain KH21, maintained in liquid cultures without antibiotic selection (23). After 100 generations, the percentage loss of plasmid per generation was 0.02, with an average of 98% of the viable cells demonstrating the clindamycin resistance phenotype. The shuttle plasmid was present in all colonies subcultured at baseline and after 100 generations, with no evidence of DNA rearrangement or deletion. Thus, pHS17 was found to be both structurally and segregationally stable in the F. nucleatum host cell background. Interestingly, both pHS17 and pFN3 were stably maintained in the transformants, indicating that these two plasmids are compatible and that pFN3 may be useful in developing plasmid vectors for use in conjunction with pFN1-derived plasmids.
The data presented here document key findings that provide a foundation for developing F. nucleatum genetic systems. These findings include the following: the occurrence of two distinct groups of plasmids native to F. nucleatum, one of which appears to be a theta-replicating iteron-regulated plasmid; the ability of the ermF-ermAM cassette to confer clindamycin resistance in F. nucleatum; the ability of F. nucleatum to be transformed by electroporation using the pFN1-derived shuttle plasmid; and the stability of the shuttle plasmid in F. nucleatum. Additional studies are in progress to examine the copy number of the native plasmids and to confirm the mode of replication of the pFN1 group of plasmids. Further, we are actively pursuing strategies to identify additional selectable markers and further optimize the structural features of the shuttle plasmid for use in molecular manipulations of fusobacteria.Nucleotide sequence accession numbers. The nucleotide sequences of pFN1 and the ermF-ermAM cassette (8) have been deposited in the GenBank database under accession no. AF159249 and AF219231, respectively.
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ACKNOWLEDGMENTS |
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Grants from the UCLA School of Dentistry Opportunity Fund and the UCLA Academic Senate and PHS grant DE12639 to S.K.H. supported this work.
The authors thank E. Lee for technical assistance, V. L. Miller and D. A. Haake for helpful discussions and review of this material, and S. Hunt Gerardo for editorial review.
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FOOTNOTES |
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* Corresponding author. Mailing address: Section of Periodontics, UCLA School of Dentistry, 10833 Le Conte Ave., Los Angeles, CA 90095-1668. Phone: (310) 794-7163. Fax: (310) 206-3282. E-mail: shaake{at}dent.ucla.edu.
We dedicate this work to the memory of Susan E. Valone in
recognition of her support and contributions to this research.
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REFERENCES |
|---|
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Abstract
Text
References
|
|---|
| 1. | Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410[CrossRef][Medline]. |
| 2. |
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 3. | Benachour, A., J. Frere, and G. Novel. 1995. pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria. FEMS Microbiol. Lett. 128:167-176[CrossRef][Medline]. |
| 4. |
Berg, T.,
N. Firth,
S. Apisiridej,
A. Hettiaratchi,
A. Leelaporn, and R. A. Skurray.
1998.
Complete nucleotide sequence of pSK41: evolution of staphylococcal conjugative multiresistance plasmids.
J. Bacteriol.
180:4350-4359 |
| 5. | Bolstad, A. I., H. B. Jensen, and V. Bakken. 1996. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin. Microbiol. Rev. 9:55-71[Abstract]. |
| 6. |
Bradshaw, D. J.,
P. D. Marsh,
G. K. Watson, and C. Allison.
1998.
Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration.
Infect. Immun.
66:4729-4732 |
| 7. | Brazier, J. S., D. M. Citron, and E. J. C. Goldstein. 1991. A selective medium for Fusobacterium spp. J. Appl. Bacteriol. 71:343-346[Medline]. |
| 8. | Fletcher, H. M., H. A. Schenkein, R. M. Morgan, K. A. Bailey, C. R. Berry, and F. L. Macrina. 1995. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene. Infect. Immun. 63:1521-1528[Abstract]. |
| 9. |
Fletcher, M.
1986.
Measurement of glucose utilization by Pseudomonas fluorescens that are free-living and that are attached to surfaces.
Appl. Environ. Microbiol.
52:672-676 |
| 10. | Helinski, D. R., A. E. Toukdarian, and R. P. Novick. 1996. Replication control and other stable maintenance mechanisms of plasmids, p. 2295-2324. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 2. ASM Press, Washington, D.C. |
| 11. | Higgins, D. G. 1994. CLUSTAL V: multiple alignment of DNA and protein sequences. Methods Mol. Biol. 25:307-318[Medline]. |
| 12. |
Holst, E.,
A. R. Goffeng, and B. Andersch.
1994.
Bacterial vaginosis and vaginal microorganisms in idiopathic premature labor and association with pregnancy outcome.
J. Clin. Microbiol.
32:176-186 |
| 13. |
Ilyina, T. V., and E. V. Koonin.
1992.
Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eukaryotes and archaebacteria.
Nucleic Acids Res.
20:3279-3285 |
| 14. | Kanatani, K., T. Tahara, M. Oshimura, K. Sano, and C. Umezawa. 1995. Identification of the replication region of Lactobacillus acidophilus plasmid pLA103. FEMS Microbiol. Lett. 133:127-130[CrossRef][Medline]. |
| 15. |
Kinder, S. A.,
J. L. Badger,
G. O. Bryant,
J. C. Pepe, and V. L. Miller.
1993.
Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a R M+ mutant.
Gene
136:271-275[CrossRef][Medline].
|
| 16. | Kinder Haake, S., and X. Wang. 1997. Cloning and expression of fomA, the major outer membrane protein gene from Fusobacterium nucleatum T18. Arch. Oral Biol. 42:19-24[CrossRef][Medline]. |
| 17. |
Kolenbrander, P. E., and J. London.
1993.
Adhere today, here tomorrow: oral bacterial adherence.
J. Bacteriol.
175:3247-3252 |
| 18. |
Leung, D. W.,
A. C. P. Lui,
H. Merilees,
B. C. McBride, and M. Smith.
1979.
A restriction enzyme from Fusobacterium nucleatum 4H which recognizes GCNGC.
Nucleic Acids Res.
6:17-25 |
| 19. |
Lui, A. C. P.,
B. C. McBride,
G. F. Vovis, and M. Smith.
1979.
Site specific endonuclease from Fusobacterium nucleatum.
Nucleic Acids Res.
6:1-15 |
| 20. | McKay, T. L., J. Ko, Y. Bilalis, and J. M. DiRienzo. 1995. Mobile genetic elements of Fusobacterium nucleatum. Plasmid 33:15-20[CrossRef][Medline]. |
| 21. | Mikamo, H., K. Kawazoe, Y. Sato, and T. Tamaya. 1999. Elastase activity of anaerobes isolated from amniotic fluid with preterm premature rupture of membranes. Am. J. Obstet. Gynecol. 180:378-380[CrossRef][Medline]. |
| 22. | Moore, W. E. C., and L. V. H. Moore. 1994. The bacteria of periodontal diseases. Periodontol. 2000 5:66-77[Medline]. |
| 23. |
Roberts, R. C., and D. R. Helinski.
1992.
Definition of the minimal plasmid stabilization system from the broad-host-range plasmid RK2.
J. Bacteriol.
174:8119-8132 |
| 24. |
Rosey, E. L.,
M. J. Kennedy,
D. K. Petrella,
R. G. Ulrich, and R. J. Yancey, Jr.
1995.
Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange.
J. Bacteriol.
177:5959-5970 |
| 25. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 26. | Schaefer, C., and W. Messer. 1991. DnaA protein/DNA interaction. Modulation of the recognition sequence. Mol. Gen. Genet. 226:34-40[CrossRef][Medline]. |
| 27. |
Sreenivasan, P. K.,
D. J. LeBlanc,
L. N. Lee, and P. Fives-Taylor.
1991.
Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.
Infect. Immun.
59:4621-4627 |
| 28. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
Journal of Bacteriology, February 2000, p. 1176-1180, Vol. 182, No. 4
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