Mobile Genetic Elements of Fusobacterium nucleatum

McKay, Terry; Ko, Julie; Bilalis, Yioryos; DiRienzo, Joseph M.

doi:10.1006/plas.1995.1003

Cited by 27 publications

(22 citation statements)

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“…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 g/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).…”

supporting

confidence: 84%

“…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.…”

mentioning

confidence: 99%

“…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.)…”

mentioning

confidence: 99%

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Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

Haake

Yoder²,

Attarian³

et al. 2000

J Bacteriol

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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.

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confidence: 84%

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confidence: 99%

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Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

Haake

Yoder²,

Attarian³

et al. 2000

J Bacteriol

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“…Genetic manipulation of F. nucleatum has been difficult, presumably due in part to its diversified restriction endonuclease systems, which differ between strains and cleave DNA irrespective of the extent of methylation (11). Although mobile genetic elements were identified in F. nucleatum nearly a decade ago (13), and several shuttle plasmids have been constructed and delivered into two strains, F. nucleatum ATCC 10953 and ATCC 23726, by electroporation (5,10), no genetic work had been successful in F. nucleatum 12230, a working strain in our laboratory known to be refractory to such manipulations. Recently, a novel FadA adhesin that encodes a small open reading frame of 387 bp was identified in F. nucleatum 12230.…”

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confidence: 99%

Sonoporation Is an Efficient Tool for Intracellular Fluorescent Dextran Delivery and One-Step Double-Crossover Mutant Construction in Fusobacterium nucleatum

Han¹,

Ikegami²,

Chung³

et al. 2007

Appl Environ Microbiol

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Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.

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“…As a result, very little is known at the molecular level about F. nucleatum properties. A homologous family of cryptic plasmids has been described to exist in F. nucleatum strains (12,23), and sequence analyses have identified a putative plasmid origin of replication as well as replication and relaxase protein homologues encoded by one member of this family, pFN1. Construction of a shuttle plasmid, pHS17, by using pFN1 has enabled the transformation of F. nucleatum (12).…”

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confidence: 99%

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Bachrach

Haake

Glick

et al. 2004

Appl Environ Microbiol

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Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.

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Mobile Genetic Elements of Fusobacterium nucleatum

Cited by 27 publications

References 0 publications

Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

Native Plasmids of Fusobacterium nucleatum : Characterization and Use in Development of Genetic Systems

Sonoporation Is an Efficient Tool for Intracellular Fluorescent Dextran Delivery and One-Step Double-Crossover Mutant Construction in Fusobacterium nucleatum

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

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