Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.