Expanding the genetic toolkit helps dissect a global stress response in the early-branching species
            <i>Fusobacterium nucleatum</i>

Ponath, Falk; Zhu, Yan; Cosi, Valentina; Vogel, Jörg

doi:10.1073/pnas.2201460119

Cited by 23 publications

(53 citation statements)

References 104 publications

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“…MazF has been used as a counterselection marker in other Gram-positive and Gram-negative bacteria (22). As we prepared this manuscript, a MazF-mediated counterselection strategy was reported in F. nucleatum (50). They used a tetracycline-inducible system to control the MazF expression in F. nucleatum .…”

Section: Discussionmentioning

confidence: 99%

A HicA toxin-based counter-selection marker for allelic exchange mutations in Fusobacterium nucleatum

Wu¹,

Gc²,

Zhou³

2023

Preprint

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The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including the galK-based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum that we recently developed. However, this method requires a host lacking the GalK gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the HicA gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counter-selective marker. Interestingly, the full-length HicA is not toxic in F. nucleatum, but a truncated HicA version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an rpsJ promoter and is controlled by its translational level using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin RadD gene within F. nucleatum rad operon and the TnaA gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This HicA-based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria.

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Section: Discussionmentioning

confidence: 99%

A HicA toxin-based counter-selection marker for allelic exchange mutations in Fusobacterium nucleatum

Wu¹,

Gc²,

Zhou³

2023

Preprint

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“…To increase the number of differentially labelled species in the model, additional fluorochromes might be looked at. Very recently, Ponath et al [52] developed several genetic tools for F. nucleatum including four distinct fluorescent proteins. Additionally, the oxygen independent HaloTag [53] or CLIP-tag [54]-a SNAP-variant [55]-seems to be worth investigating.…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal monitoring demonstrates prebiotic treatment responses in a periodontal multispecies biofilm model

Bernaerts

Ghesquière

Simoens

et al. 2023

Preprint

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In vitro periodontal model systems are required for further elucidating phenomena behind periodontal pathogenesis and treatment applications. However, most oral biofilm models apply static conditions and are restricted to end-point measurements. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic shear flow in the oral cavity. The design is re-engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a spatially uniform, dense and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features and bacterial composition of the biofilm remain stable from 3 up to 6 days. As a proof of concept for our periodontal model, the 3-day developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and co-aggregations of the pathogenic species, and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.

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“…Thus, type I SPase is considered to be an attractive target (50,51). F. nucleatum possesses a LepB homolog, and a recent study revealed by RNA-seq that lepB is controlled by σ E , a mediator of an oxygeninduced response (27). However, no genetic study of lepB in F. nucleatum has been conducted.…”

Section: Discussionmentioning

confidence: 99%

“…The F. nucleatum strain ATCC 23726 genome contains a gene locus annotated as lepB (27) (HMPREEF0397_0712, accessible at http://img.jgi.doe.gov/). We reasoned that this LepB might interact with RadD's signal peptide.…”

Section: Introductionmentioning

confidence: 99%

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Development of a Xylose-Inducible Promoter and Riboswitch Combination System for Manipulating Gene Expression inFusobacterium nucleatum

Gc¹,

Zhou²,

Naha³

et al. 2023

Preprint

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Inducible gene expression systems are important for studying bacterial gene function, yet most exhibit leakage. In this study, we engineered a leakage-free hybrid system for precise gene expression controls in Fusobacterium nucleatum by integrating the xylose-inducible expression system with the theophylline-responsive riboswitch. This innovative method enables concurrent control of target gene expression at both transcription and translation initiation levels. Using luciferase and the indole-producing enzyme tryptophanase (TnaA) as reporters, we demonstrated that the hybrid system displays virtually no observable signal in the absence of inducers. We employed this system to express FtsX, a protein related to fusobacterial cytokinesis, in an ftsX mutant strain, unveiling a dose-dependent manner in FtsX production. Without inducers, cells form long filaments, while increasing FtsX levels by increasing inducers concentrations led to a gradual reduction in cell length until normal morphology was restored. Crucially, this system facilitated essential gene investigation, identifying the signal peptidase lepB gene as vital for F. nucleatum. LepB’s essentiality stems from depletion, affecting outer membrane biogenesis and cell division. This novel hybrid system holds the potential for advancing research on essential genes and accurate gene regulation in F. nucleatum.

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Expanding the genetic toolkit helps dissect a global stress response in the early-branching species Fusobacterium nucleatum

Cited by 23 publications

References 104 publications

A HicA toxin-based counter-selection marker for allelic exchange mutations in Fusobacterium nucleatum

A HicA toxin-based counter-selection marker for allelic exchange mutations in Fusobacterium nucleatum

Spatiotemporal monitoring demonstrates prebiotic treatment responses in a periodontal multispecies biofilm model

Development of a Xylose-Inducible Promoter and Riboswitch Combination System for Manipulating Gene Expression inFusobacterium nucleatum

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