Development of SacB-Based Counterselection for Efficient Allelic Exchange in Fusobacterium nucleatum

Authors

Peng Zhou
Bibek G C
Bo Hu
Chenggang Wu

Language

English

Publication Date

1-7-2025

Journal

Microbiology Spectrum

DOI

10.1128/spectrum.02066-24

PMID

39611826

PMCID

PMC11705814

PubMedCentral® Posted Date

11-29-2024

PubMedCentral® Full Text Version

Post-print

Abstract

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.

Keywords

Fusobacterium nucleatum, Plasmids, Bacterial Proteins, Alleles, Sucrose, Humans, Bacillus subtilis, Mutation, Biofilms, Hexosyltransferases, Fusobacterium nucleatum, sacB, allelic exchange, in-frame deletion

Published Open-Access

yes

Recommended Citation

Zhou, Peng; G C, Bibek; Hu, Bo; et al., "Development of SacB-Based Counterselection for Efficient Allelic Exchange in Fusobacterium nucleatum" (2025). Faculty, Staff and Student Publications. 5879.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/5879