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

ABSTRACT 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 .