Establishment of a HicA toxin-based counterselection system for markerless genetic engineering in Veillonella atypica OK5.

Genetic manipulation in Veillonella atypica remains limited due to the scarcity of robust counterselection systems, hindering mechanistic studies of its role in oral biofilm ecology and host interactions. In this study, we adapted a highly efficient, HicA toxin–based counterselection system, originally developed for Fusobacterium nucleatum, for use in V. atypica OK5. We demonstrated that the inducible expression of a truncated Fusobacterium periodonticum HicA toxin, controlled by a theophylline-responsive riboswitch E, provides robust and reliable negative selection. The utility of this system was validated by generating a markerless deletion of the hemagglutinin gene hag1. The resulting Δhag1 mutant exhibited a complete loss of coaggregation with the early colonizer Streptococcus gordonii, confirming the essential role of Hag1 in interspecies adhesion. Furthermore, we constructed a hag1-luciferase transcriptional fusion reporter, which revealed that hag1 expression is growth-phase-dependent, peaking during early to mid-log phase. This work establishes the HicA system as a powerful genetic tool for Veillonella, enabling scarless gene deletions and promoter analysis, thereby facilitating future mechanistic studies of its role in oral biofilm development and microbial community dynamics.