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

## TL;DR

A new genetic tool using the HicA toxin was developed for Veillonella atypica, enabling markerless gene deletions and promoter analysis to study oral biofilm interactions.

## Contribution

Adaptation of the HicA toxin-based counterselection system from Fusobacterium nucleatum for use in Veillonella atypica OK5.

## Key findings

- The HicA toxin system enables robust negative selection in V. atypica OK5.
- A markerless deletion of the hemagglutinin gene hag1 was successfully generated.
- Hag1 is essential for coaggregation with Streptococcus gordonii and is growth-phase-dependent.

## Abstract

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.

The online version contains supplementary material available at 10.1186/s12866-026-04809-y.