Two transmembrane transcriptional regulators coordinate to activate chitin-induced natural transformation in Vibrio cholera
Allison C. Hullinger, Virginia E. Green, Catherine A. Klancher, Triana N. Dalia, Ankur B. Dalia
Abstract
Transcriptional regulators are a broad class of proteins that alter gene expression in response to environmental stimuli. Transmembrane transcriptional regulators (TTRs) are a subset of transcriptional regulators in bacteria that can directly regulate gene expression while remaining anchored in the membrane. Whether this constraint impacts the ability of TTRs to bind their DNA targets remains unclear. Vibrio cholerae uses two TTRs, ChiS and TfoS, to activate horizontal gene transfer by natural transformation in response to chitin by inducing the tfoR promoter (PtfoR). While TfoS was previously shown to bind and regulate PtfoR directly, the role of ChiS in PtfoR activation remains unclear.
Introduction
Bacterial species inhabit diverse environments where they encounter a wide range of stressors. To survive fluctuating conditions, they must have ways to sense and respond to environmental change. One such mechanism is the alteration of gene expression in response to specific environmental cues using a broad group of proteins called transcriptional regulators. Most commonly, transcriptional regulators are localized to the cytoplasm, where they can freely diffuse to find their DNA targets.
Materials and method
Bacterial strains and culture conditions
All mutant strains were derived from the Vibrio cholerae El Tor strain E7946 [32]. Unless otherwise indicated, V. cholerae strains were grown in LB medium or on LB agar. When necessary, LB was supplemented with chloramphenicol (1 µg/mL), kanamycin (50 µg/mL), trimethoprim (10 µg/mL), spectinomycin (200 µg/mL), carbenicillin (100 µg/mL), erythromycin (10 µg/mL), zeocin (100 µg/mL), or sulfamethoxazole (100 µg/mL). A list of all strains used in this study can be found in S1 Table.
Results
ChiS binds to P tfoR and is required for P tfoR activation
Prior work suggests that ChiS and TfoS are both required for activation of PtfoR [8,9]. To formally test this, we generated strains with a chromosomally integrated PtfoR-gfp transcriptional reporter. These strains also contained a previously described Pchb-mCherry construct [16], which serves as a reporter for ChiS activity. In addition, these strains contained a constitutively expressed [17] mTFP1 construct (Pconst2-mTFP1), which was used to normalize for intrinsic noise in gene expression.
Discussion
In this study, we uncover how ChiS and TfoS coordinate to promote activation of natural transformation in V. cholerae. Specifically, our data are most consistent with a model wherein ChiS performs two functions to facilitate PtfoR activation: (1) it recruits the PtfoR DNA locus to the membrane and (2) it recruits TfoS to the membrane-proximal promoter through a direct interaction (Fig 5).
Acknowledgments
We would like to thank the Center for Genomics and Bioinformatics at Indiana University for assistance with ChIP-seq library prep and sequencing. We would also like to thank Gabe Zentner for his assistance and advice on ChIP-seq analysis.
Citation: Hullinger AC, Green VE, Klancher CA, Dalia TN, Dalia AB (2025) Two transmembrane transcriptional regulators coordinate to activate chitin-induced natural transformation in Vibrio cholerae . PLoS Genet 21(2): e1011606. https://doi.org/10.1371/journal.pgen.1011606
Editor: David R. Hendrixson, The University of Texas Southwestern Medical Center, UNITED STATES OF AMERICA
Received: September 30, 2024; Accepted: February 3, 2025; Published: February 18, 2025
Copyright: © 2025 Hullinger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grant R35GM128674 from the National Institutes of Health to ABD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
