Functional asymmetry of the transmembrane subunits YbhS and YbhR in the tetracycline-inducible ABC transporter YbhFSR
5 affiliations
- 1College of Agronomy and Life Sciences, Zhaotong University
- 2Yunnan Engineering Research Center of Green Planting and Processing of Gastrodia, Zhaotong University
- 3Institute for Advanced Study on Ecological Agriculture and Biodiversity of the Yunnan-Guizhou Plateau, Zhaotong University
- 4Yunnan Key Laboratory of Smart Villages and Agri-Cultural-Tourism Integration, Zhaotong University
- 5College of Agronomy and Biotechnology, Yunnan Agricultural University
Abstract
Objective: The Escherichia coli ABC (ATP-binding cassette) transporter YbhFSR has an unusual subunit architecture: its two nucleotide-binding domains (NBDs) are fused within a single polypeptide YbhF, while the transmembrane domains (TMDs) are formed by two separate subunits YbhS and YbhR. This study sought to investigate the functional differences between YbhS and YbhR during tetracycline (TC) efflux, and to characterize the transcriptional regulation of the ybhFSR operon under TC stress.
Methods: A series of E. coli knockout strains (ΔybhF, ΔybhS, ΔybhR, ΔybhSR, ΔybhFSR) were generated via λ-Red homologous recombination. qRT-PCR was used to profile transcriptional responses of ybhF, ybhS, and ybhR under a gradient of TC concentrations in wild-type and ΔacrB backgrounds. Susceptibility to four tetracyclines (TC, OTC, CTC, DOX) was determined by broth microdilution and disk diffusion. Cross-species sequence alignments across seven Enterobacteriaceae genera compared evolutionary conservation between YbhS and YbhR.
Results: Tetracycline triggered dose-dependent transcriptional upregulation of ybhF, ybhS and ybhR, whereby ybhR was induced to a greater extent than ybhS across all tested concentrations. The transcriptional gap between ybhR and ybhS was markedly enlarged in the ΔacrB strain, showing a significant divergence in inducibility (23.8-fold for ybhR vs. 10.2-fold for ybhS at 8 μg/mL TC). Deletion of ybhR led to markedly increased susceptibility to all four tetracyclines (TC, OTC, CTC, DOX), while E. coli ΔybhS cells exhibited MIC (minimum inhibitory concentration) values indistinguishable from the wild type. The ΔacrB ΔybhF double mutant yielded a TC MIC of 0.125 μg/mL, considerably lower than the ΔacrB single mutant (0.25 μg/mL) and wild-type strain (1 μg/mL). Cross-species alignments across seven Enterobacteriaceae genera showed that YbhR had higher average sequence identity than YbhS (96.20% versus 93.89%); however, this difference did not reach statistical significance (Wilcoxon signed-rank test, p = 0.0625; paired t-test, p = 0.75).
Conclusion: Transcription of the ybhFSR operon is induced by TC in a dose-dependent fashion, and this induction is enhanced in the absence of AcrAB. YbhS and YbhR exhibit functional asymmetry at both transcriptional and physiological levels: YbhR acts as the core TMD subunit supporting TC export, whereas YbhS plays a dispensable role in tetracycline efflux under the tested conditions. The consistently higher sequence conservation of YbhR relative to YbhS is consistent with its dominant physiological role, although this trend did not reach statistical significance. Notably, this demonstrates TMD functional asymmetry in a Gram-negative transporter with fused NBDs, indicating that TMD specialization can occur independently of NBD architecture even when NBD asymmetry is hard-wired into the fused YbhF polypeptide.
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BibTeX
@article{feng2026functional,
title = {{Functional asymmetry of the transmembrane subunits YbhS and YbhR in the tetracycline-inducible ABC transporter YbhFSR}},
author = {Zhenyue Feng and Chaohuan Yang and Hao Wen and Meng Wu and Shengguang Feng and Wei Zheng and Defu Liu},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1959624},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1959624/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Functional asymmetry of the transmembrane subunits YbhS and YbhR in the tetracycline-inducible ABC transporter YbhFSR
AU - Zhenyue Feng
AU - Chaohuan Yang
AU - Hao Wen
AU - Meng Wu
AU - Shengguang Feng
AU - Wei Zheng
AU - Defu Liu
PY - 2026
DA - 2026/10/23
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1959624
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1959624/full
AB - Objective: The Escherichia coli ABC (ATP-binding cassette) transporter YbhFSR has an unusual subunit architecture: its two nucleotide-binding domains (NBDs) are fused within a single polypeptide YbhF, while the transmembrane domains (TMDs) are formed by two separate subunits YbhS and YbhR. This study sought to investigate the functional differences between YbhS and YbhR during tetracycline (TC) efflux, and to characterize the transcriptional regulation of the ybhFSR operon under TC stress. Methods: A series of E. coli knockout strains (ΔybhF, ΔybhS, ΔybhR, ΔybhSR, ΔybhFSR) were generated via λ-Red homologous recombination. qRT-PCR was used to profile transcriptional responses of ybhF, ybhS, and ybhR under a gradient of TC concentrations in wild-type and ΔacrB backgrounds. Susceptibility to four tetracyclines (TC, OTC, CTC, DOX) was determined by broth microdilution and disk diffusion. Cross-species sequence alignments across seven Enterobacteriaceae genera compared evolutionary conservation between YbhS and YbhR. Results: Tetracycline triggered dose-dependent transcriptional upregulation of ybhF, ybhS and ybhR, whereby ybhR was induced to a greater extent than ybhS across all tested concentrations. The transcriptional gap between ybhR and ybhS was markedly enlarged in the ΔacrB strain, showing a significant divergence in inducibility (23.8-fold for ybhR vs. 10.2-fold for ybhS at 8 μg/mL TC). Deletion of ybhR led to markedly increased susceptibility to all four tetracyclines (TC, OTC, CTC, DOX), while E. coli ΔybhS cells exhibited MIC (minimum inhibitory concentration) values indistinguishable from the wild type. The ΔacrB ΔybhF double mutant yielded a TC MIC of 0.125 μg/mL, considerably lower than the ΔacrB single mutant (0.25 μg/mL) and wild-type strain (1 μg/mL). Cross-species alignments across seven Enterobacteriaceae genera showed that YbhR had higher average sequence identity than YbhS (96.20% versus 93.89%); however, this difference did not reach statistical significance (Wilcoxon signed-rank test, p = 0.0625; paired t-test, p = 0.75). Conclusion: Transcription of the ybhFSR operon is induced by TC in a dose-dependent fashion, and this induction is enhanced in the absence of AcrAB. YbhS and YbhR exhibit functional asymmetry at both transcriptional and physiological levels: YbhR acts as the core TMD subunit supporting TC export, whereas YbhS plays a dispensable role in tetracycline efflux under the tested conditions. The consistently higher sequence conservation of YbhR relative to YbhS is consistent with its dominant physiological role, although this trend did not reach statistical significance. Notably, this demonstrates TMD functional asymmetry in a Gram-negative transporter with fused NBDs, indicating that TMD specialization can occur independently of NBD architecture even when NBD asymmetry is hard-wired into the fused YbhF polypeptide.
SN - 1664-302X
ER -