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Effects of l-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis

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Abstract

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). l-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low l-arabinose concentrations, increasing l-arabinose led to a corresponding increase in fluorophore efflux, while at higher l-arabinose concentrations, increasing l-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude l-arabinose. Further, due to the rapid export of l-arabinose, an efficient method for recombination-mediated gene knockout, the l-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of l-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that l-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of l-arabinose should facilitate the optimization of l-arabinose induction in strains with high efflux activity.

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Author notes

  1. Shi-Wei Liao and Jen-Jie Lee have contributed equally as first authors.

Authors and Affiliations

  1. Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan

    Shi-Wei Liao

  2. Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taiwan

    Jen-Jie Lee, Ying-Chen Wu, Shih-Ling Hsuan & Ter-Hsin Chen

  3. Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

    Christopher P. Ptak

  4. Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

    Chih-Jung Kuo

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  1. Shi-Wei Liao
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  2. Jen-Jie Lee
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  3. Christopher P. Ptak
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  4. Ying-Chen Wu
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  5. Shih-Ling Hsuan
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Correspondence to Chih-Jung Kuo or Ter-Hsin Chen.

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None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

Additional information

Communicated by Erko Stackebrandt.

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203_2017_1436_MOESM1_ESM.tif

Supplementary Fig. 1 Standard curve obtained by reacting different concentrations of l-arabinose with β-NAD and GalDH for 30 min and recorded by a spectrophotometer at 340 nm (TIFF 2310 kb)

Supplementary material 2 (DOCX 16 kb)

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Liao, SW., Lee, JJ., Ptak, C.P. et al. Effects of l-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis. Arch Microbiol 200, 219–225 (2018). https://doi.org/10.1007/s00203-017-1436-4

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  • DOI: https://doi.org/10.1007/s00203-017-1436-4

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