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Research Article | Spotlight

Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity

Douglas A. Higgins, John M. Gladden, Jeff A. Kimbrel, Blake A. Simmons, Steven W. Singer, Michael P. Thelen
Tina M. Henkin, Editor
Douglas A. Higgins
aThe Joint BioEnergy Institute, Emeryville, California, USA
bLawrence Livermore National Laboratory, Livermore, California, USA
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John M. Gladden
aThe Joint BioEnergy Institute, Emeryville, California, USA
cSandia National Laboratories, Livermore, California, USA
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Jeff A. Kimbrel
bLawrence Livermore National Laboratory, Livermore, California, USA
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  • ORCID record for Jeff A. Kimbrel
Blake A. Simmons
aThe Joint BioEnergy Institute, Emeryville, California, USA
dLawrence Berkeley National Laboratory, Berkeley, California, USA
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Steven W. Singer
aThe Joint BioEnergy Institute, Emeryville, California, USA
dLawrence Berkeley National Laboratory, Berkeley, California, USA
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Michael P. Thelen
aThe Joint BioEnergy Institute, Emeryville, California, USA
bLawrence Livermore National Laboratory, Livermore, California, USA
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Tina M. Henkin
Ohio State University
Roles: Editor
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DOI: 10.1128/JB.00069-19
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ABSTRACT

Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IILT) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IILT Bacillus isolates as well as a spontaneous IILT Escherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IILT strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. coli sugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes.

IMPORTANCE This study identifies bacteria that are tolerant to ionic liquid solvents used in the production of biofuels and industrial biochemicals. For industrial microbiology, it is essential to find less-harmful reagents and microbes that are resistant to their cytotoxic effects. We identified a family of small multidrug resistance efflux transporters, which are responsible for the tolerance of these strains. We also found that this resistance can be caused by mutations in the sequences of guanidine-specific riboswitches that regulate these efflux pumps. Extending this knowledge, we demonstrated that guanidine itself can promote ionic liquid tolerance. Our findings will inform genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where low concentrations of ionic liquids surpass bacterial tolerance.

FOOTNOTES

    • Received 22 January 2019.
    • Accepted 9 April 2019.
    • Accepted manuscript posted online 15 April 2019.
  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00069-19.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity
Douglas A. Higgins, John M. Gladden, Jeff A. Kimbrel, Blake A. Simmons, Steven W. Singer, Michael P. Thelen
Journal of Bacteriology Jun 2019, 201 (13) e00069-19; DOI: 10.1128/JB.00069-19

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Guanidine Riboswitch-Regulated Efflux Transporters Protect Bacteria against Ionic Liquid Toxicity
Douglas A. Higgins, John M. Gladden, Jeff A. Kimbrel, Blake A. Simmons, Steven W. Singer, Michael P. Thelen
Journal of Bacteriology Jun 2019, 201 (13) e00069-19; DOI: 10.1128/JB.00069-19
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KEYWORDS

Bacillus
biofuels
functional genomics
guanidine riboswitch
ionic liquids
quaternary ammonium compounds

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