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

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 (IIL^T) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IIL^TBacillus isolates as well as a spontaneous IIL^TEscherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IIL^T 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. colisugE 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.

Keywords: Bacillus; biofuels; functional genomics; guanidine riboswitch; ionic liquids; quaternary ammonium compounds.

FIG 1

IIL tolerance of selected bacterial strains. Growth curves (A) and maximum growth rate and maximum cell density (B) are shown for each strain cultured in IIL medium (250 mM [C₂C₁im][OAc]). The chemical structure of [C₂C₁im][OAc] is shown in the inset in panel A.

FIG 2

Identification of genome regions containing IIL tolerance genes. (A) The top blue line depicts the B. cereus loci from which the IIL^T-fosmid inserts originated. Region 2 is expanded below. (B and C) As indicated by growth curves (B) and maximum growth rate and maximum cell density (C), E. coli is protected from IIL (250 mM [C₂C₁im]Cl) by both fosmids and pBbS0c plasmids (48) containing B. cereus DNA from these loci.

FIG 3

IIL tolerance in B. thuringiensis depends upon tandem SMR pump genes. Growth curves (A) and maximum growth rate and maximum cell density (B) are shown for each strain cultured in IIL medium (360 mM [C₂C₁im][OAc]).

FIG 4

Point mutations in the guanidine II riboswitch confer IIL tolerance to E. coli. (A) The highly conserved ACGR sequence within the P1 and P2 hairpins of the guanidine II riboswitch that regulates sugE in E. coli. (B and C) Growth curves (B) and maximum growth rate and maximum cell density (C) are shown for the plasmid expression of various sugE constructs that complement ΔsugE in E. coli grown in IIL medium (250 mM [C₂C₁im]Cl).

FIG 5

Addition of guanidine protects E. coli from IIL toxicity. E. coli DH10b was cultured in medium containing either 0 or 10 mM guanidine-HCl; 250 mM [C₂C₁im][OAc] was added at 0, 30, and 180 min after the start of culture. (A) Growth curves, with the addition of IIL indicated by dashed lines; (B) maximum cell density at the initial IIL addition and after 20 h of cultivation.

FIG 6

Efflux pump library tested in E. coli. Efflux pumps from the various organisms noted were expressed in E. coli DH10b in the IPTG-inducible vector pBbS6k. Growth curves (A) and maximum growth rate and maximum cell density (B) are shown for each strain cultured in medium containing 250 mM [C₂C₁im]Cl, 0.1 mM IPTG, and 50 mg/ml kanamycin.