Letter pubs.acs.org/NanoLett
Engineering Bacterial Efflux Pumps for Solar-Powered Bioremediation of Surface Waters Vikram Kapoor and David Wendell* School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati, 2901 Woodside Drive, 705 Engineering Research Center, Cincinnati, Ohio 45221, United States S Supporting Information *
ABSTRACT: Antibiotics are difficult to selectively remove from surface waters by present treatment methods. Bacterial efflux pumps have evolved the ability to discriminately expel antibiotics and other noxious agents via proton and ATP driven pathways. Here, we describe light-dependent removal of antibiotics by engineering the bacterial efflux pump AcrB into a proteovesicle system. We have created a chimeric protein with the requisite proton motive force by coupling AcrB to the light-driven proton pump Delta-rhodopsin (dR) via a glycophorin A transmembrane domain. This creates a solar powered protein material capable of selectively capturing antibiotics from bulk solutions. Using environmental water and direct sunlight, our AcrB-dR vesicles removed almost twice as much antibiotic as the treatment standard, activated carbon. Altogether, the AcrB-dR system provides an effective means of extracting antibiotics from surface waters as well as potential antibiotic recovery through vesicle solubilization. KEYWORDS: Multidrug efflux pumps, antibiotics, bioremediation, AcrB, rhodopsin, hormones
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low abundance compared to other organic matter.11 Also, heat regeneration of the activated carbon is critical for efficient removal and increased useful lifetime of the media but this remains energy intensive.12 Clearly, there is a need for an effective and selective antibiotic removal material, capable of functioning in wastewaters rich in organics with minimal energy input. Here, we demonstrate the selective removal of ampicillin and vancomycin, commonly used human and veterinary antibiotics, and ethidium bromide, a mutagen and popular nucleic acid stain, by engineering a solar powered proteovesicle material using the multidrug efflux pump AcrB. Recent studies of bacterial drug resistance in Escherichia coli,13−15 Salmonella typhimurium,15,16 Enterobacter aerogenes15,17 and other gram-negative bacteria have provided fascinating insight into the molecular machinery enabling survival. Tripartite multidrug efflux motors expel a variety of antibiotics substrates, heavy metals, and other noxious agents out of bacterial cells via proton and ATP driven pathways. These motor proteins play a key role in cell survival and impart individual antibiotic resistance characteristics based on the type of efflux pump employed. The AcrA-AcrB-TolC efflux complex is a principal multidrug exporter in E. coli and consists of the following three components: AcrB, a proton powered motor protein, TolC, an outer membrane-anchored exit duct, and AcrA, a periplasmic adaptor protein joining TolC to AcrB.18 In this pump complex, AcrB converts the proton motive force
he extensive use of antibiotics in recent decades has contributed to an increase in antibiotic resistance among a wide variety of microorganisms. Activities such as animal husbandry, agriculture, and aquaculture, as well as human health care, have resulted in the increased release of antibiotics into terrestrial and aquatic environments, and these environments serve to proliferate antibiotic resistance in potentially pathogenic bacteria that find their way back into the community via food and water webs.1,2 Effluents containing antibiotics are of concern as there is potential to promote or maintain bacterial resistance and disrupt key processes critical to aquatic ecology or crop (soil fertility) and animal (rudimentary processes) production.1,3 In a recent study, Zeh et al.4 reported that sperm viability was significantly reduced in offspring of animals treated with the common antibiotic tetracycline, a compound that enters the environment in significant concentrations via repeated fertilizations with liquid manure, accumulating in soil and water.5 Antibiotic persistence is also driven by incomplete degradation in wastewater treatment plants6−8 and as a result, wastewater effluent and sewage have become natural reservoirs of residual antibiotics.2,5 The present technology standard for antibiotic removal is activated carbon, which is used in water treatment for removing a variety of organic contaminants such as synthetic organic chemicals (SOCs), aromatic compounds, and natural organic matter (NOM).9,10 Activated carbon is used for filtration and removal of a broad range of organic substrates, however the cost of the prolific absorbance is specificity. Despite the advantage of high carbonaceous oxygen demand removal efficiencies, antibiotics escape capture due to their relatively © 2013 American Chemical Society
Received: February 22, 2013 Revised: March 28, 2013 Published: April 13, 2013 2189
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with a pumping rate of approximately 0.01 ng/min. For continued validation of our AcrB-BR system with a common antibiotic, the proteoliposomes were mixed with fluorescently labeled vancomycin and monitored for antibiotic accumulation in the vesicles (see Supporting Information Figure 1), after which an improved rhodopsin was employed to create a coupled fusion protein system which could be expressed well in E. coli. Following initial tests with BR, we sought to improve upon our design by using an alternative bacteriorhodopsin in the form of Delta-rhodopsin (dR). dR is a light-driven H+ transporter native to Haloterrigena turkmenica and unlike its more well-studied cousin BR, dR has been shown to express well in E. coli.26 Notably, with a coupling of AcrB and dR comes the concern of future horizontal gene transfer enabling lightbased antibiotic resistance. Thus, one other essential attribute of dR critical to its selection as a power source was the exogenous retinal addition required for proper function in E. coli. This provides insurance that transgenic bacteria containing the AcrB and dR pumps cannot acquire a solar antibiotic resistance without the addition of an internal retinal biosynthesis pathway. Despite efforts to the contrary,27 almost all artificial BR-ATP synthase systems to date have suffered from a lack of control over protein orientation, since these two protein complexes are reconstituted after purification.21−23,28 Still, the energy gradient is critical to efficient coupled functionality, ideally AcrB and dR positioned in opposite directions and as close as possible to allow dR derived protons to directly power AcrB. Given that we want our membrane enclosed structures to capture antibiotics rather than expel them as bacteria do, AcrB orientation must be maintained in the opposite direction relative to native synthesis within the cell. Similarly, we want dR to be embedded in the influx direction to deliver protons useful to AcrB. A number of factors govern membrane protein insertion and final orientation, however the first several positively charged amino acid residues are essential to this process.29 Initially, we attempted traditional coreconstitution methods to create the desired coupled enzyme proteoliposomes. However, the greatest functionality was achieved with a chimeric AcrB-dR fusion protein joined via an engineered transmembrane domain. The linkage domain was derived from the glycophorin A (gA) membrane spanning α helix that forms a small but wellpacked helical interface lacking intermonomer hydrogen bonds.30 Thus, with these three components concatenated we have an AcrB-dR complex that maintains proper orientation and proximity. Given that both monomers make up larger respective trimers, the linked proteins have the potential to create a 2D protein array on the membrane surface, akin to the 2D crystals formed by BR,31 with AcrB trimers making up a new node in the array. A plot of ampicillin removal by the AcrB-dR proteoliposomes in nanopure water is given in Figure 2a. Antibiotic pumping occurred at a maximum velocity (Vmax) of 2.23 ± 0.7 nmol/ min/mg of AcrB-dR for proteoliposomes that saturated to a backflow limited rate of 0.09 nmol/min/mg after 60 min. Comparing our data with E. coli in vivo pumping experiments for the antibiotics nitrocefin and cefamandole,32 we find that our rate falls between the observed rates for these two antibiotics. Vmax for efflux of nitrocefin and cefamandole in E. coli was reported to be 1.41 and 22.2 nmol/min/mg respectively, rates that were based on the cellular content of AcrB estimated by Western blot analysis.32 Many interdepend-
(pmf) into active mechanical work expelling the toxins while the AcrA/TolC proteins provide a passive exit funnel.13 Despite pumping a broad spectrum of antibiotics and other compounds, AcrB has evolved a binding domain with inherent specificity, pumping estradiol hormones but not structurally similar sterols like testosterone19 and antibiotics like chloramphenicol but not cefazolin.16 The specificity of the AcrB pump is presumably derived from bacterial evolution and survival with natural selection producing AcrB binding domains capable of discerning noxious compounds from useful cellular contents. To engineer the AcrB pump into a material capable of exploiting this molecular specificity in an environmental setting, a power source is required. Our first choice for powering AcrB was the solar driven proton pump Bacteriorhodopsin (BR).20 BR originates from the halophile Halobacterium halobium and is capable of converting green light into a pmf. So far, the coupling of proton pumps and pmf driven motor proteins in engineered materials has been largely limited to BR and the F0-F1 ATP synthase.21−23 In this study, we extend this to AcrB with a demonstration of a novel solar-powered proteovesicle system that can capture antibiotics from water and wastewater, based on the coupled-protein activity of AcrB and BR in a membrane bound vesicle. To validate the initial design, ethidium bromide (EtBr) was used to stain DNA trapped within AcrB-BR proteoliposomes similar to previous in vivo EtBr pumping investigations.24 When exposed to UV, the orange fluorescence of EtBr is 20fold brighter in the presence of DNA,25 providing a fluorescent metric for pumping performance (see Figure 1). The percentage of EtBr removed was plotted over time by measuring the average time-dependent fluorescence intensity during the pumping process (see Methods). According to the fitted EtBr pumping curve, the fluorescent intensity increases, approaching saturation after approximately three hours. More than 90% of the total EtBr capture occurred within the first 2 h
Figure 1. Pumping of ethidium bromide by AcrB-BR proteoliposomes (black square) and a control experiment in dark (red circle) for comparison. The pumping activity of AcrB was measured by adding 1 μL of 1 μg/mL EtBr solution to 400 μL of proteoliposomes containing DNA and then exposed to green light (500−580 nm). The fluorescence intensity of the solution was measured every 30 min at an excitation wavelength of 510 nm and emission wavelength of 595 nm. All error bars refer to standard deviation (n = 3). 2190
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Figure 2. (A) Removal of ampicillin by AcrB-dR proteoliposomes (black square) and a control experiment in dark for comparison. The Vmax calculated from the slope for 0 to 15 min was 2.23 ± 0.7 nmol/min/mg of AcrB-dR. (B) Light-induced ΔpH formed by AcrB-dR proteoliposomes (black square). (Inset) Increased proton permeability coinciding with asymptotic approach of AcrB-dR pumping to steady state. All error bars refer to standard deviation (n = 3).
Figure 3. Comparison of removal of antibiotic by activated carbon and AcrB-dR proteoliposomes. Activated carbon and AcrB-dR proteoliposomes were used for removing antibiotic from environmental water spiked with 10 μg/mL of ampicillin. AcrB-dR proteoliposomes were used for removing antibiotic from nanopure water spiked with 10 μg/mL of ampicillin. Samples were exposed to an average temperature of 15 °C and 4 h of sunlight in March 2012. A control experiment with AcrB-dR proteoliposomes and nanopure water spiked with 10 μg/mL of ampicillin was performed in dark. All error bars refer to standard deviation (n = 3). (RW, river water; NPW, nanopure Water).
Figure 2a was around 9 μg/mL. The minimal inhibitory concentration (MIC) of ampicillin for E. coli cells has been reported as 4−6 μg/mL,33,34 which is lower than the observed capacity of our system; however, the largest tolerated ampicillin concentration for E. coli cells overexpressing AcrB has been reported as high as 10 μg/mL,33 which is consistent with our results. Taken together these findings indicate that the bacterial MIC values observed in vivo may be the result of a simple
ent proteoliposome formation factors influence this conversion, including vesicle size, composition, formation method, protein activity, and orientation,23 as well as antibiotic molecular weight; however, in light of the in vivo comparison, our engineered chimera appears as good or better than natural implementation. The maximum observed ampicillin concentration within the proteovesicles during the steady-state pumping phase shown in 2191
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the required retinal during growth, even dead cells could act as a reservoir for bioremediation given that at least some of the protons appear to be used locally. In addition to future in vitro biophysical investigations offered by the AcrB-dR chimera, a cellular or liposome-based material harnessing this construct provides an environmental friendly means for extracting antibiotics, hormones, and heavy metals from surface waters, as well as potential cost-effective antibiotic recovery and reuse through vesicle solubilization.
concentration driven backflow mechanism rather than a more complex cellular process, such as methylation of rRNA residues.35 Indeed, it has been shown that the MIC of ampicillin decreased strikingly in AcrB-deletion strains and increased significantly in the AcrB overexpression strain33 pointing toward this direct relationship. Figure 2b shows the light-driven intravesicular pH change of AcrB-dR proteoliposomes. The proteoliposomes rapidly formed a light-induced pH gradient of more than 0.5 unit followed by increased proton permeability (see Figure 2b inset) after approximately 30 min, which appears to coincide with the asymptotic approach of AcrB to steady-state. These results would indicate that there is a relationship between the stalled pumping in the presence of the back-flow concentration and proton leakage. Using the maximum observed ΔpH after the initial pmf is formed (∼2.9 pmol H+/ ml of vesicles) and the total amount of antibiotic pumped over this change (∼21 nmol ampicillin/ml of vesicles), we can see that picomoles of protons are lost in exchange for nanomoles of antibiotic, indicating a large number of active protons remain undetected by pyranine. This suggests that protons could be used locally, as proposed elsewhere.36 The maximum antibiotic concentration pumped into the vesicles appears identical to the MIC values observed for bacterial cells. A high concentration of antibiotics inside the vesicles would generate back pressure effects that limit the maximum attainable pumping rate, analogous to BR37,38 and this limit appears to coincide with the increased proton permeability as antibiotic pumping begins to stall. To test the specificity and real-world application of our system against the present standard in antibiotic water treatment technology, activated carbon, we added the two removal materials to environmental water (Little Miami River, Ohio +39° 7′ 37.49″, −84° 24′ 36.39″) and exposed them to 4 h of direct, unobstructed sunlight (3 μmol/m2/sec of green light) after spiking both samples with 10 μg/mL of ampicillin. Remarkably, we found that our AcrB-dR vesicle material system showed almost the same efficiency in antibiotic capture as observed in nanopure water containing antibiotic (Figure 3). When comparing our system to an equivalent amount of activated carbon (by weight), the vesicle system showed almost double the removal of antibiotic from river water (Figure 3). The total removal per mass was about 40% for activated carbon and 64% for the solar powered AcrB-dR vesicles. Previously, activated carbon has been shown to remove 44% of incoming antibiotics in river water by mass,11 which is consistent with our results. With access to sunlight, our AcrB-dR proteovesicles can be used as artificial organelles to capture antibiotics. We have repurposed a normally deleterious biological antibiotic removal process into an engineered system, demonstrating feasibility with environmental water and functionality better than the present treatment standard. Our initial attempts with AcrB and BR reconstituted separately suffered from a lack of control over protein orientation. Our chimeric AcrB-dR results suggest that control over both specific orientation and optimal composition in proteoliposomes can be similarly optimized for other in vitro pmf driven motor proteins like ATP synthase. While the potential of our artificial organelle system is presently limited in scale, effective antibiotic removal from water and wastewater as well as removal of a variety of estradiols and heavy metals could be realized with E. coli efflux pumps engineered with a chimeric dR attachment in a non-native orientation. By supplementing
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ASSOCIATED CONTENT
S Supporting Information *
A detailed methods section including preparation of proteoliposomes, light exposure, pumping experiments, and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
The manuscript was written by V.K. and D.W. Experiments were conceived by D.W. and performed by V.K. and D.W.. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Hiroshi Nikaido for donating pUC151A plasmid containing AcrB and Dr. Jacob Schmidt for critically reading the manuscript. We also thank Dan Li for initial experiments, as well as Elizabeth Wurtzler and Jacob Todd for laboratory assistance.
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