Abstract
ABSTRACT Experimental evolution was conducted with Escherichia coli K-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations in emrAB or in its negative repressor mprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion of emrA abolished the CCCP resistance of these strains. One CCCP-evolved isolate lacked emrA or mprA mutations; this strain (C-B11-1) showed mutations in drug efflux regulators cecR (ybiH) (upregulates drug pumps YbhG and YbhFSR) and gadE (upregulates drug pump mdtEF). A cecR∷kanR deletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations in ybhR (MDR efflux). Another isolate showed acrB (MDR efflux pump). The acrB isolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains include adhE (alcohol dehydrogenase), rng (ribonuclease G), and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).
IMPORTANCE The genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.
INTRODUCTION
The proton motive force (PMF) is diminished or abolished by uncouplers of oxidative phosphorylation such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (1). Uncouplers such as CCCP are generally hydrophobic compounds with an acidic proton that reside in the inner membrane of the cell. These cations then shuttle protons across the membrane via protonation/deprotonation (1–3). The transmembrane proton flux equilibrates both ΔpH and Δψ, and thus depletes PMF (1, 2). Respiration then runs a futile cycle, as protons continue to be pumped but PMF is not maintained (4–6). The uncoupler effect is most pronounced during growth at low external pH (pH 5.0-5.7) where the electron transport system is upregulated and a higher PMF is maintained (7).
Evolution experiments show how E. coli evolve over generations of pH-related stress and increase fitness under low pH, high pH, and benzoic acid exposure (8–11). Such experiments often reveal surprising fitness tradeoffs. Evolution under continual low pH exposure leads to loss of activity of amino acid decarboxylases that are highly induced under short-term acid stress (8, 10). Evolution in benzoic acid, a membrane permeant aromatic acid, yields unexpected loss of multidrug resistance (MDR) efflux pumps and antibiotic resistance (9). For example, benzoate stress selects for mutations that delete or downregulate the Gad acid fitness island including the mdtEF drug efflux system (9, 12). At high concentration, benzoic acid partly uncouples PMF and thus could increase the fitness cost of efflux pumps driven by proton flux.
It was of interest therefore to test the fitness effect of long-term exposure to a strong uncoupler, CCCP, that more completely abolishes PMF. One system of interest for CCCP tolerance is EmrAB-TolC. EmrA, EmrB, and TolC form a multidrug efflux pump that exports CCCP and various ionophores and antibiotics (13–15). The emrAB operon is regulated by MprA, a repressor of the operon under the same promoter control as emrAB (16). MprA binds CCCP and becomes inactivated, allowing for higher expression and activity of EmrA and EmrB. It is unknown whether long-term CCCP exposure would select for increased activity of the pump, or its regulators; or for loss of this CCCP-responsive system, as is found for the loss of acid-inducible decarboxylases following long-term exposure to acid (10).
We performed experimental evolution to test the long-term effects of exposure to a full uncoupler, and the role of external pH in CCCP tolerance. We conducted serial dilution of E. coli at pH 6.5 and 8.0 with increasing concentrations of CCCP.
RESULTS
CCCP-evolved populations show increased relative fitness in the presence of CCCP
To investigate the selection effects of CCCP on E. coli, populations of strain W3110 were subcultured daily with CCCP in medium buffered at pH 6.5 or at pH 8.0 (Table 1). The initial CCCP concentrations, 20 μM for low pH and 50 μM for high pH, respectively, were determined by culturing W3110 in a range of CCCP concentrations at pH 6.5 and pH 8.0 (Figs. 1A and 1B). The ancestral strain W3110 failed to grow consistently above 40 μM CCCP (pH 6.5) or above 60 μM CCCP (pH 8.0). Culture densities at 16 h showed that 20 μM CCCP at pH 6.5 and 50 μM CCCP at pH 8.0 resulted in significant decrease of growth without full loss of viability. From this point forward, CCCP concentration was increased in a stepwise fashion, reaching 150 μM CCCP by generation 1,000. Figure 1C compares the 16-h endpoint culture densities attained by the evolving populations versus the ancestral strain W3110, using samples from populations frozen at succeeding generations up to 1,000. The pH 8.0 populations showed a steeper increase in fitness than those exposed to CCCP at pH 6.5, where fitness leveled off after 600 generations.
After 1,000 generations, isolates were obtained from selected microplate populations. Isolated strains are named by the position on the plate and isolate number; for example, strain C-A1-1 was the first CCCP-evolved strain from the well in row A and column 1. Strain names for each strain are listed in Table 2. Figure 2 shows growth curves obtained for isolates from populations following evolution at pH 8.0 (panels A, B) or at pH 6.5 (panels C, D). For each isolate, eight replicate curves were obtained. Panels A and C show the curve exhibiting median density at 16 h for each strain and condition; panels B and D show all eight replicate curves. Isolates that had evolved at pH 8.0 (C-B11-1, C-D11-1, C-F9-1, C-G7-1) as well as isolates that had evolved at pH 6.5 (C-A1-1, C-DA3-1, C-G5-1) showed an increase in tolerance to 150 μM CCCP.
Genomes of CCCP-evolved strains show independent recurring mutations in common genes
The genomes of CCCP-evolved strains were resequenced and analyzed using the computational pipeline breseq to characterize mutations acquired over the course of the evolution (Table 3, selected mutations; Table S1, all mutations). Isolate C-B3-1 behaved as a mutator, showing approximately 20-fold higher mutation rate than the other strains; this strain contained a mutation to mutS (Table S2). Isolates C-E1-1 and C-A1-1 were genetically highly similar, indicating that the strains were nearly clones. For these reasons, isolates C-B3-1 and C-E1-1 were excluded from further study.
For eight CCCP-evolved populations at pH 6.5, and five at pH 8.0, all mutations predicted by breseq are shown in Table S1. Selected mutations, including all for genes that showed mutations in more than one population and for genes of the emrAB operon or its repressor, are shown in Table 3. The table is organized by condition, with mutations found in populations cultured at pH 6.5 are shaded blue, whereas mutations found at high pH are shaded red. Mutations that occurred in both the low pH and high pH project are shaded purple.
All populations that evolved with CCCP at pH 6.5, and five out of six that evolved at pH 8.0, showed mutations in mprA, which encodes the repressor of emrAB (Table 3) (13, 17). Of the 12 strains with mutations in mprA, there were a total of 10 unique mutations to the coding region of MprA and 6 mutations to the intergenic region between mprA and emrA. Two mutations caused early stop codons in MprA, and two other mutations caused deletions of 50 bp or greater (Table 3). MprA represses the emrAB operon which encodes a multidrug efflux pump which exports CCCP from the cell (14, 15, 18). Thus, mprA repressor knockouts could be associated with increased production of the EmrAB ad TolC multidrug efflux pump, and could mediate the increased CCCP tolerance in the CCCP-evolved strains. Nine of the thirteen strains show additional mutations in emrAB or in the intergenic region between mprA and emrA, which includes promoter control sequences. No knockout insertions or deletions were seen for emrAB.
Deletion of mprA enhances growth in CCCP
Given the fitness selection for mutations in mprA, we tested the role of MprA in CCCP tolerance by deleting mprA from the ancestral strain E. coli W3110. Both at pH 6.5 and at pH 8.0, the ΔmprA∷kanR deletant grew at higher concentration of CCCP than the ancestral strain W3110 (Fig. 3). In addition, loss of MprA causes no growth difference in the absence of CCCP. Thus, the growth improvements due to ΔmprA∷kanR are associated with CCCP, probably by efflux via EmrAB complex. The increased CCCP tolerance of ΔmprA∷kanR is consistent with the hypothesis that loss of MprA repressor function in CCCP-evolved isolates increases fitness in the presence of CCCP.
Deletion of emrA in CCCP-evolved strains decreases CCCP fitness
Three of the CCCP-evolved strains (C-E3-1, C-A5-1, C-F9-1) showed missense mutations in emrA. We therefore tested whether emrA activity was required for these and other CCCP-evolved strains. Deletion of emrA (by transduction of ΔemrA∷kanR) in strains C-A1-1, C-A3-1, C-G5-1, and C-D11-1 substantially decreased growth compared to the strains with emrA intact, both at pH 6.5 and at pH 8.0 with CCCP (Fig. 4). Thus, emrA appears to be required for most if not all of the fitness advantage of these evolved strains.
The one CCCP-evolved strain that lacked any emrA or mprA mutation (strain C-B11-1) showed a fitness advantage that was independent of emrA, during culture at pH 8.0 (Fig. 4B). Strain C-D11-1 ΔemrA∷kanR also showed partial CCCP tolerance. However, all strains including C-B11-1 required emrA for growth at pH 6.5, where the uncoupler effect of CCCP is greatest.
Mutations affecting cecR in strain C-B11-1 increase CCCP tolerance
The CCCP-evolved isolate C-B11-1 contains no mutations in mprA or emrAB, so we sought to identify other major components of CCCP tolerance in this strain. C-B11-1 notably sustained an insH-mediated deletion of nearly 20 kb which includes phoE, proA and proB, perR, pepD, gpt, frsA, and crl (Table S1). In addition to this large deletion, C-B11-1 also showed a mutation to cecR (ybiH) and a mutation in the intergenic region between cecR and rhlE. Mutations to this region were also present in another high pH CCCP-evolved strain, C-D11-1. CecR regulates genes that affect sensitivity to cefoperazone and chloramphenicol (19). We found that W3110 ΔcecR∷kanR showed increased CCCP tolerance at pH 6.5 (Fig. 5). In the presence of 30 μM CCCP, the cecR deletion strain reached a cell density comparable to that of isolate C-B11-1, whereas the ancestral strain W3110 grew significantly less. Thus, a cecR defect could contribute the major part of the CCCP fitness advantage for C-B11-1.
Several additional genes that showed mutations in strain C-B11-1 were tested for effects on CCCP tolerance. A C-B11-1 rng+ construct made by recombineering showed no difference in CCCP tolerance compared to the parental strain C-B11-1, cultured in 150 μM CCCP pH 8.0. Other deletions were tested in the ancestral strain background: W3110 ΔacrB, (30 μM CCCP pH 6.5), W3110 ΔcyaA (50 μM CCCP pH 6.5) and W3110 ΔnhaB (0-50 μM CCCP pH 7.0). None of these constructs showed significant difference in growth compared to strain W3110.
Additional mutations in population C-B11
The strain C-B11-1 showed additional mutations of interest for drug efflux, most notably gadE (20, 21). GadE activates expression of the Gad acid fitness island, which includes drug efflux mdtFE (22). The MdtFE efflux pump is not known to transport CCCP. Negative mutation of gadE would decrease expression of mdtFE, an effect seen in evolution under benzoate stress (9).
We pursued the effect of the C-B11-1 genotype by sequencing two later isolates from the C-B11 population (C-B11-3, C-B11-4) after a total of 2,000 generations (Table S3). Cultures of the later isolates showed growth curves comparable to those for C-B11-1. These later isolates retained most of the mutations found in C-B11-1, including the nearly 20-kb deletion of 28 genes that was unique to population C-B11. Between 1,000 and 2,000 generations, the C-B11 strains acquired additional mutations affecting genes ybhR, lptD, tsx, gltA, cfa, mgrB, pykA, cpsB, ydhN and yjjU. Gene ybhR encodes a subunit of the cefoperazone efflux complex regulated by CecR (19). This result is thus consistent with the enhanced fitness shown by cecR mutation found in the C-B11 lineage. The mgrB gene is acid-inducible, via PhoPQ response (23) and shows mutations selected after high-pH evolution (11). The gene lptD forms part of the lipopolysaccharide transport slide (24), which could be relevant for CCCP membrane interactions.
CCCP-evolved strains show altered resistance to antibiotics
Exposure to the partial uncoupler benzoate selects for strains sensitive to chloramphenicol and tetracycline (9). We therefore tested the growth of the CCCP-evolved strains in the presence of various antibiotics (Figure 6). Nalidixic acid is target of the EmrAB TolC multidrug efflux pump (14), so mutations increasing expression of function of this pump might increase resistance to the substrate antibiotics. In the presence of nalidixic acid, several CCCP-evolved strains grew to higher levels than the ancestor (Figure 6A). Strain C-B11-1 does not contain mutations known to affect EmrAB, but it contains a mutation affecting CecR, which may increase resistance to nalidixic acid (19).
Other antibiotics tested for resistance include chloramphenicol (4 μg/ml) (Figure 6B) tetracycline (1 μg/ml) (Figure 6C), ampicillin (1 μg/ml) (Figure 6D). Chloramphenicol, tetracycline and ampicillin showed varied effects in different CCCP-evolved isolates, including marginal gain or loss of resistance. Strain C-A1-1 was very sensitive to chloramphenicol and tetracycline, similar to the result seen for benzoate-evolved strains (9). This strain contained mutations in acrB (25, 26), a drug pump conferring resistance to chloramphenicol and tetracycline; also mutations in sohA and in rpoB that are similar to mutations selected in the benzoate-evolved strains.
Evolved strains show small pH growth effects independent of CCCP
Serial culture at low or high pH is known to shift the growth range (8, 11). Thus, it was important to test the effect of pH alone during CCCP evolution at low or high pH. The CCCP-evolved strains showed marginal changes in pH dependence, as represented by culture density at 16 h. Strains evolved at pH 6.5 showed growth indistinguishable from that of the ancestral W3110 at pH 6.5 (Fig. 7A). At pH 8.0, we saw a small significant increase in density during stationary phase (Fig. 7B). Strains evolved at pH 8.0 consistently showed a small increase in stationary phase growth at either pH 6.5 (Fig. 7C) or at pH 8.0 (Fig. 7D).
In order to test whether these improvements in growth reflect some other factor associated with the stress of growing in a microplate, we conducted an aerated growth curve in baffled flasks. We selected two evolved strains from the low pH and high pH conditions and measured their grown compared to the ancestor at pH 6.5 100 mM PIPES and pH 8.0 100 mM TAPS, respectively, over the course of 8 hours using a traditional growth assay conducted in comparatively more aerobic baffled flasks. We saw no significant growth differences between ancestral and evolved populations (data not shown).
To determine whether conferred growth advantages to pH stress would occur under more extreme acidic and basic conditions, growth of CCCP-evolved strains was tested at pH 5.0 100 mM MES (Fig. 8A) and pH 9.0 100 mM AMPSO (Fig. 8B). Tests at pH 5.0 showed no significant differences. A pH 9.0, the pH 8.0-evolved strains (C-B11-1, C-D11-1, C-F9-1, C-G7-1) grew to a higher density than the ancestor while the pH 6.5-evolved strains (C-A1-1, C-A3-1, C-G5-1) grew less well than the ancestor (Fig. 8B). These results show some shifting of growth range in favor of the pH during serial culture. At pH 7.0, CCCP-evolved strains showed marginal differences from the ancestor (Fig. 9).
Evolved strains show many mutations to adhE
Multiple mutations were found in adhE (acetaldehyde-CoA dehydrogenase) in the strains that evolved in CCCP at pH 8.0. The CCCP stock had been dissolved in ethanol, leading to 1.5% ethanol at the highest concentration used in growth media (150 μM CCCP for both pH 8.0 and pH 6.5). We tested the ability of the high pH evolved strains to grow in 1.5% ethanol, and found that evolved strains grew to a significantly higher optical density than the ancestor (Fig. 10A). We also tested the CCCP tolerance of our strains using a stock dissolved in DMSO, instead of ethanol. The CCCP fitness advantage remained in the absence of ethanol (Fig. 10B).
DISCUSSION
A major finding of our CCCP experimental evolution of E. coli was that all strains but one (C-B11-1) showed mutations to mprA, the repressor of emrAB. The emrAB genes also showed point mutations in many of the strains. The emrAB genes encode a multidrug efflux transport system that has been previously linked to CCCP resistance (13). Deleting the repressor may increase expression or activity of CCCP efflux via EmrAB-TolC (13, 15). The upregulation of EmrAB in our strains is consistent with their increased resistance to nalidixic acid (Fig. 6A), another substrate of the efflux pump (13, 27).
Nevertheless, the strain C-B11-1 that lacked mutations in emrA or mprA attained comparable fitness increase without increasing CCCP export. This strain showed downregulation of other MDR pumps, via mutations in regulators gadE (activates mtdEF) or cecR (represses ybhG; resistance to chloramphenicol and cefoperazone). Other strains showed MDR mutations in MDR components acrB and ybhR. Table 4 compiles all the MDR-related mutations we have found to be selected during evolution in CCCP or in benzoate (9). By contrast, microplate experimental evolution under other conditions does not incur loss of multiple drug pumps (10, 11). The one exception would be mutations in the Gad acid fitness island during evolution in near-extreme acid; but acid response components of Gad are the likely targets.
Overall, our results add to a pattern of selection against drug efflux pumps and their regulators under evolution with an uncoupler or a partial uncoupler (benzoate). However, unlike CCCP, benzoate acts at relatively high aqueous concentrations that equilibrate across the membrane. These concentrations cannot be effluxed by MDR pumps; therefore, adaptation to the presence of benzoate and other permeant acids may be more dependent upon mutations that decrease transmembrane proton flux through unneeded pumps. We are following up this work with more extended comparison of the relative fitness costs of MDR genes, using flow cytometry competition assays (28).
MATERIALS AND METHODS
CCCP Experimental Evolution a pH 6.5 and 8.0
For experimental evolution, E. coli K-12 W3110 populations were cultured at 37°C in a 96-well microplate in buffered LBK media (8). To compare the effects of CCCP at low versus high pH, 24 populations with 20 μM CCCP were buffered at pH 6.5 with 100 mM Na-PIPES (which contained 0.55 M PIPES buffer and 1.4 M NaOH), and 24 populations with 50 μM CCCP were buffered at pH 8.0 with 100 mM TAPS. The populations grew to stationary phase, and after a total of 22 h were diluted 1:100 daily. A multi-channel pipettor with filter tips was used for daily transfer of 2-μl culture into 200-μl fresh medium. Over the course of their serial dilutions, the populations’ concentration of CCCP was increased, with the pH 6.5 condition increasing from 20 μM to 150 μM and the pH 8.0 condition increasing from 50 μM to 150 μM CCCP (Table 1).
After the populations had grown for 1,000 generations, clonal isolates were obtained from selected well populations. Of the populations evolved at pH 6.5, seven isolates from different wells were selected for full genome sequencing and of those at pH 8.0, six isolates were selected for whole genome sequencing. Clonal isolates were generated by streaking samples from all wells three times on LBK plates. Each isolate was given a population designation: “C” for CCCP evolution, the alphanumeric position in the well plate, and a digit for the isolate number from the given well population.
Whole Genome Sequencing and Sequence Analysis. The genomes of fourteen isolates from generation 1,000 and the ancestor were sequenced (Table S1). DNA extraction was performed using the Epicentre Masterpure Purification Kit. The genomes were validated and quantitated using the Illumina TruSeq Nano DNA Library Preparation Kit by the Research and Technology Support Facility at Michigan State University. Sequencing was then performed utilizing the Illumina MiSeq platform in 2x300 bp format, resulting in 20-25 million read pairs. Read alignment and mutation predictions were performed using the computational pipeline breseq (v.0.28.1, v.0.30.0, and v.0.30.2) with default parameters. The reads were mapped to the E. coli K-12 W3110 reference (NC_007779.1). Mutations found in both the ancestor and evolved strain were ignored.
Knockouts in genes of interest
To determine the fitness impact of specific genes on growth in CCCP, certain genes were deleted from both the ancestor and evolved strains. Donor E. coli strains containing kanR insertion knockouts were obtained from the Keio collection (29). Deletion strains of W3110 background were constructed by P1 phage transduction (9). Knockout constructs were confirmed by PCR amplification of the kanR insert and were checked against the recipient and donor strains using X-Gal for lacZ+ (W3110 background). Colony PCR was performed using Lucigen CloneID Colony PCR Master Mix to determine whether the kanR cassette had been inserted into the correct gene and that the recipient strain was as expected. Primers included gene of interest forward direction, gene of interest reverse direction, internal KAN gene forward, internal KAN gene reverse, and Primer KT for within the KAN marker.
Recombineering
In order to generate strain C-B11-1 rng+, a section of gene rng containing the non-sense mutation in CCCP-evolved isolate C-B11-1 was replaced with the wild-type sequence using recombineering (30, 31). A counter-selectable cat-sacB cassette was amplified from colonies of E. coli strain XLT241 using primers with 5’ homology to regions surrounding the rng mutation site in C-B11-1 (5’-CAGCGCCGAAAAACCATTAACGCTGGTTTTCACCCGGTCTTGTGACGGAAGATCACT TCGCAGAATA-3’ and 5’-ATAATGAAGATCACCGCCGCCGAGTGCTGCACTCGCTGGAGCAATCAAAGGGAAAA CTGTCCATAT-3’). Isolate C-B11-1 transformed with the heat-inducible pSIM6∷ampR plasmid was cultured to mid-log phase at 32°C then transferred to 42°C to induce recombineering proteins. Cells were then made electrocompetent and electroporated with the cat-sacB PCR product containing edges homologous to regions flanking the rng mutation site in B11-1. The cells were outgrown at 32°C for 3 to 5 hours and plated on LB with 10 μg/ml chloramphenicol. Colonies were then screened for chloramphenicol resistance and sucrose sensitivity. To replace the cat-sacB cassette with the wild-type rng sequence, successful colonies were made electrocompetent and electroporated with a DNA oligonucleotide containing the wild-type sequence of the region replaced by cat-sacB as well as homology to the flanking regions (5’-CCATTAACGCTGGTTTTCACCCGGTCTTTGCTCAACGCCTGCTCCAGCGAGTGCAGC ACTCGGCGGCGGTG-3’). The cells were then plated on LB lacking NaCl and containing 6% sucrose. Colonies were further screened for resistance to sucrose and chloramphenicol sensitivity. The rng sequence of the new construct was then confirmed by PCR sequencing (5’-GCATGGTGGACACGAACAAT-3’ and 5’-CGCTGGAACGCAAAGTAGAA-3’).
Growth Assays
Batch culture growth was assayed under semiaerobic conditions based on previous procedures (9–11). A 96-well microplate was filled with the desired media and inoculated at a concentration of 1:200 from an overnight culture in each test. Eight replicates from two independent overnight cultures were performed for each strain within a single plate, and growth curves were then run in triplicate. Microplates were placed in the Spectramax 384 spectrophotometer and cultured for 22 hours at 37°C with OD600 measured every 15 min.
Growth curves for antibiotic tests were inoculated from overnight cultures that were grown in LBK 100 mM MOPS pH 7.0. These antibiotic growth curves were tested in LBK 100 mM MOPS pH 7.0 with 1 μg/ml ampicillin, 1 μg/ml tetracycline, 4 μg/ml chloramphenicol or 6 μg/ml nalidixic acid.
For growth curves under aeration, 125-ml baffled flasks containing 20 ml of LBK (pH 8.0 100 mM TAPS or pH 6.5 100 mM Na-PIPES) were inoculated 1:200 from overnight cultures. The cultures were grown in a water bath shaker set to 37 °C 200 rpm. 200-μl aliquots were removed from the baffled flasks and read in a 96 well plate (wavelength 600 nm) every thirty minutes for eight hours in order to construct the growth curve.
Statistics
Means are reported with standard error of the mean (SEM). Statistics were computed using R packages. Samples were compared using Analysis of Variance tests (ANOVA) with Tukey post hoc tests.
Accession number
Sequence data have been uploaded in the NCBI Sequence Read Archive (SRA) under accession number SRP157768.
ACKNOWLEDGMENTS
This work was supported by award MCB-1613278 from the National Science Foundation and the support of Kenyon College. We thank Ellen Broeren for excellent technical support.