Figure 1.
Overview of furfural selection and SCALEs analysis.
A) 1, 2, 4, and 8 kb fragments were prepared from E. coli genomic DNA and ligated into pSMART-LCK vector. Each sized genomic library was transformed into BW25113 ΔrecA::FRT host cells, recovered, mixed together, and then grown on minimal medium plates (control) or solid minimal medium with 0.75 g l−1 furfural. Cells were harvested from the plates and microarrays (square boxes) were run with plasmid extracts from both the furfural and control plates in order to determine individual gene fitness scores (W). The fitness vs. position plot illustrates how different clones (stacked rectangles) can contribute to an individual gene's fitness. The red “triangle” has contribution from various sized clones, but is centered around a specific locus, whereas the blue “rectangle” represents a high fitness score from the presence of one single sized clone (e.g., requiring a large operon where smaller library sizes would not be found). B) Genome plot depicting clone fitnesses for the different library sizes. Loci corresponding to the top gene fitness scores are labeled. C) Histogram of log-transformed gene fitness scores, where increased fitness corresponds to ln(W)>0.
Table 1.
Gene(s) cloned for confirmation studies.
Figure 2.
Plating assay of hypothesized tolerance-conferring clones identified in SCALEs selection.
Cells (104) were streaked onto solid minimal medium with 0, 0.75, or 1.5 g l−1 furfural (0, 1, or 2× selection pressure) and growth was observed for 72 hours.
Figure 3.
Growth curve analysis of tolerant clones grown in minimal medium with 0.75−1 furfural.
A) Seed cultures were inoculated into furfural at the same initial density and grown for 24 hours. Optical density was recorded every 3–6 hours. Error bars represent one standard error (n = 3). Double asterisks denote p<0.05.
Figure 4.
Phenotypic analysis of tolerant clones for furfural reduction and DNA mutation frequency.
A) Samples were collected for measuring furfural in growth curve cultures with 0.75 g l−1 furfural initial concentration. Furfural concentrations were normalized to cell number (optical density) for each value and disappearance rate was calculated during the transition from lag to exponential phase (n = 3). B) Frequency of rifampin resistance of cells treated with furfural (n = 4). Error bars represent one standard error. Double asterisks denote p<0.05.
Figure 5.
Mutational studies on lpcA and groESL clones.
Mutations were introduced onto the plasmids within the coding sequence targeted for (A) lpcA or (B) groESL. Cultures were grown in 0.75 g l−1 furfural for 20 hr. (n = 3; error bars represent standard error). Percentage improvement was calculated as the difference of the test strain subtracted from the control, divided by the control.