Summary and location of mutation sites.

<p><b>A:</b> Summary of mutation sites in the amino acid sequence of all the mutants. The full sequence is divided into three parts by two dashed lines. Each part represents a separate domain of irrE (from left to right: N-terminal domain, HTH domain, C-terminal domain). <b>B:</b> Location of mutation sites of all the mutants in a modeled structure of irrE from <i>D. radiodurans</i>. Domains of irrE: N-terminal domain, cyan; HTH domain, green; C-terminal domain, pink. Mutation sites of: ethanol-tolerant mutants, yellow; butanol-tolerant mutant, blue; acetate-tolerant mutant, red.</p

Plasmid map for <i>irrE</i> library construction and expression of <i>irrE</i> mutants.

<p>The expression of <i>irrE</i> mutants was under the control of a GroESL promoter from <i>D. radiodurans</i>.</p

Intracellular ROS assay of alcohol-tolerant mutants.

<p><b>A:</b> Relative fluorescence unit (RFU) per OD of cells of the strain harboring plasmid pMD18T (grey), wild-type (diagonal) and ethanol-tolerant mutant E1 (black) cultivated in LB medium supplemented with 0% or 1.5% ethanol. <b>B:</b> RFU per OD of cells of the strain harboring plasmid pMD18T (grey), wild-type (diagonal) and butanol-tolerant mutant B29 (black) cultivated in LB medium supplemented with 0% or 0.25% butanol. The cells were dyed with H₂DCFDA before the RFU assay, as described. All assays were performed in triplicate.</p

Growth of ethanol-tolerant mutants under ethanol stress.

<p>OD₆₀₀ of controls and ethanol-tolerant mutants after cultivation in LB medium supplemented with 0% (<b>A</b>), 3% (<b>B</b>), 4% (<b>C</b>) or 5% (<b>D</b>) ethanol for 10 hours and 23 hours. Strains were pMD: strain harboring plasmid pMD18T, WT: strain harboring wild-type <i>irrE</i>, E1, E28, E30, E79 and E80: ethanol-tolerant mutants. All data represent the mean values from three independent experiments.</p

Butanol shock experiment of butanol-tolerant mutant.

<p>The viabilities of the strains were tested after shocking with 2.1% butanol for 1 hour (right panel) or without shock (left panel). Strains were pMD: strain harboring plasmid pMD18T, WT: strain harboring wild-type <i>irrE</i>, B29: butanol-tolerant mutant. Triangles below each panel indicate tenfold serial dilutions of plated cells (1∶1 to 1∶1000, from left to right).</p

Growth of strains in LB medium supplemented with butanol.

<p><b>A:</b> OD₆₀₀ of the strain harboring plasmid pMD18T (grey), wild-type (diagonal) and butanol-tolerant mutant B29 (black) after cultivation in LB medium supplemented with butanol of different concentrations for 24 hours. <b>B:</b> Fold increase in OD₆₀₀ for wild-type (filled squares) and mutant B29 (filled circles) compared with control at different butanol concentrations. All data represent the mean values from three independent experiments.</p

Tolerances of butanol-tolerant mutant toward other C4 or C5 alcohols.

<p>OD₆₀₀ of the strain harboring plasmid pMD18T (grey), wild-type (diagonal) and butanol-tolerant mutant B29 (black) was assayed after cultivation in LB medium supplemented with 0.5% isobutanol, 0.25% pentanol or 0.25% isopentanol for 24 hours. All data represent the mean values from three independent experiments.</p

Significant Rewiring of the Transcriptome and Proteome of an <em>Escherichia coli</em> Strain Harboring a Tailored Exogenous Global Regulator IrrE

<div><p>Cell reprogramming for microorganisms via engineered or artificial transcription factors and RNA polymerase mutants has presented a powerful tool for eliciting complex traits that are practically useful particularly for industrial strains, and for understanding at the global level the regulatory network of gene transcription. We previously further showed that an exogenous global regulator IrrE (derived from the extreme radiation-resistant bacterium <em>Deinococcus radiodurans</em>) can be tailored to confer <em>Escherichia coli</em> (<em>E. coli</em>) with significantly enhanced tolerances to different stresses. In this work, based on comparative transcriptomic and proteomic analyses of the representative strains E1 and E0, harboring the ethanol-tolerant IrrE mutant E1 and the ethanol-intolerant wild type IrrE, respectively, we found that the transcriptome and proteome of <em>E. coli</em> were extensively rewired by the tailored IrrE protein. Overall, 1196 genes (or approximately 27% of <em>E. coli</em> genes) were significantly altered at the transcriptomic level, including notably genes in the nitrate-nitrite-nitric oxide (NO) pathway, and genes for non-coding RNAs. The proteomic profile revealed significant up- or downregulation of several proteins associated with syntheses of the cell membrane and cell wall. Analyses of the intracellular NO level and cell growth under reduced temperature supported a close correlation between NO and ethanol tolerance, and also suggests a role for membrane fluidity. The significantly different omic profiles of strain E1 indicate that IrrE functions as a global regulator in <em>E. coli</em>, and that IrrE may be evolved for other cellular tolerances. In this sense, it will provide synthetic biology with a practical and evolvable regulatory “part” that operates at a higher level of complexity than local regulators. This work also suggests a possibility of introducing and engineering other exogenous global regulators to rewire the genomes of microorganism cells.</p> </div

Differentially expressed genes associated with tryptophan metabolism and transport.

<p>The number after each gene is the Log₂ value (fold change in E1 compared with E0). Red values: upregulated in E1; green values: downregulated in E1; black values: no difference in expression.</p

Differentially expressed non-coding RNAs.

<p>Differentially expressed non-coding RNAs.</p