Multiomics Analysis Provides Insight into the Laboratory Evolution of Escherichia coli toward the Metabolic Usage of Fluorinated Indoles

Abstract

Organofluorine compounds are known to be toxic to a broad variety of living beings in different habitats, and chemical fluorination has been historically exploited by mankind for the development of therapeutic drugs or agricultural pesticides. On the other hand, several studies so far have demonstrated that, under appropriate conditions, living systems (in particular bacteria) can tolerate the presence of fluorinated molecules (e.g., amino acids analogues) within their metabolism and even repurpose them as alternative building blocks for the synthesis of cellular macromolecules such as proteins. Understanding the molecular mechanism behind these phenomena would greatly advance approaches to the biotechnological synthesis of recombinant proteins and peptide drugs. However, information about the metabolic effects of long-term exposure of living cells to fluorinated amino acids remains scarce. Hereby, we report the long-term propagation of Escherichia coli (E. coli) in an artificially fluorinated habitat that yielded two strains naturally adapted to live on fluorinated amino acids. In particular, we applied selective pressure to force a tryptophan (Trp)-auxotrophic strain to use either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of Trp analogues, followed by their incorporation in the cellular proteome. We found that full adaptation to both fluorinated Trp analogues requires a low number of genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity, and quality control of protein folding. These findings highlight the cellular mechanisms behind the adaptation to unnatural amino acids and provide the molecular foundation for bioengineering of novel microbial strains for synthetic biology and biotechnology.

Figure 1

(A) ALE experimental setup and overview of the changes observed in the E. coli strains adapted to fluoroindoles that marked a divergence from TUB00. Abbreviations include the following: trpA (Trp synthase α subunit); trpB (Trp synthase β subunit); trpR (trp operon transcriptional repressor); TrpRS (tryptophanyl-tRNA synthetase); Mtr (Trp permease); MdtF, MdtO, and MdtK (multidrug efflux pumps); and CpxA (sensor histidine kinase). Under natural conditions, cells can uptake extracellular Trp. However, during the ALE cultivation, Trp was not supplied; hence, its structure is crossed out with a red “×”. (B) trp operon of E. coli in the parent strain MG1655 and in the Trp-auxotrophic derivative TUB00 (used for ALE cultivation) after deletion of the genes trpLEDC. The reaction catalyzed by TrpS (which requires pyridoxal-5′- phosphate, PLP, as cofactor) is shown below.

Figure 2

Cultivation scheme of E. coli ALE toward usage of (A) 4-fluoroindole; (B) 5-fluoroindole as precursors for the synthesis of Trp analogues. Optical density (OD₆₀₀) at the reinoculation step (“passage”) is plotted against days of incubation and number of passages. The color code refers to the composition of minimal medium (NMM, Tables S3 and S4), NMMa/b/c where a is the number of amino acids supplied, b the concentration of fluoroindole, and c the concentration of indole, both μM. Black arrows indicate the isolates selected for multiomics analysis, referred to as early (4TUB34 and 5TUB23), intermediate (4TUB81 and 5TUB48), and final time points (4TUB93 and 5TUB83).

Figure 3

Deconvoluted mass spectra of His-tagged enhanced green fluorescent protein (EGFP-H6) expressed in (A) 4TUB93 and (B) 5TUB83. Structure of the enhanced green fluorescent protein from Aequorea victoria (EGFP, structure 2Y0G deposited in PDB) figure generated with the EzMol interface. Trp57 is highlighted in pink.

Figure 4

Analysis of metabolites produced during 4- and 5-fluoroindole ALE. Principal coordinate analysis (PCoA) plots with Canberra distance metric of the metabolomes from (A) 4-fluoroindole ALE and (B) 5-fluoroindole ALE. Each point represents the metabolome extracted from three independent cultures at early (yellow), intermediate (green), and final (red) time points of ALE and TUB00 (blue). The spatial distance in the plot is proportional to the chemical diversity between the samples, and evolutionary trajectories are shown (dashed arrows). Global Natural Products Social Molecular Networking (GNPS) visual output in the form of molecular subnetworks of (C) Trp and fluorotryptophan and of (D) lipids and biotin, i.e., the two groups of metabolites which were most pronounced among the isolates adapted to fluoroindoles. The nodes represent metabolites with unique retention time and m/z identifiers: (1) Trp; (2) fluorotryptophan; (3) 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)); (4) 1-palmitoleoyl-sn-glycero-3-phosphoethanolamine (PE(16:1/0:0)); (5a) 1-oleyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PE(18:0/0:0)); (5b) 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PE(16:0/0:0)); (6) 1-oleoyl-2-acetyl-sn-glycerol (DG(18:1/2:0/0:0)); (7) monopalmitolein (MG(16:0/0:0/0:0)); and (8) biotin. The pie chart representation illustrates the relative abundance of each feature across the samples. (E) Normalized abundance of the annotated metabolites.

Figure 5

Cell membrane rearrangement during 4- and 5-fluoroindole ALE. (A, B) The hydrophobicity of E. coli membranes before and after the adaptation to fluoroindoles was probed by Nile Red staining, and the permeability properties were investigated by assessing the susceptibility to the antibiotic vancomycin. (C) Fluorescent micrographs of the ancestor strain TUB00 and of the final time points of 4- and 5-fluoroindole ALE (4TUB93 and 5TUB83, respectively) stained with Nile red. The last panel reports 5TUB83 irradiated with white light (WL) for cell count comparison. (D) Total fluorescence normalized to TUB00. (E) Minimal inhibitory concentration (MIC) of vancomycin.