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. 2020 Apr 21;5(2):e00232-20.
doi: 10.1128/mSystems.00232-20.

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia Coli

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Free PMC article

Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia Coli

Cong Chen et al. mSystems. .
Free PMC article

Abstract

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs.IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

Keywords: CREATE; antibiotic resistance; global regulators; mutation; transcriptome.

Figures

FIG 1
FIG 1
Library tolerance to the different antibiotics. (A) The mixed culture was used for inoculation of LB medium with doxycycline at 10 μg/ml, and then the resulting culture was transferred into fresh LB medium with doxycycline at a concentration of 15 μg/ml and then 25 μg/ml. (B) The mixed culture was used for inoculation of LB medium with 60-μg/ml thiamphenicol, and then the resulting culture was transferred into fresh LB medium with thiamphenicol at a concentration of 100 μg/ml. (C) The mixed culture was used for inoculation of LB medium with 10-μg/ml gentamicin, and then the resulting culture was transferred into fresh LB medium with gentamicin at a concentration of 20 μg/ml and then 30 μg/ml. OD600, optical density at 600 nm.
FIG 2
FIG 2
Mutations in SoxR confer resistance to doxycycline. (A) Verification of mutations enriched in the regulator library for resistance to doxycycline at a 25-μg/ml concentration. (B) The regulation network of SoxR control genes involved in multidrug efflux pumps and expression of porins. (C) Fold change in the levels of expression of SoxR-regulated genes in the SoxR G121K, I120E, G121N, and G121P mutants relative to their expression in strain MG1655 under a doxycycline concentration of 15 μg/ml. (D) The positions of mutated residues in the SoxR structure (blue). The ligand [2Fe-2S] is shown in red (PDB accession number 2ZHH).
FIG 3
FIG 3
Mutations in SoxR are pleiotropic for resistance to different antibiotics. (A) Verification of the mutations enriched in the global regulator library for resistance to thiamphenicol at a 100-μg/ml concentration. (B) Evaluation of the extent of synergistic pleiotropy for the mutations in soxR conferring resistance to doxycycline for resistance to thiamphenicol at a 100-μg/ml concentration. (C) Evaluation of the extent of synergistic pleiotropy for all mutations in soxR conferring resistance to doxycycline for resistance to azithromycin at a 120-μg/ml concentration. The colored lines indicate the variants, and the black lines indicate wild-type E. coli.
FIG 4
FIG 4
Scatterplot of unigenes mapped to the KEGG database. (A) Strain DC and the SoxR G121P mutant. (B) Strain GC and the CRP V140W mutant. Rich factor, the ratio of the number of genes with significant differences in transcription levels to the total number of all annotated genes in the pathway. Q value, the P value after correction by the multiple-hypothesis test.
FIG 5
FIG 5
Heat map of the expression levels of genes for carbohydrate metabolism and oxidative phosphorylation in the SoxR G121P mutant. The fold change in expression for DEGs is represented by the color code on the heat map. The red code indicates that the gene was upregulated, and the blue code indicates that the gene was downregulated. Glc, glucose; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 1,3-DPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenol pyruvate; PYR, pyruvate; Acetyl-CoA, acetyl coenzyme A; Cit, citrate; ICit, isocitric acid; AKG, oxoglutarate; Suc-CoA, succinyl coenzyme A; Suc, succinic acid; Fum, fumarate; Mal, malic acid; Glyox, glyoxylate; OAA, oxaloacetate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; E4P, erythrose-4-phosphate; S7P, seduheptulose-7-phosphate; PPP, pentose phosphate pathway.
FIG 6
FIG 6
Mutations in CRP confer resistance to gentamicin and are pleiotropic for resistance to other antibiotics. (A) Verification of mutations enriched in the regulator library for resistance to gentamicin at a 30-μg/ml concentration. (B) Position of mutated residues (blue) on the CRP structure (gray) and their proximity to DNA (red). (C) Evaluation of the extent of synergistic pleiotropy for the mutation in crp conferring resistance to gentamicin for resistance to thiamphenicol at a 100-μg/ml concentration. (D) Evaluation of the extent of synergistic pleiotropy for the mutation in crp conferring resistance to gentamicin for resistance to azithromycin at a 120-μg/ml concentration. The blue lines indicate variants, and the black lines indicate wild-type E. coli. All strains used to verify resistance and pleiotropy were cultured in LB medium (10-g/liter tryptone, 5-g/liter yeast extract, 10-g/liter NaCl) with the corresponding antibiotic at 37°C and 220 rpm in 250-ml conical flasks with a 50-ml final volume of the medium.

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