High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12 - PubMed (original) (raw)

Comparative Study

. 2004 Dec;14(12):2495-502.

doi: 10.1101/gr.2977704.

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Comparative Study

High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12

Christiane Honisch et al. Genome Res. 2004 Dec.

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.

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Figures

Figure 1.

Figure 1.

The universal primer system: Scheme of base-specific cleavage and MALDI-TOF MS based resequencing. PCR amplification of the DNA region of interest is followed by T7-mediated in vitro transcription and base-specific cleavage. RNA transcripts of the target regions are cleaved in four reactions corresponding to virtually each of the four bases. This corresponds to an RNAse-specific cleavage at U and C of the forward as well as of the reverse RNA transcript. MALDI-TOF spectra of all four cleavage reactions are subject to subsequent mutation analysis.

Figure 2.

Figure 2.

Simulated cumulative frequencies of all detectable single base sequence variation events in the 362 amplicons of the _E. coli_-K-12 reference set. 97%–100% of all possible deletions are detectable in a total of 319 amplicons, 97%–100% of all possible insertions are detectable in 314 amplicons, and 97%–100% of all single nucleotide substitutions can be detected in 355 amplicons. (Black) Total single base variation events; (gray) SNPs, insertions, and deletions.

Figure 3.

Figure 3.

Base-specific cleavage and MALDI-TOF based discovery of the mutation G→A at position 356 in a 748-bp amplicon of the glycerol kinase gene (glpK). Mutation-specific signal changes as described in the text are highlighted and enlarged. (A) Overlay of mass spectra resulting from the T-specific cleavage reaction of the forward RNA transcript of the wild-type _E. coli_-K12 strain and the mutant isolate G1-A. Mutation-specific signal changes at 6813.2 Da and 6829.2 Da. (B) Overlay of mass spectra resulting from the T-specific cleavage reaction of the reverse RNA transcript of the wild-type _E. coli_-K12 strain and the mutant isolate G1-A. Mutation-specific signal changes at 1594.0 Da and 2830.8 Da. (C) Overlay of mass spectra resulting from the C-specific cleavage reaction of the forward RNA transcript of the wild-type _E. coli_-K12 strain and a mutant isolate G1-A. Intensity changes of mutation-specific mass signals at 1936.2 Da and 1952.2 Da. (D) Verification of the mutation by MALDI-TOF-based genotyping. A G-allele-specific extend product is detected in the wild-type _E. coli_-K12 strain at 6125.2 Da. The corresponding mutant C allele is detected at 6398.2 Da in clone G1-A.

Figure 3.

Figure 3.

Base-specific cleavage and MALDI-TOF based discovery of the mutation G→A at position 356 in a 748-bp amplicon of the glycerol kinase gene (glpK). Mutation-specific signal changes as described in the text are highlighted and enlarged. (A) Overlay of mass spectra resulting from the T-specific cleavage reaction of the forward RNA transcript of the wild-type _E. coli_-K12 strain and the mutant isolate G1-A. Mutation-specific signal changes at 6813.2 Da and 6829.2 Da. (B) Overlay of mass spectra resulting from the T-specific cleavage reaction of the reverse RNA transcript of the wild-type _E. coli_-K12 strain and the mutant isolate G1-A. Mutation-specific signal changes at 1594.0 Da and 2830.8 Da. (C) Overlay of mass spectra resulting from the C-specific cleavage reaction of the forward RNA transcript of the wild-type _E. coli_-K12 strain and a mutant isolate G1-A. Intensity changes of mutation-specific mass signals at 1936.2 Da and 1952.2 Da. (D) Verification of the mutation by MALDI-TOF-based genotyping. A G-allele-specific extend product is detected in the wild-type _E. coli_-K12 strain at 6125.2 Da. The corresponding mutant C allele is detected at 6398.2 Da in clone G1-A.

Figure 4.

Figure 4.

Mutation detection in the glycerol kinase gene (glpK) by base-specific cleavage and MALDI-TOF MS. Discriminating mass signals in the T-cleavage reaction of the reverse RNA transcript (highlighted in boxes) at 2541.6 Da, 2581.6 Da, 2886.8 Da, 3256.0 Da, and 3272.0 Da allow for unambiguous identification of the substitution T→G at position 160, G→T at position 184, A→C at position 218, and A→T at position 218.

Figure 5.

Figure 5.

Enzyme kinetics of the glycerol kinase variant (G692A, Gly232Asp) of E. coli G1-A in comparison to the wild-type enzyme (wt). Enzyme activities were determined as a decrease in absorbance at 340 nm monitoring ADP release by coupling pyruvate kinase and lactate dehydrogenase activity as detailed in Methods. (A) A 12-fold increase in enzyme activity in the recombinant mutant clone as opposed to the wild type. (B) Enzyme inhibition by an allosteric inhibitor Fructose-1,6-bisphosphate (FBP) was determined by addition of FBP to a final concentration of 2 mM. Inhibitory effects decreased by ∼33% in the enzyme variant when compared to the wild-type enzyme.

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