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.
Affiliations
- PMID: 15574828
- PMCID: PMC534674
- DOI: 10.1101/gr.2977704
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.
Figures
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.
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.
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.
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.
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.
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.
Similar articles
- Rapid identification of protein biomarkers of Escherichia coli O157:H7 by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight mass spectrometry and top-down proteomics.
Fagerquist CK, Garbus BR, Miller WG, Williams KE, Yee E, Bates AH, Boyle S, Harden LA, Cooley MB, Mandrell RE. Fagerquist CK, et al. Anal Chem. 2010 Apr 1;82(7):2717-25. doi: 10.1021/ac902455d. Anal Chem. 2010. PMID: 20232878 - Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium.
LaCroix RA, Sandberg TE, O'Brien EJ, Utrilla J, Ebrahim A, Guzman GI, Szubin R, Palsson BO, Feist AM. LaCroix RA, et al. Appl Environ Microbiol. 2015 Jan;81(1):17-30. doi: 10.1128/AEM.02246-14. Epub 2014 Oct 10. Appl Environ Microbiol. 2015. PMID: 25304508 Free PMC article. - Mass-spectrometry DNA sequencing.
Edwards JR, Ruparel H, Ju J. Edwards JR, et al. Mutat Res. 2005 Jun 3;573(1-2):3-12. doi: 10.1016/j.mrfmmm.2004.07.021. Mutat Res. 2005. PMID: 15829234 Review. - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens.
Garritsen HS, Fan AX, Bosse N, Hannig H, Kelsch R, Kroll H, Holzgreve W, Zhong XY. Garritsen HS, et al. Transfusion. 2009 Feb;49(2):252-8. doi: 10.1111/j.1537-2995.2008.01953.x. Epub 2008 Oct 29. Transfusion. 2009. PMID: 18980617 - DNA analysis by MALDI-TOF mass spectrometry.
Gut IG. Gut IG. Hum Mutat. 2004 May;23(5):437-41. doi: 10.1002/humu.20023. Hum Mutat. 2004. PMID: 15108274 Review.
Cited by
- Experimental Evolution of Anticipatory Regulation in Escherichia coli.
Mahilkar A, Venkataraman P, Mall A, Saini S. Mahilkar A, et al. Front Microbiol. 2022 Jan 11;12:796228. doi: 10.3389/fmicb.2021.796228. eCollection 2021. Front Microbiol. 2022. PMID: 35087497 Free PMC article. - Transposon-mediated directed mutation controlled by DNA binding proteins in Escherichia coli.
Saier MH Jr, Zhang Z. Saier MH Jr, et al. Front Microbiol. 2014 Aug 1;5:390. doi: 10.3389/fmicb.2014.00390. eCollection 2014. Front Microbiol. 2014. PMID: 25136335 Free PMC article. No abstract available. - Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: the role of extra copies of glpK, glpX, and tktA genes.
Gottlieb K, Albermann C, Sprenger GA. Gottlieb K, et al. Microb Cell Fact. 2014 Jul 11;13(1):96. doi: 10.1186/s12934-014-0096-1. Microb Cell Fact. 2014. PMID: 25012491 Free PMC article. - Comprehensive detection of genes causing a phenotype using phenotype sequencing and pathway analysis.
Harper M, Gronenberg L, Liao J, Lee C. Harper M, et al. PLoS One. 2014 Feb 26;9(2):e88072. doi: 10.1371/journal.pone.0088072. eCollection 2014. PLoS One. 2014. PMID: 24586303 Free PMC article. - Two combinatorial optimization problems for SNP discovery using base-specific cleavage and mass spectrometry.
Chen X, Wu Q, Sun R, Zhang L. Chen X, et al. BMC Syst Biol. 2012;6 Suppl 2(Suppl 2):S5. doi: 10.1186/1752-0509-6-S2-S5. Epub 2012 Dec 12. BMC Syst Biol. 2012. PMID: 23282116 Free PMC article.
References
- Bjedov, I., Tenaillon, O., Gérard, B., Souza, V., Denamur, E., Radman, M., Taddei, F., and Matic, I. 2003. Stress-induced mutagenesis in bacteria. Science 300: 1404-1409. - PubMed
- Blattner, F.R., Plunket III, G., Bloch, C.A., Perna, T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., et al. 1997. The complete sequence of Escherichia coli K-12. Science 277: 1453-1462. - PubMed
- Boecker, S. 2003. SNP and mutation discovery using base-specific cleavage and MALDI-TOF mass spectrometry. Bioinformatics 19: 44-53. - PubMed
Web site references
- http://www.premierbiosoft.com/netprimer/; NetPrimer.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Miscellaneous