A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species
Edited by Roy Curtiss III, University of Florida, Gainesville, FL, and approved January 26, 2016 (received for review October 9, 2015)
Significance
Current tools for bacterial genome engineering suffer from major limitations. They have been optimized for a few laboratory model strains, lead to the accumulation of numerous undesired, off-target modifications, and demand extensive modification of the host genome prior to large-scale editing. Herein, we address these problems and present a simple, all-in-one solution. By utilizing a highly conserved mutant allele of the bacterial mismatch-repair system, we were able to gain unprecedented precision in the control over the generation of desired modifications in multiple bacterial species. These results have broad implications with regards to both biotechnological and clinical applications.
Abstract
Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.
Acknowledgments
We thank Donald L. Court for providing the λ Red recombinase expression plasmids; Tamás Fehér for donating pZA31tetR and pZA31YFPtetR; and Andrea Tóth for her technical assistance. This work was supported by grants from the European Research Council (to C.P.), the Wellcome Trust (to C.P.), and the Lendület Program of the Hungarian Academy of Sciences (to C.P.); Hungarian Scientific Research Fund Grants OTKA PD 109572 (to B.C.) and OTKA PD 106231 (to K.U.); Hungarian Academy of Sciences Postdoctoral Fellowship Program Grant SZ-039/2013 (to B. Bogos); the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (I.N.); and a PhD fellowship from the Boehringer Ingelheim Fonds (to Á.N.).
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References
1
KM Esvelt, HH Wang, Genome-scale engineering for systems and synthetic biology. Mol Syst Biol 9, 641 (2013).
2
Y Li, et al., Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab Eng 31, 13–21 (2015).
3
Y Jiang, et al., Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microiol 81, 2506–2514 (2015).
4
RR Gallagher, Z Li, AO Lewis, FJ Isaacs, Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA. Nat Protoc 9, 2301–2316 (2014).
5
HH Wang, et al., Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).
6
DL Court, JA Sawitzke, LC Thomason, Genetic engineering using homologous recombination. Annu Rev Genet 36, 361–388 (2002).
7
HH Wang, et al., Genome-scale promoter engineering by coselection MAGE. Nat Methods 9, 591–593 (2012).
8
S Raman, JK Rogers, ND Taylor, GM Church, Evolution-guided optimization of biosynthetic pathways. Proc Natl Acad Sci USA 111, 17803–17808 (2014).
9
NR Sandoval, et al., Strategy for directing combinatorial genome engineering in Escherichia coli. Proc Natl Acad Sci USA 109, 10540–10545 (2012).
10
MJ Lajoie, et al., Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).
11
AJ Rovner, et al., Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).
12
DJ Mandell, et al., Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).
13
JP van Pijkeren, RA Britton, Precision genome engineering in lactic acid bacteria. Microb Cell Fact 13, S10 (2014).
14
S Binder, S Siedler, J Marienhagen, M Bott, L Eggeling, Recombineering in Corynebacterium glutamicum combined with optical nanosensors: A general strategy for fast producer strain generation. Nucleic Acids Res 41, 6360–6369 (2013).
15
Z Sun, et al., A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35. Appl Microbiol Biotechnol 99, 5151–5162 (2015).
16
N Costantino, DL Court, Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants. Proc Natl Acad Sci USA 100, 15748–15753 (2003).
17
FJ Isaacs, et al., Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).
18
Á Nyerges, et al., Conditional DNA repair mutants enable highly precise genome engineering. Nucleic Acids Res 42, e62 (2014).
19
RM Lennen, et al., Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects. Nucleic Acids Res, 2015).
20
A Aronshtam, MG Marinus, Dominant negative mutator mutations in the mutL gene of Escherichia coli. Nucleic Acids Res 24, 2498–2504 (1996).
21
YY Polosina, J Mui, P Pitsikas, CG Cupples, The Escherichia coli mismatch repair protein MutL recruits the Vsr and MutH endonucleases in response to DNA damage. J Bacteriol 191, 4041–4043 (2009).
22
C Ban, W Yang, Crystal structure and ATPase activity of MutL: Implications for DNA repair and mutagenesis. Cell 95, 541–552 (1998).
23
DJ Baumler, B Ma, JL Reed, NT Perna, Inferring ancient metabolism using ancestral core metabolic models of enterobacteria. BMC Syst Biol 7, 46 (2013).
24
R Lutz, H Bujard, Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res 25, 1203–1210 (1997).
25
T Fehér, et al., Competition between transposable elements and mutator genes in bacteria. Mol Biol Evol 29, 3153–3159 (2012).
26
PL Foster, Methods for determining spontaneous mutation rates. Methods Enzymol 409, 195–213 (2006).
27
S Datta, N Costantino, DL Court, A set of recombineering plasmids for gram-negative bacteria. Gene 379, 109–115 (2006).
28
HH Wang, G Xu, AJ Vonner, G Church, Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion. Nucleic Acids Res 39, 7336–7347 (2011).
29
B Lehner, Molecular mechanisms of epistasis within and between genes. Trends Genet 27, 323–331 (2011).
30
V Lázár, et al., Genome-wide analysis captures the determinants of the antibiotic cross-resistance interaction network. Nat Commun 5, 4352 (2014).
31
V Lázár, et al., Bacterial evolution of antibiotic hypersensitivity. Mol Syst Biol 9, 700 (2013).
32
ME Cullen, AW Wyke, R Kuroda, LM Fisher, Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones. Antimicrob Agents Chemother 33, 886–894 (1989).
33
J Ruiz, et al., High frequency of mutations at codon 83 of the gyrA gene of quinolone-resistant clinical isolates of Escherichia coli. J Antimicrob Chemother 36, 737–738 (1995).
34
MN Alekshun, SB Levy, The mar regulon: Multiple resistance to antibiotics and other toxic chemicals. Trends Microbiol 7, 410–413 (1999).
35
MT Bonde, et al., MODEST: A web-based design tool for oligonucleotide-mediated genome engineering and recombineering. Nucleic Acids Res 42, W408–W415 (2014).
36
MT Bonde, et al., Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides. ACS Synth Biol 4, 17–22 (2015).
37
AB Dalia, E McDonough, A Camilli, Multiplex genome editing by natural transformation. Proc Natl Acad Sci USA 111, 8937–8942 (2014).
38
DK Jacquelín, A Filiberti, CE Argaraña, JL Barra, Pseudomonas aeruginosa MutL protein functions in Escherichia coli. Biochem J 388, 879–887 (2005).
39
B Quaresima, et al., Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: A simple genetic system to assay MLH1 function. Biochem J 371, 183–189 (2003).
40
JE DiCarlo, et al., Yeast oligo-mediated genome engineering (YOGE). ACS Synth Biol 2, 741–749 (2013).
41
X Rios, et al., Stable gene targeting in human cells using single-strand oligonucleotides with modified bases. PLoS One 7, e36697 (2012).
42
K Xu, AF Stewart, ACG Porter, Stimulation of oligonucleotide-directed gene correction by Redβ expression and MSH2 depletion in human HT1080 cells. Mol Cells 38, 33–39 (2015).
43
S Datta, N Costantino, X Zhou, DL Court, Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc Natl Acad Sci USA 105, 1626–1631 (2008).
44
A Lopes, J Amarir-Bouhram, G Faure, M-A Petit, R Guerois, Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res 38, 3952–3962 (2010).
45
Y Guo, BB Kragelund, MF White, X Peng, Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities. J Mol Biol 427, 2179–2191 (2015).
46
M Kushwaha, HM Salis, A portable expression resource for engineering cross-species genetic circuits and pathways. Nat Commun 6, 7832 (2015).
47
RT Ranallo, S Barnoy, S Thakkar, T Urick, MM Venkatesan, Developing live Shigella vaccines using λ Red recombineering. FEMS Immunol Med Microbiol 47, 462–469 (2006).
48
C Yang, et al., Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol. Biochem Eng J 57, 55–62 (2011).
49
PI Nikel, E Martínez-García, V de Lorenzo, Biotechnological domestication of pseudomonads using synthetic biology. Nat Rev Microbiol 12, 368–379 (2014).
50
C Pál, B Papp, G Pósfai, The dawn of evolutionary genome engineering. Nat Rev Genet 15, 504–512 (2014).
51
M Schirmer, et al., Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res 43, e37 (2015).
52
J Felton, S Michaelis, A Wright, Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J Bacteriol 142, 221–228 (1980).
53
KA Datsenko, BL Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640–6645 (2000).
54
I Wiegand, K Hilpert, REW Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3, 163–175 (2008).
55
K Zhou, et al., Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol 12, 18 (2011).
56
BM Hall, C-X Ma, P Liang, KK Singh, Fluctuation analysis CalculatOR: A web tool for the determination of mutation rate using Luria-Delbruck fluctuation analysis. Bioinformatics 25, 1564–1565 (2009).
57
H Kim, J-S Kim, A guide to genome engineering with programmable nucleases. Nat Rev Genet 15, 321–334 (2014).
58
HH Wang, GM Church, Multiplexed genome engineering and genotyping methods applications for synthetic biology and metabolic engineering. Methods Enzymol 498, 409–426 (2011).
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Published online: February 16, 2016
Published in issue: March 1, 2016
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Acknowledgments
We thank Donald L. Court for providing the λ Red recombinase expression plasmids; Tamás Fehér for donating pZA31tetR and pZA31YFPtetR; and Andrea Tóth for her technical assistance. This work was supported by grants from the European Research Council (to C.P.), the Wellcome Trust (to C.P.), and the Lendület Program of the Hungarian Academy of Sciences (to C.P.); Hungarian Scientific Research Fund Grants OTKA PD 109572 (to B.C.) and OTKA PD 106231 (to K.U.); Hungarian Academy of Sciences Postdoctoral Fellowship Program Grant SZ-039/2013 (to B. Bogos); the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (I.N.); and a PhD fellowship from the Boehringer Ingelheim Fonds (to Á.N.).
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This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species, Proc. Natl. Acad. Sci. U.S.A.
113 (9) 2502-2507,
https://doi.org/10.1073/pnas.1520040113
(2016).
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