Skip to main content
SpringerLink
Log in

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions

  • Protocol
  • First Online:
Metabolic Network Reconstruction and Modeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1716))

Abstract

The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArrayâ„¢ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. diCenzo GC, Finan TM (2015) Genetic redundancy is prevalent within the 6.7 Mb Sinorhizobium meliloti genome. Mol Genet Genomics 290:1345–1356. https://doi.org/10.1007/s00438-015-0998-6

    Article  CAS  PubMed  Google Scholar 

  2. Hutchison CA, Chuang R-Y, Noskov VN et al (2016) Design and synthesis of a minimal bacterial genome. Science 351:aad6253–aad6253. https://doi.org/10.1126/science.aad6253

    Article  PubMed  Google Scholar 

  3. Biondi EG, Tatti E, Comparini D et al (2009) Metabolic capacity of Sinorhizobium (Ensifer) meliloti strains as determined by Phenotype MicroArray analysis. Appl Environ Microbiol 75:5396–5404. https://doi.org/10.1128/AEM.00196-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. diCenzo GC, Checcucci A, Bazzicalupo M et al (2016) Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti. Nat Commun 7:12219. https://doi.org/10.1038/ncomms12219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Charles TC, Finan TM (1991) Analysis of a 1600-kilobase Rhizobium meliloti megaplasmid using defined deletions generated in vivo. Genetics 127:5–20

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Milunovic B, diCenzo GC, Morton RA, Finan TM (2014) Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti. J Bacteriol 196:811–824. https://doi.org/10.1128/JB.01104-13

    Article  PubMed  PubMed Central  Google Scholar 

  7. Chain PS, Hernández-Lucas I, Golding B, Finan TM (2000) oriT-directed cloning of defined large regions from bacterial genomes: identification of the Sinorhizobium meliloti pExo megaplasmid replicator region. J Bacteriol 182:5486–5494. https://doi.org/10.1128/JB.182.19.5486-5494.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. MacLean AM, White CE, Fowler JE, Finan TM (2009) Identification of a hydroxyproline transport system in the legume endosymbiont Sinorhizobium meliloti. Mol Plant Microbe Interact 22:1116–1127. https://doi.org/10.1094/MPMI-22-9-1116

    Article  CAS  PubMed  Google Scholar 

  9. MacLean AM, MacPherson G, Aneja P, Finan TM (2006) Characterization of the β-ketoadipate pathway in Sinorhizobium meliloti. Appl Environ Microbiol 72:5403–5413. https://doi.org/10.1128/AEM.00580-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yurgel SN, Mortimer MW, Rice JT et al (2013) Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library. Appl Environ Microbiol 79:2081–2087. https://doi.org/10.1128/AEM.02974-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheng J, Poduska B, Morton RA, Finan TM (2011) An ABC-type cobalt transport system is essential for growth of Sinorhizobium meliloti at trace metal concentrations. J Bacteriol 193:4405–4416. https://doi.org/10.1128/JB.05045-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. diCenzo G, Milunovic B, Cheng J, Finan TM (2013) The tRNAarg gene and engA are essential genes on the 1.7-Mb pSymB megaplasmid of Sinorhizobium meliloti and were translocated together from the chromosome in an ancestral strain. J Bacteriol 195:202–212. https://doi.org/10.1128/JB.01758-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ying B-W, Seno S, Kaneko F et al (2013) Multilevel comparative analysis of the contributions of genome reduction and heat shock to the Escherichia coli transcriptome. BMC Genomics 14:25. https://doi.org/10.1186/1471-2164-14-25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dominguez-Ferreras A, Perez-Arnedo R, Becker A et al (2006) Transcriptome profiling reveals the importance of plasmid pSymB for osmoadaptation of Sinorhizobium meliloti. J Bacteriol 188:7617–7625. https://doi.org/10.1128/JB.00719-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. diCenzo GC, MacLean AM, Milunovic B et al (2014) Examination of prokaryotic multipartite genome evolution through experimental genome reduction. PLoS Genet 10:e1004742. https://doi.org/10.1371/journal.pgen.1004742

    Article  PubMed  PubMed Central  Google Scholar 

  16. Ullrich S, Schüler D (2010) Cre-lox-based method for generation of large deletions within the genomic magnetosome island of Magnetospirillum gryphiswaldense. Appl Environ Microbiol 76:2439–2444. https://doi.org/10.1128/AEM.02805-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. diCenzo GC, Zamani M, Milunovic B, Finan TM (2016) Genomic resources for identification of the minimal N2-fixing symbiotic genome. Environ Microbiol 18:2534–2547. https://doi.org/10.1111/1462-2920.13221

    Article  CAS  PubMed  Google Scholar 

  18. Döhlemann J, Brennecke M, Becker A (2016) Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination. J Biotechnol 233:160–170. https://doi.org/10.1016/j.jbiotec.2016.06.033

    Article  PubMed  Google Scholar 

  19. Bochner BR (2001) Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. Genome Res 11:1246–1255. https://doi.org/10.1101/gr.186501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Finan TM, Kunkel B, De Vos GF, Signer ER (1986) Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167:66–72

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vincze T, Pósfai J, Roberts RJ (2003) NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res 31:3688–3691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Leprince A, de Lorenzo V, Völler P et al (2012) Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida. Environ Microbiol 14:1444–1453. https://doi.org/10.1111/j.1462-2920.2012.02730.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang Y, Wang Z, Cao J et al (2014) FLP-FRT-based method to obtain unmarked deletions of CHU_3237 (porU) and large genomic fragments of Cytophaga hutchinsonii. Appl Environ Microbiol 80:6037–6045. https://doi.org/10.1128/AEM.01785-14

    Article  PubMed  PubMed Central  Google Scholar 

  24. Lambert JM, Bongers RS, Kleerebezem M (2007) Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol 73:1126–1135. https://doi.org/10.1128/AEM.01473-06

    Article  CAS  PubMed  Google Scholar 

  25. Harrison CL, Crook MB, Peco G et al (2011) Employing site-specific recombination for conditional genetic analysis in Sinorhizobium meliloti. Appl Environ Microbiol 77:3916–3922. https://doi.org/10.1128/AEM.00544-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yu BJ, Sung BH, Koob MD et al (2002) Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20:1018–1023. https://doi.org/10.1038/nbt740

    Article  CAS  PubMed  Google Scholar 

  27. Heil JR, Cheng J, Charles TC (2012) Site-specific bacterial chromosome engineering: ΦC31 integrase mediated cassette exchange (IMCE). J Vis Exp 61:e3698. https://doi.org/10.3791/3698

    Google Scholar 

  28. Jones JD, Gutterson N (1987) An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a. Gene 61:299–306

    Article  CAS  PubMed  Google Scholar 

  29. Posfai G, Koob MD, Kirkpatrick HA, Blattner FR (1997) Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. J Bacteriol 179:4426–4428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Posfai G, Koob M, Hradecná Z et al (1994) In vivo excision and amplification of large segments of the Escherichia coli genome. Nucleic Acids Res 22:2392–2398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wilson JW, Figurski DH, Nickerson CA (2004) VEX-capture: a new technique that allows in vivo excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments. J Microbiol Methods 57:297–308. https://doi.org/10.1016/j.mimet.2004.01.007

    Article  CAS  PubMed  Google Scholar 

  32. Galardini M, Mengoni A, Biondi EG et al (2014) DuctApe: a suite for the analysis and correlation of genomic and OmniLog™ Phenotype Microarray data. Genomics 103:1–10. https://doi.org/10.1016/j.ygeno.2013.11.005

    Article  CAS  PubMed  Google Scholar 

  33. Vaas LAI, Sikorski J, Michael V et al (2012) Visualization and curve-parameter estimation strategies for efficient exploration of phenotype microarray kinetics. PLoS One 7:e34846. https://doi.org/10.1371/journal.pone.0034846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vehkala M, Shubin M, Connor TR et al (2015) Novel R pipeline for analyzing biolog phenotypic microarray data. PLoS One 10:e0118392. https://doi.org/10.1371/journal.pone.0118392

    Article  PubMed  PubMed Central  Google Scholar 

  35. Schellenberger J, Que R, Fleming RMT et al (2011) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat Protoc 6:1290–1307. https://doi.org/10.1038/nprot.2011.308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ebrahim A, Lerman JA, Palsson BØ, Hyduke DR (2013) COBRApy: COnstraints-based reconstruction and analysis for python. BMC Syst Biol 7:74. https://doi.org/10.1186/1752-0509-7-74

    Article  PubMed  PubMed Central  Google Scholar 

  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  38. Fei F, diCenzo GC, Bowdish DME et al (2016) Effects of synthetic large-scale genome reduction on metabolism and metabolic preferences in a nutritionally complex environment. Metabolomics 12:23. https://doi.org/10.1007/s11306-015-0928-y

    Article  Google Scholar 

  39. Spini G, Decorosi F, Cerboneschi M et al (2015) Effect of the plant flavonoid luteolin on Ensifer meliloti 3001 phenotypic responses. Plant Soil 399:159–178. https://doi.org/10.1007/s11104-015-2659-2

    Article  Google Scholar 

  40. Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318

    Article  CAS  PubMed  Google Scholar 

  41. Zhang Y, Werling U, Edelmann W (2014) Seamless ligation cloning extract (SLiCE) cloning method. Methods Mol Biol 1116:235–244. https://doi.org/10.1007/978-1-62703-764-8_16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jeong J-Y, Yim H-S, Ryu J-Y et al (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443. https://doi.org/10.1128/AEM.00844-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, McMaster University, Hamilton, ON, Canada

    George C. diCenzo & Turlough M. Finan

Authors
  1. George C. diCenzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Turlough M. Finan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Turlough M. Finan .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Florence, Sesto Fiorentino, Florence, Italy

    Marco Fondi

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

diCenzo, G.C., Finan, T.M. (2018). Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions. In: Fondi, M. (eds) Metabolic Network Reconstruction and Modeling. Methods in Molecular Biology, vol 1716. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7528-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7528-0_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7527-3

  • Online ISBN: 978-1-4939-7528-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation