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Volume 168, Issue 6

Markerless gene editing in Open Access

Abstract

, the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of as a counter-selectable marker that can be used for markerless mutagenesis in . encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in . We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and , that efficiently rendered susceptible to growth on 2-DOG. We then adapted the -based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for has a high success rate, and should be a valuable gene editing tool in the future.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): counter-selection , galK , markerless and Neisseria gonorrhoeae
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-06-28
2024-04-27
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References

  1. WHO Global health sector strategy on Sexually Transmitted Infections, 2016-2021; 2016 https://www.who.int/publications/i/item/WHO-RHR-16.09 accessed 21 March 2022
  2. Cehovin A, Jolley KA, Maiden MCJ, Harrison OB, Tang CM. Association of Neisseria gonorrhoeae plasmids with distinct lineages and the economic status of their country of origin. J Infect Dis 2020; 222:1826–1836 [View Article] [PubMed]
     [Google Scholar]
  3. Unemo M, Del Rio C, Shafer WM. Antimicrobial resistance expressed by Neisseria gonorrhoeae: a major global public health problem in the 21st century. Microbiol Spectr 2016; 4: [View Article] [PubMed]
     [Google Scholar]
  4. Cehovin A, Simpson PJ, McDowell MA, Brown DR, Noschese R et al. Specific DNA recognition mediated by a type IV pilin. Proc Natl Acad Sci U S A 2013; 110:3065–3070 [View Article] [PubMed]
     [Google Scholar]
  5. Callaghan MM, Dillard JP. Transformation in Neisseria gonorrhoeae. Methods Mol Biol 2019; 1997:143–162 [View Article] [PubMed]
     [Google Scholar]
  6. Hobbs MM, Duncan JA. Experimental human infection with Neisseria gonorrhoeae. Methods Mol Biol 2019; 1997:431–452 [View Article] [PubMed]
     [Google Scholar]
  7. Ball LM, Criss AK. Constitutively Opa-expressing and Opa-deficient neisseria gonorrhoeae strains differentially stimulate and survive exposure to human neutrophils. J Bacteriol 2013; 195:2982–2990 [View Article] [PubMed]
     [Google Scholar]
  8. Pachulec E, Siewering K, Bender T, Heller E-M, Salgado-Pabon W et al. Functional analysis of the gonococcal genetic island of Neisseria gonorrhoeae. PLoS One 2014; 9:e109613 [View Article] [PubMed]
     [Google Scholar]
  9. Reyrat JM, Pelicic V, Gicquel B, Rappuoli R. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect Immun 1998; 66:4011–4017 [View Article] [PubMed]
     [Google Scholar]
  10. Johnston DM, Cannon JG. Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection. Gene 1999; 236:179–184 [View Article] [PubMed]
     [Google Scholar]
  11. Kohler PL, Cloud KA, Hackett KT, Beck ET, Dillard JP. Characterization of the role of LtgB, a putative lytic transglycosylase in Neisseria gonorrhoeae. Microbiology 2005; 151:3081–3088 [View Article] [PubMed]
     [Google Scholar]
  12. Ueki T, Inouye S, Inouye M. Positive-negative KG cassettes for construction of multi-gene deletions using a single drug marker. Gene 1996; 183:153–157 [View Article] [PubMed]
     [Google Scholar]
  13. Weickert MJ, Adhya S. The galactose regulon of Escherichia coli. Mol Microbiol 1993; 10:245–251 [View Article] [PubMed]
     [Google Scholar]
  14. Alper MD, Ames BN. Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions. J Bacteriol 1975; 121:259–266 [View Article] [PubMed]
     [Google Scholar]
  15. Biswas K, Stauffer S, Sharan SK. Using recombineering to generate point mutations:galK-based positive-negative selection method. Methods Mol Biol 2012; 852:121–131 [View Article] [PubMed]
     [Google Scholar]
  16. Barkan D, Stallings CL, Glickman MS. An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose. Gene 2011; 470:31–36 [View Article] [PubMed]
     [Google Scholar]
  17. Gregoire SA, Byam J, Pavelka MS. galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus. Microbiology 2017; 163:1399–1408 [View Article]
     [Google Scholar]
  18. Jennings MP, van der Ley P, Wilks KE, Maskell DJ, Poolman JT et al. Cloning and molecular analysis of the galE gene of Neisseria meningitidis and its role in lipopolysaccharide biosynthesis. Mol Microbiol 1993; 10:361–369 [PubMed]
     [Google Scholar]
  19. Robertson BD, Frosch M, van Putten JP. The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide. Mol Microbiol 1993; 8:891–901 [View Article] [PubMed]
     [Google Scholar]
  20. Cornelissen CN. Subversion of nutritional immunity by the pathogenic Neisseriae. Pathog Dis 2018; 76: [View Article] [PubMed]
     [Google Scholar]
  21. Marsay L, Dold C, Green CA, Rollier CS, Norheim G et al. A novel meningococcal outer membrane vesicle vaccine with constitutive expression of FetA: A phase I clinical trial. J Infect 2015; 71:326–337 [View Article] [PubMed]
     [Google Scholar]
  22. Maharjan S, Saleem M, Feavers IM, Wheeler JX, Care R et al. Dissection of the function of the RmpM periplasmic protein from Neisseria meningitidis. Microbiology 2016; 162:364–375 [View Article] [PubMed]
     [Google Scholar]
  23. Gulati S, Mu X, Zheng B, Reed GW, Ram S et al. Antibody to reduction modifiable protein increases the bacterial burden and the duration of gonococcal infection in a mouse model. J Infect Dis 2015; 212:311–315 [View Article] [PubMed]
     [Google Scholar]
  24. Joiner KA, Scales R, Warren KA, Frank MM, Rice PA. Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae. J Clin Invest 1985; 76:1765–1772 [View Article] [PubMed]
     [Google Scholar]
  25. Pachulec E, van der Does C. Conjugative plasmids of Neisseria gonorrhoeae. PLoS One 2010; 5:e9962 [View Article] [PubMed]
     [Google Scholar]
  26. Roberts MC, Knapp JS. Transfer of beta-lactamase plasmids from Neisseria gonorrhoeae to Neisseria meningitidis and commensal Neisseria species by the 25.2-megadalton conjugative plasmid. Antimicrob Agents Chemother 1988; 32:1430–1432 [View Article] [PubMed]
     [Google Scholar]
  27. Chen J, Li Y, Zhang K, Wang H. Whole-genome sequence of phage-resistant strain Escherichia coli DH5α. Genome Announc 2018; 6:10 [View Article] [PubMed]
     [Google Scholar]
  28. Dillard JP. Genetic manipulation of Neisseria gonorrhoeae. Curr Protoc Microbiol 2011; Chapter 4:2 [View Article] [PubMed]
     [Google Scholar]
  29. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410 [View Article] [PubMed]
     [Google Scholar]
  30. Ramsey ME, Hackett KT, Kotha C, Dillard JP. New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol 2012; 78:3068–3078 [View Article] [PubMed]
     [Google Scholar]
  31. Rosenqvist E, Musacchio A, Aase A, Høiby EA, Namork E et al. Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis. Infect Immun 1999; 67:1267–1276 [View Article] [PubMed]
     [Google Scholar]
  32. Cehovin A, Harrison OB, Lewis SB, Ward PN, Ngetsa C et al. Identification of novel Neisseria gonorrhoeae lineages harboring resistance plasmids in coastal Kenya. J Infect Dis 2018; 218:801–808 [View Article] [PubMed]
     [Google Scholar]
  33. Rudel T, van Putten JP, Gibbs CP, Haas R, Meyer TF. Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells. Mol Microbiol 1992; 6:3439–3450 [View Article] [PubMed]
     [Google Scholar]
  34. Leuzzi R, Nesta B, Monaci E, Cartocci E, Serino L et al. Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells. BMC Microbiol 2013; 13:251 [View Article] [PubMed]
     [Google Scholar]
  35. Hollander A, Mercante AD, Shafer WM, Cornelissen CN. The iron-repressed, AraC-like regulator MpeR activates expression of fetA in Neisseria gonorrhoeae. Infect Immun 2011; 79:4764–4776 [View Article] [PubMed]
     [Google Scholar]
  36. Wi T, Lahra MM, Ndowa F, Bala M, Dillon J-AR et al. Antimicrobial resistance in Neisseria gonorrhoeae: global surveillance and a call for international collaborative action. PLoS Med 2017; 14:e1002344 [View Article] [PubMed]
     [Google Scholar]
  37. Debouck C, Riccio A, Schumperli D, McKenney K, Jeffers J et al. Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon. Nucleic Acids Res 1985; 13:1841–1853 [View Article] [PubMed]
     [Google Scholar]
  38. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 2005; 33:e36 [View Article] [PubMed]
     [Google Scholar]
  39. Ji X, Lu P, van der Veen S. Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes. Appl Microbiol Biotechnol 2019; 103:1465–1474 [View Article] [PubMed]
     [Google Scholar]
  40. DeVito JA. Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli. Nucleic Acids Res 2008; 36:e4 [View Article] [PubMed]
     [Google Scholar]
  41. Gurung I, Berry JL, Hall AMJ, Pelicic V. Cloning-independent markerless gene editing in Streptococcus sanguinis: novel insights in type IV pilus biology. Nucleic Acids Res 2017; 45:e40 [View Article] [PubMed]
     [Google Scholar]
  42. Petousis-Harris H, Paynter J, Morgan J, Saxton P, McArdle B et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. Lancet 2017; 390:1603–1610 [View Article] [PubMed]
     [Google Scholar]
  43. Matthias KA, Connolly KL, Begum AA, Jerse AE, Macintyre AN et al. Meningococcal detoxified outer membrane vesicle vaccines enhance gonococcal clearance in a murine infection model. J Infect Dis 2022; 225:650–660 [View Article] [PubMed]
     [Google Scholar]
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Markerless gene editing in Neisseria gonorrhoeae
Microbiology 168, 001201 (2022); https://doi.org/10.1099/mic.0.001201
/content/journal/micro/10.1099/mic.0.001201
/content/journal/micro/10.1099/mic.0.001201
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