Genetic Manipulation of Neisseria gonorrhoeae

Joseph P. Dillard1

1 Medical Microbiology and Immunology, University of Wisconsin‐Madison, Madison, Wisconsin
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 4A.2
DOI:  10.1002/9780471729259.mc04a02s23
Online Posting Date:  November, 2011
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Abstract

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and to the panmictic structure of the gonococcal population. However, high‐frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high‐frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Techniques are described for genetic manipulation of N. gonorrhoeae, as well as for growth of this fastidious organism. Curr. Protoc. Microbiol. 23:4A.2.1‐4A.2.24. © 2011 by John Wiley & Sons, Inc.

Keywords: Neisseria gonorrhoeae; natural transformation; electroporation; complementation; genetic transformation; heterologous gene expression

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Spot Transformation of Neisseria gonorrhoeae on Agar Plates
  • Basic Protocol 2: Transformation of Neisseria gonorrhoeae in Liquid Culture
  • Basic Protocol 3: Electroporation of Neisseria gonorrhoeae
  • Basic Protocol 4: Creation of Unmarked Mutations Using a Positive and Negative Selection Cassette
  • Basic Protocol 5: Shuttle Mutagenesis of Neisseria gonorrhoeae DNA Cloned into PHSS6 Plasmids Using mTnCmNS
  • Basic Protocol 6: Chemical Mutagenesis
  • Basic Protocol 7: Complementation on the Neisseria gonorrhoeae Chromosome
  • Alternate Protocol 1: Complementation with Replicating Plasmids
  • Basic Protocol 8: Preparation of Chromosomal DNA from Neisseria gonorrhoeae Grown on Solid Medium
  • Alternate Protocol 2: Preparation of Chromosomal DNA from Neisseria gonorrhoeae Grown in Broth
  • Support Protocol 1: Preparing PCR Templates from Neisseria gonorrhoeae Colonies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spot Transformation of Neisseria gonorrhoeae on Agar Plates

  Materials
  • Recipient N. gonorrhoeae strain, frozen
  • GCB plates (see recipe), room temperature and 37°C
  • ≥10 ng/µl plasmid DNA
  • Appropriate restriction enzyme and buffer (for double‐crossover only)
  • GCB plate containing appropriate antibiotic (Table 4.2.1; for fragments containing selectable markers only)
  • Dacron swabs (Fisher Scientific), sterile
  • Additional reagents and equipment for PCR (see protocol 11).

Basic Protocol 2: Transformation of Neisseria gonorrhoeae in Liquid Culture

  Materials
  • Recipient N. gonorrhoeae strain, frozen
  • GCB plate (see recipe)
  • ≥10 ng/µl plasmid DNA
  • GCBL medium (see recipe) containing 5 mM MgSO 4, room temperature and 37°C
  • GCBL medium containing Kellogg's supplements (see recipe)
  • 60% (v/v) glycerol
  • 1.5‐ml microcentrifuge tubes
  • Dacron swabs, sterile
  • 25‐cm2 tissue culture flask, 37°C

Basic Protocol 3: Electroporation of Neisseria gonorrhoeae

  Materials
  • Recipient N. gonorrhoeae strain, frozen
  • GCB plates (see recipe) with and without antibiotic (Table 4.2.1)
  • 0.3 M sucrose: pass through a 0.22‐µm filter to sterilize; store up to 1 year at room temperature
  • 10 ng/µl DNA solution
  • GCBL medium with Kellogg's supplements (see recipe)
  • GCBL medium (see recipe)
  • Dacron swabs (Fisher Scientific), sterile
  • Microcentrifuge
  • Electroporation cuvette, 2‐mm gap length
  • Electroporator

Basic Protocol 4: Creation of Unmarked Mutations Using a Positive and Negative Selection Cassette

  Materials
  • Gene of interest
  • pKH6, pUP1 (Elkins et al., ), or similar plasmid carrying a DUS and a marker conferring resistance other than erythromycin
  • pKC1 (Cloud and Dillard, ) or similar plasmid carrying an ermC/rpsL cassette
  • PCR primers for introducing an unmarked mutation into gene of interest
  • E. coli
  • N. gonorrhoeae, streptomycin‐resistant strain
  • GCB plate (see recipe) containing erythromycin (Table 4.2.1)
  • GCB plate (see recipe) containing streptomycin (Table 4.2.1)
  • Dacron swab, sterile (Fisher Scientific)
  • Additional reagents and equipment for preparing a miniprep (Engebrecht et al., ), spot transformation ( protocol 1), and PCR ( protocol 11)

Basic Protocol 5: Shuttle Mutagenesis of Neisseria gonorrhoeae DNA Cloned into PHSS6 Plasmids Using mTnCmNS

  Materials
  • E. coli strains RDP146(pTCA), RDP146(pOX38::mTnCmNS), and NS2114Sm (Boyle‐Vavra and Seifert, )
  • LB medium ( appendix 4A)
  • 1 mM HEPES, pH 7 (optional): sterilize by autoclaving; store up to 1 year at room temperature
  • 10 ng/µl target plasmid
  • LB plates ( appendix 4A) containing 40 µg/ml kanamycin
  • LB plates containing 40 µg/ml kanamycin and 12 µg/ml tetracycline
  • LB plates containing 40 µg/ml kanamycin and 25 µg/ml chloramphenicol
  • LB plates containing 100 µg/ml streptomycin, 40 µg/ml kanamycin, and 25 µg/ml chloramphenicol
  • GCB plates (see recipe) containing chloramphenicol (Table 4.2.1)
  • Electroporation cuvette, 2‐mm gap length
  • Electroporator
  • 37°C water bath
  • Rotator
  • Additional reagents and solutions for PCR ( protocol 11; optional) and spot transformation with N. gonorrhoeae ( protocol 1)

Basic Protocol 6: Chemical Mutagenesis

  Materials
  • N. gonorrhoeae strain, frozen
  • GCB plates (see recipe)
  • GCBL medium with Kellogg's supplements and sodium bicarbonate (see recipe), 37°C
  • Ethyl methanesulfonate (EMS)
  • GCBL medium
  • GCBL medium containing 15% glycerol
  • GCB plates containing nalidixic acid (Table 4.2.1)
  • Dacron swab

Basic Protocol 7: Complementation on the Neisseria gonorrhoeae Chromosome

  Materials
  • Gene of interest
  • pKH35 (Hamilton et al., ) or similar plasmid
  • E. coli
  • N. gonorrhoeae mutant of interest
  • GCB plate (see recipe) containing chloramphenicol (Table 4.2.1)
  • 1 M IPTG solution
  • Dacron swab, sterile (Fisher Scientific)
  • Additional reagents and equipment for preparing a miniprep (Engebrecht et al., ), spot transformation ( protocol 1), and PCR ( protocol 11)

Alternate Protocol 1: Complementation with Replicating Plasmids

  Materials
  • N. gonorrhoeae, overnight culture
  • GCB plates (see recipe)
  • TES buffer (see recipe)
  • 10 mg/ml RNase A
  • 10% SDS
  • Tris‐buffered phenol ( appendix 2A)
  • Chloroform
  • 3 M sodium acetate
  • 95% and 70% ethanol
  • Dacron swab, sterile
  • 13‐ml snap‐cap polypropylene tubes
  • Centrifuge
  • 1‐ml pipet tips with wide bore

Basic Protocol 8: Preparation of Chromosomal DNA from Neisseria gonorrhoeae Grown on Solid Medium

  Materials
  • N. gonorrhoeae, frozen stock
  • GCB plate (see recipe)
  • GCBL medium with Kellog's supplements and sodium bicarbonate (see recipe), 37°C
  • TE buffer ( appendix 2A)
  • 10% SDS
  • 5 M potassium acetate
  • 95% and 70% ethanol
  • Dacron swab
  • Centrifuge
  • 13‐ml polypropylene tube
  • 65°C water bath
  • Glass rod

Alternate Protocol 2: Preparation of Chromosomal DNA from Neisseria gonorrhoeae Grown in Broth

  Materials
  • N. gonorrhoeae growing on solid medium
  • Colony lysis solution (see recipe)
  • Appropriate restriction enzyme(s) and buffer(s) (optional)
  • Thermal cycler and appropriate PCR tubes
  • Additional reagents and equipment for PCR (Kramer and Coen, )
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Figures

Videos

Literature Cited

   Ambur, O.H., Frye, S.A., and Tonjum, T. 2007. New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J. Bacteriol. 189:2077‐2085.
   Belland, R.J. 1991. H‐DNA formation by the coding repeat elements of neisserial opa genes. Mol. Microbiol. 5:2351‐2360.
   Black, C.G., Fyfe, J.A., and Davies, J.K. 1998. Absence of an SOS‐like repair system in Neisseria gonorrhoeae. Gene 208:61‐66.
   Bloch, K.D. 1995. Mapping by multiple endonuclease digestions. Curr. Protoc. Mol. Biol. 13:3.2.1‐3.2.5.
   Boyle‐Vavra, S. and Seifert, H.S. 1993. Shuttle mutagenesis: Two mini‐transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae. Gene 129:51‐57.
   Boyle‐Vavra, S. and Seifert, H.S. 1994. Shuttle mutagenesis: A minitransposon for producing PhoA fusions in Neisseria gonorrhoeae. Gene 155:101‐106.
   Cahoon, L.A. and Seifert, H.S. 2009. An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae. Science 325:764‐767.
   Campbell, L.A. and Yasbin, R.E. 1984. Mutagenesis of Neisseria gonorrhoeae: Absence of error prone repair. J. Bacteriol. 160:288‐293.
   Chen, I. and Dubnau, D. 2004. DNA uptake during bacterial transformation. Nat. Rev. Microbiol. 2:241‐249.
   Cloud, K.A. and Dillard, J.P. 2002. A lytic transglycosylase of Neisseria gonorrhoeae is involved in peptidoglycan‐derived cytotoxin production. Infect. Immun. 70:2752‐2757.
   Cooke, S.J., Jolley, K., Ison, C.A., Young, H., and Heckels, J.E. 1998. Naturally occurring isolates of Neisseria gonorrhoeae, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules. FEMS Microbiol. Lett. 162:75‐82.
   Dillard, J.P. and Yother, J. 1994. Genetic and molecular characterization of capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 3. Mol. Microbiol. 12:959‐972.
   Elkins, C., Thomas, C.E., Seifert, H.S., and Sparling, P.F. 1991. Species‐specific uptake of DNA by gonococci is mediated by a 10‐base‐pair sequence. J. Bacteriol. 173:3911‐3913.
   Engebrecht, J., Brent, R., and Kaderbhai, M.A. 1991. Minipreps of plasmid DNA. Curr. Protoc. Mol. Biol. 15:1.6.1‐1.6.10.
   Genco, C.A., Chen, C.Y., Arko, R., Kapczynski, D., and Morse, S.A. 1991. Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers. J. Gen. Microbiol. 137:1313‐1321.
   Gibbs, C.P. and Meyer, T.F. 1996. Genome plasticity in Neisseria gonorrhoeae. FEMS Microbiol. Lett. 145:173‐179.
   Goodman, S.D. and Scocca, J.J. 1988. Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. U.S.A. 85:6982‐6986.
   Gunn, J.S. and Stein, D.C. 1996. Use of a nonselective transformation technique to construct a multiply restriction/modification‐deficient mutant of Neisseria gonorrhoeae. Mol. Gen. Genet. 251:509‐517.
   Haas, R., Kahrs, A., Facius, D., Allmeier, H., Schmitt, R., and Meyer, T.F. 1993. TnMax—a versatile mini‐transposon for the analysis of cloned genes and shuttle mutagenesis. Gene 130:23‐31.
   Hamilton, H.L. and Dillard, J.P. 2006. Natural transformation of Neisseria gonorrhoeae: From DNA donation to homologous recombination. Mol. Microbiol. 59:376‐385.
   Hamilton, H.L., Schwartz, K.J., and Dillard, J.P. 2001. Insertion‐duplication mutagenesis of Neisseria: Use in characterization of DNA transfer genes in the gonococcal genetic island. J. Bacteriol. 183:4718‐4726.
   Hamilton, H.L., Dominguez, N.M., Schwartz, K.J., Hackett, K.T., and Dillard, J.P. 2005. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol. Microbiol. 55:1704‐1721.
   Hebeler, B.H. and Young, F.E. 1975. Autolysis of Neisseria gonorrhoeae. J. Bacteriol. 122:385‐392.
   Johnston, D. M. and Cannon, J.G. 1999. Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic markers using a two gene cassette with positive and negative selection. Gene 236:179‐184.
   Kado, C.I. and Liu, S. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365‐1373.
   Kellogg, D.S. Jr., Peacock, W.L. Jr., Deacon, W.E., Brown, L., and Pirkle, C.L. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274‐1279.
   Kohler, P.L., Cloud, K.A., Hackett, K.T., Beck, E.T., and Dillard, J.P. 2005. Characterization of the role of LtgB, a putative lytic transglycosylase in Neisseria gonorrhoeae. Microbiology 151:3081‐3088.
   Koomey, J.M., Gill, R.E., and Falkow, S. 1982. Genetic and biochemical analysis of gonococcal IgA1 protease: Cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity. Proc. Natl. Acad. Sci. U.S.A. 79:7881‐7885.
   Koomey, M., Gotschlich, E.C., Robbins, K., Bergstrom, S., and Swanson, J. 1987. Effects of recA mutations on pilus antigenic variation and phase transitions in Neisseria gonorrhoeae. Genetics 117:391‐398.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Kupsch, E.M., Aubel, D., Gibbs, C.P., Kahrs, A.F., Rudel, T., and Meyer, T.F. 1996. Construction of Hermes shuttle vectors: A versatile system useful for genetic complementation of transformable and nontransformable Neisseria mutants. Mol. Gen. Genet. 250:558‐569.
   LaScolea, L.J. Jr. and Young, F.E. 1974. Development of a defined minimal medium for the growth of Neisseria gonorrhoeae. Appl. Microbiol. 28:70‐76.
   Lederberg, J. 1951. Streptomycin resistance; a genetically recessive mutation. J. Bacteriol. 61:549‐550.
   Mehr, I.J. and Seifert, H.S. 1997. Random shuttle mutagenesis: Gonococcal mutants deficient in pilin antigenic variation. Mol. Microbiol. 23:1121‐1131.
   Mehr, I.J., Long, C.D., Serkin, C.D., and Seifert, H.S. 2000. A homologue of the recombination‐dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation. Genetics 154:523‐532.
   Morse, S.A. and Bartenstein, L. 1974. Factors affecting autolysis of Neisseria gonorrhoeae. Proc. Soc. Exp. Biol. Med. 145:1418‐1421.
   Morse, S.A. and Bartenstein, L. 1980. Purine metabolism in Neisseria gonorrhoeae: The requirement for hypoxanthine. Can. J. Microbiol. 26:13‐20.
   O'Dwyer, C.A., Langford, P.R., and Kroll, J.S. 2005. A novel neisserial shuttle plasmid: A useful new tool for meningococcal research. FEMS Microbiol. Lett. 251:143‐147.
   Pagotto, F.J., Salimnia, H., Totten, P.A., and Dillon, J.R. 2000. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication. Gene 244:13‐19.
   Pelicic, V., Morelle, S., Lampe, D., and Nassif, X. 2000. Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner. J. Bacteriol. 182:5391‐5398.
   Ramsey, M.E. and Dillard, J.P. 2010. Characterization of putative surface‐exposed proteins of the type IV secretion system in Neisseria gonorrhoeae. International Pathogenic Neisseria Conference P155. Banff, Canada.
   Salgado‐Pabón, W., Du, Y., Hackett, K.T., Lyons, K.M., Grove‐Arvidson, C., and Dillard, J.P. 2010. Increased expression of the type IV secretion system in piliated Neisseria gonorrhoeae variants. J. Bacteriol. 192:1912‐1920.
   Sechman, E.V., Rohrer, M.S., and Seifert, H.S. 2005. A genetic screen identifies genes and sites involved in pilin antigenic variation in Neisseria gonorrhoeae. Mol. Microbiol. 57:468‐483.
   Sechman, E.V., Kline, K.A., and Seifert, H.S. 2006. Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation. Mol. Microbiol. 61:185‐193.
   Seifert, H.S. 1996. Questions about gonococcal pilus phase‐ and antigenic variation. Mol. Microbiol. 21:433‐440.
   Seifert, H.S. 1997. Insertionally inactivated and inducible recA alleles for use in Neisseria. Gene 188:215‐220.
   Seifert, H.S., Ajioka, R.S., Paruchuri, D., Heffron, F., and So, M. 1990. Shuttle mutagenesis of Neisseria gonorrhoeae: Pilin null mutations lower DNA transformation competence. J. Bacteriol. 172:40‐46.
   Smith, H.O., Gwinn, M.L., and Salzberg, S.L. 1999. DNA uptake signal sequences in naturally transformable bacteria. Res. Microbiol. 150:603‐616.
   Sparling, P.F. 1966. Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92:1364‐1371.
   Stein, D.C. 1991. Transformation of Neisseria gonorrhoeae: Physical requirements of the transforming DNA. Can. J. Microbiol. 37:345‐349.
   Stein, D.C., Silver, L.E., Clark, V.L., and Young, F.E. 1983. Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae. Gene 25:241‐247.
   Stern, A., Brown, M., Nickel, P., and Meyer, T.F. 1986. Opacity genes in Neisseria gonorrhoeae: Control of phase and antigenic variation. Cell 47:61‐71.
   Stohl, E.A., Brockman, J.P., Burke, K.L., Morimatsu, K., Kawalczykowski, S.C., and Seifert, H.S. 2003. Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo. J. Biol. Chem. 278:2278‐2285.
   Swanson, J., Kraus, S.J., and Gotschlich, E.C. 1971. Studies on gonococcus infection I. Pili and zones of adhesion: Their relation to gonococccal growth patterns. J. Exp. Med. 134:886‐906.
   Wade, J.J. and Graver, M.A. 2007. A fully defined, clear and protein‐free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula. FEMS Microbiol. Lett. 273:35‐37.
   Wegener, W.S., Hebeler, B.H., and Morse, S.A. 1977. Cell envelope of Neisseria gonorrhoeae: Relationship between autolysis in buffer and the hydrolysis of peptidoglycan. Infect. Immun. 18:210‐219.
Key Reference
   Gunn, J.S. and Stein, D.C. 1996. See above.
  This article describes the spot transformation method that has greatly increased the frequency of obtaining nonselected mutations and also reviews the data on the many restriction systems of N. gonorrhoeae.
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