Affiliation:
1. Medical Microbiology and Immunology University of Wisconsin‐Madison Madison Wisconsin
2. Division of Infection and Immunity University College London London
Abstract
AbstractThe sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and 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. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar platesBasic Protocol 2: Spot transformation of commensal Neisseria on agar platesBasic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid cultureBasic Protocol 4: Electroporation of Neisseria gonorrhoeaeBasic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassetteBasic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ‐Tn5Basic Protocol 7: Chemical mutagenesisBasic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosomeAlternate Protocol 1: Complementation with replicating plasmidsAlternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosomeBasic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid mediumAlternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in brothSupport Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies