Genetic Manipulation of Corynebacterium diphtheriae and Other Corynebacterium Species

Curr Protoc Microbiol. 2020 Sep;58(1):e111. doi: 10.1002/cpmc.111.

Abstract

This article describes several established approaches for genetic manipulation of Corynebacterium diphtheriae, the causative agent of diphtheria that is known to have provided key evidence for Koch's postulates on the germ theory. First, it includes a detailed gene deletion method that generates nonpolar, in-frame, markerless deletion mutants, utilizing the levansucrase SacB as a counter-selectable marker. Second, it provides a thorough protocol for rescuing deletion mutants using Escherichia coli-Corynebacterium shuttle vectors. Finally, a Tn5 transposon mutagenesis procedure is described. In principle, these protocols can be used for other Corynebacterium species, including Corynebacterium glutamicum and Corynebacterium matruchotii. © 2020 Wiley Periodicals LLC Basic Protocol 1: Gene deletion in Corynebacterium diphtheriae Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of Corynebacterium diphtheriae.

Keywords: Corynebacterium; Tn5 transposon; gene deletion; genetic manipulation; mutagenesis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Corynebacterium / genetics*
  • Corynebacterium diphtheriae / genetics*
  • DNA Transposable Elements
  • DNA, Bacterial
  • Diphtheria / microbiology
  • Escherichia coli / genetics
  • Gene Deletion
  • Genetic Complementation Test / methods*
  • Genetic Vectors / genetics
  • Hexosyltransferases / genetics
  • Mutagenesis, Insertional / methods*

Substances

  • DNA Transposable Elements
  • DNA, Bacterial
  • Hexosyltransferases
  • levansucrase

Supplementary concepts

  • Corynebacterium matruchotii