Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae

Mol Gen Genet. 1996 Jul 19;251(5):509-17. doi: 10.1007/BF02173639.

Abstract

A technique that allows for easy identification of transformants of Neisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.N goI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • DNA-Cytosine Methylases / genetics*
  • DNA-Cytosine Methylases / metabolism
  • Deoxyribonucleases, Type II Site-Specific / genetics*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Genes, Bacterial / genetics
  • Genetic Vectors / genetics
  • Molecular Sequence Data
  • Neisseria gonorrhoeae / enzymology
  • Neisseria gonorrhoeae / genetics*
  • Restriction Mapping
  • Sequence Deletion / genetics
  • Transformation, Bacterial*

Substances

  • DNA, Bacterial
  • DNA-Cytosine Methylases
  • Deoxyribonucleases, Type II Site-Specific

Associated data

  • GENBANK/U42459
  • GENBANK/U42460
  • GENBANK/U43735
  • GENBANK/U43736