Molecular and General Genetics MGG

, Volume 251, Issue 5, pp 509–517

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

  • J. S. Gunn
  • D. C. Stein
Original Paper

DOI: 10.1007/BF02173639

Cite this article as:
Gunn, J.S. & Stein, D.C. Molec. Gen. Genet. (1996) 251: 509. doi:10.1007/BF02173639

Abstract

A technique that allows for easy identification of transformants ofNeisseria 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.NgoI, 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.

Key words

DNA methyltransferase Restriction endonuclease Neisseria gonorrhoeae Transformation Mutagenesis 

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • J. S. Gunn
    • 1
  • D. C. Stein
    • 1
  1. 1.Department of MicrobiologyUniversity of MarylandCollege ParkUSA
  2. 2.Infectious Disease UnitMassachusetts General Hospital, Fruit St.BostonUSA

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