Functional expression of the Flp recombinase in Mycobacterium bovis BCG

Academic Article

Abstract

  • Mycobacteria contain a large number of redundant genes whose functions are difficult to analyze in mutants because there are only two efficient antibiotic resistance genes available for allelic exchange experiments. Sequence-specific recombinbases such as the Flp recombinase can be used to excise resistance markers. Expression of the flpe gene from Saccharomyces cerevisiae is functional for this purpose in fast-growing Mycobacterium smegmatis but not in slow-growing mycobacteria such as M. bovis BCG or M. tuberculosis. We synthesized the flpm gene by adapting the codon usage to that preferred by M. tuberculosis. This increased the G + C content from 38% to 61%. Using the synthetic flpm gene, the frequency of removal of FRT-hyg-FRT cassette from the chromosome by the Flp recombinase was increased by more than 100-fold in M. smegmatis. In addition, 40% of all clones of M. bovis BCG had lost the hyg resistance cassette after transient expression of the flpm gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flpm encoded Flp recombinase is not only an improved genetic tool for M. smegmatis, but can also be used in slow growing mycobacteria such as M. tuberculosis for constructing unmarked mutations. Other more sophisticated applications in mycobacterial genetics would also profit from the improved Flp/FRT system. © 2007 Elsevier B.V. All rights reserved.
  • Published In

  • Gene  Journal
  • Digital Object Identifier (doi)

    Author List

  • Song H; Niederweis M
  • Start Page

  • 112
  • End Page

  • 119
  • Volume

  • 399
  • Issue

  • 2