Functional expression of the Flp recombinase in Mycobacterium bovis BCG.
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 flp(e) 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 flp(m) 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 flp(m) 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 flp(m) gene. Sequencing of the chromosomal DNA showed that excision of the FRT-hyg-FRT cassette by Flp was specific. These results show that the flp(m) 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.
Song
Houhui
H
Department of Microbiology, University of Alabama at Birmingham, 609 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294, USA.
Niederweis
Michael
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D009163:Mycobacterium bovis / Q000235:genetics / Q000502:physiology*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase