Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 ha s been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcu s faecalis to serve as a donor for the conjugative transfer of transposon T n916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of greater than or equal to 6 x 10(-8) per recipient CFU. To determine the transposon insertion site. an oligonucleotide primer corresponding to the 3' end of Tn 916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown t o insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepti cum genome. The ability to determine transposon insertion sites in mycoplas mas by genomic sequencing has not been previously described and allows rapi d sequence analysis of transposon-generated mutants. (C) 2000 Academic Pres s.