Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid

摘要: While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma have unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able transform three separate serovars of parvum Tn4001-based mini-transposon plasmid containing gentamicin resistance selection marker. Despite the large degree homology between urealyticum, all latter in parallel failed, exception single clinical U. urealyticum isolate. PCR probing sequencing confirm insertion into bacterial genome identify disrupted genes. Transformation prototype serovar 3 consistently resulted transfer only sequence inverted repeats, but some strains showed additional transfer. Transposon occurred randomly resulting unique disruption genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 clones from panel screened clones. An intergenic UU187 UU188 was also characterised. Two phenotypic alterations observed mutated strains: Disruption DEAD-box RNA helicase (UU582) altered growth kinetics, while strain lost serum attack coincident gene UUR10_137 loss expression 41 kDa protein. insert copies leading changes strains. This method can now be deliver exogenous essential replication culture experimental models.