title: Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.
abstract: While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.
authors.affiliation: Cardiff University, School of Medicine, Department of Child Health, 5th floor University Hospital of Wales, Cardiff CF14 4XN, UK; Sebha University, Faculty of Engineering and Technology, Medical Laboratory Sciences Department, PO Box 68, Libya.
authors.forename: Ali F
authors.initials: AF
authors.lastname: Aboklaish
authors.affiliation: INRA, UMR 1225, IHAP, 31076 Toulouse, France; Université de Toulouse, INP, ENVT, UMR1225, IHAP, 31076 Toulouse, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Dordet-Frisoni
authors.affiliation: INRA, UMR 1225, IHAP, 31076 Toulouse, France; Université de Toulouse, INP, ENVT, UMR1225, IHAP, 31076 Toulouse, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Citti
authors.affiliation: Cardiff University, School of Medicine, Institute of Infection and Immunity, Cardiff CF14 4XN, UK.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Toleman
authors.affiliation: J. Craig Venter Institute, Rockville, MD, USA.
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.affiliation: Cardiff University, School of Medicine, Department of Child Health, 5th floor University Hospital of Wales, Cardiff CF14 4XN, UK. Electronic address: SpillerB@cardiff.ac.uk.
authors.forename: O Brad
authors.initials: OB
authors.lastname: Spiller
mesh_terms: D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques; D005827:Genetics, Microbial / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D014169:Transformation, Bacterial; D014509:Ureaplasma / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements
keywords: Gene disruption; RNA helicase; Transposon mutagenesis; Ureaplasma parvum; Ureaplasma urealyticum