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.

Cervical epithelial damage promotes Ureaplasma parvum ascending infection, intrauterine inflammation and preterm birth induction in mice

“…mice (aliquots were never refrozen). In contrast to the previous sheep studies, the HPA5 strain of serovar 3 U. parvum was genetically manipulated to express the Promega NanoLuc® luciferase gene by site-directed mutagenesis with minitransposon pMT85 using methods previously described 70 . The altered pMT85 plasmid contained two copies of NanoLuc® luciferase, preceded by the promoter from U. parvum tufA gene (synthesised by GenScript, Piscataway, NJ) subcloned into the XbaI site in pMT85; the sequence of the entire plasmid is available at GenBank (accession number MN275117).…”

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies

“…These antibodies included clones 8A1.2 (specific for serovar 10), 10C6.6 (specific for serovar 3), 5B1.1 (specific for serovars 3 and 14 only) and 8B5.2 (specific for serovars 1, 3, 6 and 14; all U. parvum strains). [14], as were U. parvum strains HPA2 (SV6), HPA5 (SV3) and HPA32 (SV14) which have been further characterised in other investigations [15,16].…”

SUSCEPTIBILIDADE ANTIMICROBIANA DE CEPAS DE Escherichia coli ISOLADAS DE AVES CONDENADAS POR COLIBACILOSE

“…Com base nas tabelas anteriores, pode se observar uma grande variação no perfil de sensibilidade, seja pela procedência da amostra ou a origem das cepas. Em parte essa grande variação pode ser explicada pela heterogeneidade dos sistemas de produção adotado no Brasil, clima, manejo e características de mercado (Aboklaish et al, 2014;Hsu et al, 2014;Oltramari et al, 2012). Entretanto, existe uma variação normal filogenética de cepas de E. coli, que podem ser resistente a um tipo de antimicrobianos, ou mesmo a um grupo de antimicrobianos (Dens, 2003;Hammerum & Lester & Heuer, 2010).…”