Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.
Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.
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DOI：
10.1016/j.ijmm.2014.09.003
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发表时间：
2014-11
期刊：
影响因子：
--
通讯作者：
Spiller OB
中科院分区：
文献类型：
--
作者：
Aboklaish AF;Dordet-Frisoni E;Citti C;Toleman MA;Glass JI;Spiller OB
而转座子诱变已成功应用于支原体的诱变。为了干扰和确定非必需基因，以前用解脲支原体进行的尝试。都没有成功。利用聚乙二醇转化增强方案，我们能够将含有庆大霉素抗性选择标记的基于Tn4001的微型转座子质粒转化为三个不同的微小解脲支原体血清型。尽管细小解脲原体和解脲支原体之间有很大程度的同源性，但所有平行转化解脲支原体的尝试都失败了，除了一个临床分离的解脲支原体。用聚合酶链式反应探测和测序来确认转座子插入到细菌基因组中并鉴定被破坏的基因。原型血清型3的转化始终只导致微型转座子反向重复序列之间的序列转移，但一些菌株表现出额外的序列转移。转座子插入在基因组中随机发生，导致一组筛选克隆中的单个克隆的UU047、UU390、UU440、UU450、UU520、UU526、UU582基因的独特中断。基因UU187和UU188之间的基因间插入也是特征。在突变菌株中观察到两种表型变化：死亡盒RNA解旋酶(UU582)的破坏改变了生长动力学，而解脲支原体菌株失去了对血清攻击的抵抗力，这与UUR10_137基因的破坏和41 kDa蛋白的表达丧失一致。转座子突变被成功地用于将微型转座子的单拷贝插入基因组并干扰导致微小解脲支原体株表型变化的基因。这种方法现在可以用来运送外源基因进行表达，并在培养和实验模型中确定微小解脲支原体复制的必要基因。
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.
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影响因子：
4.5
作者：
Pereyre S;Sirand-Pugnet P;Beven L;Charron A;Renaudin H;Barré A;Avenaud P;Jacob D;Couloux A;Barbe V;de Daruvar A;Blanchard A;Bébéar C
通讯作者：
Bébéar C
影响因子：
4.9
作者：
Algire, Mikkel A.;Lartigue, Carole;Merryman, Chuck
通讯作者：
Merryman, Chuck
影响因子：
3.7
作者：
Sharma S;Markham PF;Browning GF
通讯作者：
Browning GF
影响因子：
2.6
作者：
DYBVIG, K;ALDERETE, J
通讯作者：
ALDERETE, J
影响因子：
5.2
作者：
Govender, S.;Gqunta, K.;Chalkley, L. J.
通讯作者：
Chalkley, L. J.