Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival.
Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival.
复制标题
DOI：
10.1186/1471-2180-6-102
复制
发表时间：
2006-12-18
期刊：
影响因子：
4.2
通讯作者：
Ficht TA
中科院分区：
文献类型：
--
作者：
Wu Q;Pei J;Turse C;Ficht TA
用于鉴定与布鲁氏菌毒力和存活相关的细胞功能的随机基因失活在很大程度上依赖于在G/C碱基对上整合的转座子Tn5的使用。水手家族的转座子不需要物种特异性宿主因子进行有效转座，非特异性地整合在T/A碱基对上，并且至少为基因发现提供了另一种方法。本研究利用含有高活性转座酶C9和卡那霉素抗性基因侧转座子末端倒置重复序列的质粒载体pSC189，将Himar1转座因子传递到白螺旋藻基因组中。在不到一代的时间内，为了最大限度地减少兄弟姐妹的形成，同时确保最高水平的基因组覆盖率，有效而迅速地进行了偶联。虽然先前鉴定的毒力和存活所需的基因组或类别在筛选中得到了代表，但其他新的鉴定被揭示，这可能归因于两个转座子插入序列偏差的差异。利用基于荧光的巨噬细胞筛选鉴定出的突变体，通过基于庆大霉素的巨噬细胞保护试验、小鼠脾清除模型中的存活和体外生长进一步评估，以鉴定生长速率降低的突变体。在先前描述的群体中发现新基因是预料之中的，95个基因中有近三分之二以前没有报道过使用随机tn5诱变对生存和毒力有贡献。这项工作的结果为有效的细胞内生长和生物体存活所需的调控元件、营养需求和机制提供了更多的见解。
Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage. Although previously identified groups or classes of genes required for virulence and survival were represented in the screen, additional novel identifications were revealed and may be attributable to the difference in insertion sequence biases of the two transposons. Mutants identified using a fluorescence-based macrophage screen were further evaluated using gentamicin-based protection assay in macrophages, survival in the mouse splenic clearance model and growth in vitro to identify mutants with reduced growth rates. The identification of novel genes within previously described groups was expected, and nearly two-thirds of the 95 genes had not been previously reported as contributing to survival and virulence using random Tn5-based mutagenesis. The results of this work provide added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism.
登录
查看更多内容
影响因子：
3.1
作者：
Burkhardt, S;de Bagüés, MPJ;Köhler, S
通讯作者：
Köhler, S
DOI：
10.1073/pnas.221575398
发表时间：
2002-01-08
影响因子：
11.1
作者：
DelVecchio, VG;Kapatral, V;Overbeek, R
通讯作者：
Overbeek, R
影响因子：
3.1
作者：
Haine, V;Sinon, A;De Bolle, X
通讯作者：
De Bolle, X
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
3.3
作者：
Essenberg, RC;Seshadri, R;Paulsen, I
通讯作者：
Paulsen, I