Hunt Tracey A, Kooi Cora, Sokol Pamela A, Valvano Miguel A
Department of Microbiology and Immunology, Dental Sciences Building, Rm. 3014, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Infect Immun. 2004 Jul;72(7):4010-22. doi: 10.1128/IAI.72.7.4010-4022.2004.
Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.
洋葱伯克霍尔德菌（以前称为洋葱伯克霍尔德菌复合体基因变种III）可导致囊性纤维化患者发生慢性肺部感染。在本研究中，我们采用了一种改良的标志标签诱变（STM）策略来分离无法在体内存活的洋葱伯克霍尔德菌突变体。构建了37个特殊的转座子，每个转座子携带一个独特的寡核苷酸标签标志，并用于检测2627个洋葱伯克霍尔德菌转座子突变体（以37个独特突变体为一组）在通过琼脂珠模型感染大鼠肺部10天后的存活情况。通过实时PCR对回收的突变体进行筛选，从而鉴定出260个可能未在肺内存活的突变体。将这些突变体重新分组为较小的组，并重复感染实验。经过第二轮筛选，我们分离出102个无法在大鼠模型中存活的突变体。将每个突变体中转座子的位置定位到洋葱伯克霍尔德菌染色体上。我们鉴定出参与细胞代谢、全局调控、DNA复制和修复的基因中的突变，以及那些编码细菌表面结构（包括跨膜蛋白和细胞表面多糖）的基因中的突变。此外，我们还发现了18个功能未知但在其他细菌中保守的基因。使用大鼠模型与野生型菌株竞争单独检测的12个代表性突变体的一个子集显示出存活率降低，证实了我们STM筛选的预测价值。本研究提供了一个蓝图，用于在分子水平上研究洋葱伯克霍尔德菌在气道内存活和持续存在的基础。
