Liberati Nicole T, Urbach Jonathan M, Miyata Sachiko, Lee Daniel G, Drenkard Eliana, Wu Gang, Villanueva Jacinto, Wei Tao, Ausubel Frederick M
Department of Genetics, Harvard Medical School, Boston, MA 02114, USA.
Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2833-8. doi: 10.1073/pnas.0511100103. Epub 2006 Feb 13.
Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.
随机转座子插入文库已被证明在研究细菌基因组方面具有极高价值。接近饱和的文库必须规模庞大，每个基因有多个插入，这使得全面的全基因组扫描变得困难。为便于对机会性人类病原体铜绿假单胞菌PA14菌株进行全基因组规模研究，我们构建了一个PA14转座子突变体的非冗余文库（PA14NR集），其中非必需的PA14基因由从插入突变体综合文库中选择的单个转座子插入来代表。PA14转座子插入突变体的亲本文库是通过使用MAR2xT7构建的，MAR2xT7是一种与转座子位点杂交兼容且基于水手座转座子的转座子。转座子位点杂交遗传足迹特征通过允许在不同实验条件下对混合的MAR2xT7突变体进行单独追踪，拓宽了文库的用途。开发了一个可通过互联网访问的公共数据库（PA14转座子插入突变体数据库，http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi），以促进PA14NR集的构建、分发和使用。PA14NR集在全基因组扫描表型突变体方面的实用性在筛选非生物表面附着的实验中得到了验证。将PA14转座子插入文库中被破坏的基因与在铜绿假单胞菌PAO1菌株中独立构建的插入文库进行比较，可估算铜绿假单胞菌必需基因的数量。
