Fernandes Paula J, Powell Justin A C, Archer John A C
Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK1.
Microbiology (Reading). 2001 Sep;147(Pt 9):2529-2536. doi: 10.1099/00221287-147-9-2529.
The ability to generate tagged mutants of Rhodococcus spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn5-based DNA-protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A Rhodococcus rhodochrous CW25 Tn5 insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn5 was confirmed by the presence of 9 bp direct repeats of Rhodococcus DNA flanking the transposon insertion site. To further test this system, a Tn5 insertion library was constructed in a wild-type soil isolate of Rhodococcus spp. This is the first viable transposon knockout system reported for Rhodococcus.
生成红球菌属标记突变体的能力将有助于更深入地了解这个在医学和商业上都很重要的属。这些生物体中缺乏有效的转座子系统，在此通过使用基于Tn5的DNA-蛋白质转座复合物得以克服，该复合物能够高效转座。为实现这一点，对电穿孔效率和抗生素选择进行了优化。创建了一个包含1500个突变体的红平红球菌CW25 Tn5插入文库。对23个代表性突变体进行Southern印迹分析表明插入是随机的。分离出了一些营养缺陷型突变体，并通过反向PCR和后续测序确定了涉及的 disrupted 区域。转座子插入位点两侧存在红球菌DNA的9 bp直接重复序列，证实了Tn5的转座。为进一步测试该系统，在红球菌属的野生型土壤分离株中构建了一个Tn5插入文库。这是报道的第一个适用于红球菌的可行转座子敲除系统。
