新型mucA基因突变对铜绿假单胞菌生物被膜形成的影响

The effect of a new mucA gene mutation on the biofilm formation of Pseudomonas aeruginosa

目的研究新型突变的mucA基因对黏液型铜绿假单胞菌PA17生物被膜形成过程和成熟生物被膜形态的影响。方法将PAO1的mucA基因全长克隆到铜绿假单胞菌表达质粒pUCP20上，转化到含新型mucA基因突变的黏液型铜绿假单胞菌PA17，酶切、测序鉴定；异丙基-B—D-硫代半乳糖苷（IPTG）诱导重组质粒表达，半定量逆转录（RT）-PCR检测IPTG诱导前后藻酸盐合成关键基因algD的表达水平；改良平板培养法建立PA17、含重组质粒的PA17及PAO1的生物被膜模型，扫描电镜观察其生物被膜形成过程。结果IPTG诱导后，表达重组质粒的PA17浮游状态时algD表达下降，证实PAO1的mucA基因转化PA17成功，其生物被膜形成速度居PAO1和PA17之间，8h时即可出现不可逆性黏附，6d形成成熟生物被膜，PA17、PAO1和含重组质粒的PA17所形成的成熟生物被膜形态相似。结论PA17所含的新型mucA基因突变造成PA17生物被膜形成初始阶段的不可逆黏附过程延迟，但对成熟生物被膜形态无影响。

Objective To study the effect of a new mucA gene mutation on the biofilm formation process and the morphology of matured biofilm of Pseudomonas aeruginosa. Methods The mucA gene of PAO1 was cloned into the Pseudomonas aeruginosa expression plasmid pUCP20. The recombinant plasmid was transformed to mucoid PA17 which contained a new mucA mutation. Positive clones of the plasmids were identified by enzyme digestion and sequencing. The expression levels of algD in the positive clones were assessed by semi-quantitative RT-PCR. The modified plate culture method was used to establish the biofilm models of PA17, PA17 with recombinant plasmid and PAO1 in vitro. Results Transformation was identified by the decreased expression of algD in positive clones. The rate of biofilm formation of the positive clones was between those of PAO1 and PA17. The irreversible adhesion occurred after 8 h and the matured biofilm was observed on day 6. The morphologies of PA17, PAO1 and PA17 with recombinant plasmid were the same. Conclusion The mucA gene mutation of PA17 delays the formation of irreversible adhesion of PA17 biofilm, but it has no effect on the morphology of matured biofihn.