铜绿假单胞菌蹭行运动相关基因的研究

应用Mu转座重组技术研究铜绿假单胞菌 (Pseudomonasaeruginosa)蹭行运动 (Twitchingmotility)的相关基因。通过转座突变、表型筛选 ,得到 8个Twitchingmotility缺陷或减弱的突变子。经过基因克隆、核苷酸测序研究 ,鉴定转座子插入到基因组中的位置。结果表明 ,在其中 4个突变子中 ,转座子分别插入到与IV型菌毛生物合成和功能相关的 3个已知基因中 (其中有两个突变子转座子插入到同一基因的不同位置 ) ,它们是pilV ,pilQ ,algR。另外 4个突变子中 ,有 3个是转座子分别插入到基因pilL基因的前端 ,中部和后端 ,均引起Twitchingmotility功能缺失。另一个突变子中 ,转座子插入到基因PA1 82 1中 ,引起Twitchingmotility功能减弱。PilL和PA1 82 1的编码产物均属于 3 类蛋白质 ,它们的功能是根据其保守的氨基酸基序或基因序列与已知功能基因的相似性推测得出的。但缺乏详细的试验证据。研究结果为pilL控制Twichingmotility提供了有力的证据。并证实基因PA1 82 1与Twitchingmotility有关。将Mu转座重组技术应用到假单胞菌的研究中 ,国内外均未见报道。由于该技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点。保证了表型的改变与转座子插入位点的基因突变的一一对应关系。为进一步研究铜绿假

Study on Genes Involved in Twitching Motility of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen of humans. Twitching motility mediated by type IV pili plays an important role in the pathogenesis of this organism. In this paper, Mu transposition recombination technique was utilized to study a cluster of genes required for twitching motility of P. aeruginosa. Mu DNA transposition complexes were formed in vitro, and then were electroporated into P. aeruginosa. The complexes were integrated into bacterial genomes randomly resulting in gene inactivation and phenotypical change. Eight mutants deficient in twitching motility were obtained. Gene cloning and sequencing of the flanking region of the inserted artificial Mu transposon revealed that five genes cause loss of twitching motility. Among them, the function of gene pilV, pilQ and algR has been previously experimentally demonstrated in P. aeruginosa. They are required for biogenesis and function of type IV pili. Gene pilL (PA0413) encodes a probable component of a chemotactic signal transduction system. Its function was predicted according to its conserved amino acid motif. This result suggests that PA0413 is involved in regulation of twitching motility. PA1821 is also a class 3 gene, its putative product is enoyl-CoA hydratase/isomerase.